Tim Stearns is an Emeritus Professor who, as of Sept. 2022, is Professor and Head of Laboratory at Rockefeller University and Dean of the David Rockefeller Graduate School. He previously held the Frank Lee and Carol Hall Professorship in the Department of Biology at Stanford and was Senior Associate Vice Provost of Research. He also held appointments in the Department of Genetics in Stanford Medical School, the Stanford Cancer Institute, and Bio-X, and was a Faculty Fellow in Chem-H, and an affiliated faculty member of the Center for International Security and Cooperation. Stearns is a member of JASON, a group that advises the government on matters of science, technology and national security and has also been an advisor to the National Academies of Science, President's Council of Advisors on Science and Technology, and the Defense Science Board. Stearns received a B.S. from Cornell University, a Ph.D. from MIT, and did a postdoctoral fellowship at the University of California, San Francisco. His research concerns the mechanism and regulation of cell division, the organization of signaling pathways within cells, and cell biology of fungal pathogens. Stearns was named an HHMI Professor in 2002, for his work in science education, and has taught international laboratory workshops in South Africa, Chile, Ghana, and Tanzania. He was chair of the NCSD Study Section at NIH, and served on the editorial boards of several journals.

Academic Appointments

Administrative Appointments

  • Acting Dean of Research, VPDoR (2022 - 2022)
  • Senior Associate Vice Provost of Research, VPDoR (2020 - 2022)
  • Chair, Dept. of Biology (2014 - 2020)

Current Research and Scholarly Interests

The central question behind our work is how the centrosome and primary cilium control cell function and influence development, and how defects in these structures cause a remarkable range of human disease, ranging from cancer, polycystic kidney disease, and obesity, to neurocognitive defects including mental retardation, schizophrenia, and dyslexia.

The centrosome consists of a pair of centrioles and pericentriolar material and organizes the cytoplasmic microtubules of most animal cells. Most importantly, the mother centriole (the older of the two in the pair) nucleates the formation of a primary cilium in most cells in the body. First seen by cell biologists in the 1950's, the primary cilium was ignored for many years until a combination of human and model organism genetics revealed that it is a critical sensory organelle with functions in many important processes. Defects in primary cilium structure and function cause a set of human conditions, called ciliopathies, that share a set of phenotypes that reflect the importance of the cilium in signaling pathways.

There are three main projects in the lab:

1) Ciliary biogenesis and function. In addition to the microtubules making up the interphase array and the mitotic spindle, many animal cells make a specialized microtubule structure, the primary cilium. This is a single, non-motile cilium that is able to act as a transducer of mechanical and chemical signals - sort of a cellular antenna. The microtubules of the ciliary axoneme grow directly from a centriole at their base, this centriole is often called a basal body. Some epithelial cells in the trachea, oviduct and brain produce hundreds of motile cilia on their surface, each with a centriole at their base. We are studying both the primary cilium and multi-ciliated cells for clues into ciliary structure and function, and centriole formation.

2) Cell cycle control of centrosome duplication. We have shown that duplication of the centrosome, the microtubule organizing center of animal cells, is dependent on the cell cycle kinase cdk2, and on cell cycle-specific proteolysis. We are working to determine the molecular mechanisms of centrosome duplication and to understand how centrosome duplication is controlled so that it happens once and only once per cell cycle. Cancer cells often have aberrant centrosome numbers, and we are investigating the relationship between aberrant centrosome number and the genome instability that is common in cancer cells.

3) Microtubule nucleation and organization. Microtubules are polymers of tubulin, which is a heterodimer of alpha-tubulin and beta-tubulin. We have identified a remarkable complex of proteins associated with a third type of tubulin, gamma-tubulin. Gamma-tubulin and its associated proteins are localized to the centrosome and are critical for initiation, or nucleation, of microtubule assembly. The gamma-tubulin complex (gammaTuRC) is a very large, ring-shaped complex and contains at least 6 proteins in addition to gamma-tubulin. We are determining the role of gamma-tubulin and its associated proteins in microtubule nucleation and organization.

2023-24 Courses

Stanford Advisees

Graduate and Fellowship Programs

All Publications

  • MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement. Developmental cell Tran, M. V., Khuntsariya, D., Fetter, R. D., Ferguson, J. W., Wang, J. T., Long, A. F., Cote, L. E., Wellard, S. R., Vazquez-Martinez, N., Sallee, M. D., Genova, M., Magiera, M. M., Eskinazi, S., Lee, J. D., Peel, N., Janke, C., Stearns, T., Shen, K., Lansky, Z., Magescas, J., Feldman, J. L. 2023


    Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained invivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C.elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

    View details for DOI 10.1016/j.devcel.2023.12.001

    View details for PubMedID 38159567

  • Calcineurin associates with centrosomes and regulates cilia length maintenance. Journal of cell science Tsekitsidou, E., Wong, C. J., Ulengin-Talkish, I., Barth, A. I., Stearns, T., Gingras, A. C., Wang, J. T., Cyert, M. S. 2023


    Calcineurin, or PP2B, the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain undiscovered. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal/ciliary proteins. These include POC5, which binds centrin in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT-type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin co-localizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes primary cilia elongation without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in cilia length maintenance, a process frequently disrupted in ciliopathies.

    View details for DOI 10.1242/jcs.260353

    View details for PubMedID 37013443

  • Single-molecule imaging in the primary cilium. Methods in cell biology Weiss, L. E., Love, J. F., Yoon, J., Comerci, C. J., Milenkovic, L., Kanie, T., Jackson, P. K., Stearns, T., Gustavsson, A. 2023; 176: 59-83


    The primary cilium is an important signaling organelle critical for normal development and tissue homeostasis. Its small dimensions and complexity necessitate advanced imaging approaches to uncover the molecular mechanisms behind its function. Here, we outline how single-molecule fluorescence microscopy can be used for tracking molecular dynamics and interactions and for super-resolution imaging of nanoscale structures in the primary cilium. Specifically, we describe in detail how to capture and quantify the 2D dynamics of individual transmembrane proteins PTCH1 and SMO and how to map the 3D nanoscale distributions of the inversin compartment proteins INVS, ANKS6, and NPHP3. This protocol can, with minor modifications, be adapted for studies of other proteins and cell lines to further elucidate the structure and function of the primary cilium at the molecular level.

    View details for DOI 10.1016/bs.mcb.2023.01.003

    View details for PubMedID 37164543

  • Post-mitotic centriole disengagement and maturation leads to centrosome amplification in polyploid trophoblast giant cells. Molecular biology of the cell Buss, G., Stratton, M. B., Milenkovic, L., Stearns, T. 2022: mbcE22050182


    DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Prior to differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication, or four centrioles after. We find that the average nuclear area increases approximately 8-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inhibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number, but to a limited extent compared with DNA reduplication. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

    View details for DOI 10.1091/mbc.E22-05-0182

    View details for PubMedID 36001376

  • Long-range migration of centrioles to the apical surface of the olfactory epithelium. eLife Ching, K., Wang, J. T., Stearns, T. 2022; 11


    Olfactory sensory neurons (OSNs) in vertebrates detect odorants using multiple cilia, which protrude from the end of the dendrite and require centrioles for their formation. In mouse olfactory epithelium, the centrioles originate in progenitor cells near the basal lamina, often 50 to 100 mum from the apical surface. It is unknown how centrioles traverse this distance or mature to form cilia. Using high-resolution expansion microscopy, we found that centrioles migrate together, with multiple centrioles per group and multiple groups per OSN, during dendrite outgrowth. Centrioles were found by live imaging to migrate slowly, with a maximum rate of 0.18 m/min. Centrioles in migrating groups were associated with microtubule nucleation factors, but acquired rootletin and appendages only in mature OSNs. The parental centriole had preexisting appendages, formed a single cilium before other centrioles, and retained its unique appendage configuration in the mature OSN. We developed an air-liquid interface explant culture system for OSNs and used it to show that centriole migration can be perturbed ex vivo by stabilizing microtubules. We consider these results in the context of a comprehensive model for centriole formation, migration, and maturation in this important sensory cell type.

    View details for DOI 10.7554/eLife.74399

    View details for PubMedID 35420544

  • Investigate the origins of COVID-19. Science (New York, N.Y.) Bloom, J. D., Chan, Y. A., Baric, R. S., Bjorkman, P. J., Cobey, S., Deverman, B. E., Fisman, D. N., Gupta, R., Iwasaki, A., Lipsitch, M., Medzhitov, R., Neher, R. A., Nielsen, R., Patterson, N., Stearns, T., van Nimwegen, E., Worobey, M., Relman, D. A. 2021; 372 (6543): 694

    View details for DOI 10.1126/science.abj0016

    View details for PubMedID 33986172

  • Hedgehog signaling and the primary cilium: implications for spatial and temporal constraints on signaling. Development (Cambridge, England) Ho, E. K., Stearns, T. 2021; 148 (9)


    The mechanisms of vertebrate Hedgehog signaling are linked to the biology of the primary cilium, an antenna-like organelle that projects from the surface of most vertebrate cell types. Although the advantages of restricting signal transduction to cilia are often noted, the constraints imposed are less frequently considered, and yet they are central to how Hedgehog signaling operates in developing tissues. In this Review, we synthesize current understanding of Hedgehog signal transduction, ligand secretion and transport, and cilia dynamics to explore the temporal and spatial constraints imposed by the primary cilium on Hedgehog signaling in vivo.

    View details for DOI 10.1242/dev.195552

    View details for PubMedID 33914866

  • A not-so-simple twist of fate. Developmental cell Long, A. F., Stearns, T. 2021; 56 (4): 402–4


    Multiciliated cells are considered terminally differentiated, yet tissues bearing them are remodeled during development and after injury. In this issue of Developmental Cell, Tasca etal. (2021) show that multiciliated epithelial cells are lost via two different Notch-dependent processes, apoptosis and transdifferentiation, during developmental remodeling of the Xenopus epidermis.

    View details for DOI 10.1016/j.devcel.2021.02.003

    View details for PubMedID 33621489

  • Assaying Cell Cycle Progression via Flow Cytometry in CRISPR/Cas9-Treated Cells. Methods in molecular biology (Clifton, N.J.) Geisinger, J. M., Stearns, T. 2021; 2329: 195-204


    CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors. This assay can also easily determine the effect of various interventions on obtaining a larger pool of Cas9-treated cells.

    View details for DOI 10.1007/978-1-0716-1538-6_14

    View details for PubMedID 34085224

  • The nucleus serves as the pacemaker for the cell cycle. eLife Afanzar, O., Buss, G. K., Stearns, T., Ferrell, J. E. 2020; 9


    Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, the cellular location with the fastest intrinsic rhythm should act as a pacemaker for the process. Here we traced the waves of tubulin polymerization and depolymerization that occur at mitotic entry and exit in Xenopus egg extracts back to their origins. We found that mitosis was commonly initiated at sperm-derived nuclei and their accompanying centrosomes. The cell cycle was ~20% faster at these initiation points than in the slowest regions of the extract. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.

    View details for DOI 10.7554/eLife.59989

    View details for PubMedID 33284106

  • Cilium Axoneme Internalization and Degradation in Chytrid Fungi. Cytoskeleton (Hoboken, N.J.) Venard, C. M., Vasudevan, K. K., Stearns, T. 2020


    Loss of the cilium is important for cell cycle progression and certain developmental transitions. Chytrid fungi are a group of basal fungi that have retained centrioles and cilia, and they can disassemble their cilia via axoneme internalization as part of the transition from free-swimming spores to sessile sporangia. While this type of cilium disassembly has been observed in many single-celled eukaryotes, it has not been well characterized because it is not observed in common model organisms. To better characterize cilium disassembly via axoneme internalization, we focused on chytrids Rhizoclosmatium globosum and Spizellomyces punctatus to represent two lineages of chytrids with different motility characteristics. Our results show that each chytrid species can reel in its axoneme into the cell body along its cortex on the order of minutes, while S. punctatus has additional faster ciliary compartment loss and lash-around mechanisms. S. punctatus retraction can also occur away from the cell cortex and is partially actin dependent. Post-internalization, the tubulin of the axoneme is degraded in both chytrids over the course of about 2 hours. Axoneme disassembly and axonemal tubulin degradation are both partially proteasome dependent. Overall, chytrid cilium disassembly is a fast process that separates axoneme internalization and degradation. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/cm.21637

    View details for PubMedID 33103844

  • Systematic Discovery of Short Linear Motifs Decodes Calcineurin Phosphatase Signaling. Molecular cell Wigington, C. P., Roy, J. n., Damle, N. P., Yadav, V. K., Blikstad, C. n., Resch, E. n., Wong, C. J., Mackay, D. R., Wang, J. T., Krystkowiak, I. n., Bradburn, D. A., Tsekitsidou, E. n., Hong, S. H., Kaderali, M. A., Xu, S. L., Stearns, T. n., Gingras, A. C., Ullman, K. S., Ivarsson, Y. n., Davey, N. E., Cyert, M. S. 2020


    Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

    View details for DOI 10.1016/j.molcel.2020.06.029

    View details for PubMedID 32645368

  • CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells. Nucleic acids research Geisinger, J. M., Stearns, T. n. 2020


    While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus. Cell lines with functionally wild-type TP53 exhibited higher levels of cell cycle arrest compared to lines without. Chemical inhibition of TP53 protein combined with TP53 and RB1 transcript silencing alleviated induced arrest in TP53+/+ cells. Using dCas9, we determined this arrest is driven in part by Cas9 binding to DNA. Additionally, wild-type Cas9 induced fewer 53BP1 foci in TP53+/+ cells compared to TP53-/- cells and DD-Cas9, suggesting that differences in break sensing are responsible for cell cycle arrest variation. We conclude that CRISPR/Cas9 treatment induces a cell cycle arrest dependent on functional TP53 as well as Cas9 DNA binding and cleavage. Our findings suggest that transient inhibition of TP53 may increase genome editing recovery in primary and TP53+/+ cell lines.

    View details for DOI 10.1093/nar/gkaa603

    View details for PubMedID 32687165

  • Centrioles are amplified in cycling progenitors of olfactory sensory neurons. PLoS biology Ching, K. n., Stearns, T. n. 2020; 18 (9): e3000852


    Olfaction in most animals is mediated by neurons bearing cilia that are accessible to the environment. Olfactory sensory neurons (OSNs) in chordates usually have multiple cilia, each with a centriole at its base. OSNs differentiate from stem cells in the olfactory epithelium, and how the epithelium generates cells with many centrioles is not yet understood. We show that centrioles are amplified via centriole rosette formation in both embryonic development and turnover of the olfactory epithelium in adult mice, and rosette-bearing cells often have free centrioles in addition. Cells with amplified centrioles can go on to divide, with centrioles clustered at each pole. Additionally, we found that centrioles are amplified in immediate neuronal precursors (INPs) concomitant with elevation of mRNA for polo-like kinase 4 (Plk4) and SCL/Tal1-interrupting locus gene (Stil), key regulators of centriole duplication. These results support a model in which centriole amplification occurs during a transient state characterized by elevated Plk4 and Stil in early INP cells. These cells then go on to divide at least once to become OSNs, demonstrating that cell division with amplified centrioles, known to be tolerated in disease states, can occur as part of a normal developmental program.

    View details for DOI 10.1371/journal.pbio.3000852

    View details for PubMedID 32931487

  • Transient Primary Cilia Mediate Robust Hedgehog Pathway-Dependent Cell Cycle Control. Current biology : CB Ho, E. K., Tsai, A. E., Stearns, T. n. 2020


    The regulation of proliferation is a primary function of Hedgehog (Hh) signaling in development. Hh signal transduction requires the primary cilium for several steps in the pathway [1-5]. Many cells only build a primary cilium upon cell cycle exit, in G0. In those proliferating cells that do make a cilium, it is a transient organelle, being assembled in G1 and disassembled sometime prior to mitosis [6-9]. Thus, the requirement for primary cilia presents a conundrum: how are proliferative signals conveyed through an organelle that is present for only part of the cell cycle? Here, we investigate this question in a mouse medulloblastoma cell line, SMB55, that requires cilium-mediated Hh pathway activity for proliferation [10]. We show that SMB55 cells, and the primary cerebellar granule neuron precursors (GNPs) from which they derive, are often ciliated beyond G1 into S phase, and the presence of the cilium in SMB55 cells determines the periods of Hh pathway activity. Using live imaging over multiple cell cycles, we demonstrate that Hh pathway activity in either G1-S of the previous cell cycle or G1 of the cell cycle in which the decision is made is sufficient for cell cycle entry. We also show that cyclin D1 contributes to the persistent effects of pathway activity over multiple cell cycles. Together, our results reveal that, even though the signaling organelle itself is transient, Hh pathway control of proliferation is remarkably robust. Further, primary cilium transience may have implications for other Hh-mediated events in development.

    View details for DOI 10.1016/j.cub.2020.05.004

    View details for PubMedID 32531277

  • Growth disadvantage associated with centrosome amplification drives population-level centriole number homeostasis. Molecular biology of the cell Sala, R. n., Farrell, K. C., Stearns, T. n. 2020: mbcE19040195


    The centriole duplication cycle normally ensures that centriole number is maintained at two centrioles per G1 cell. However, some circumstances can result in an aberrant increase in centriole number-a phenotype that is particularly prevalent in several types of cancer. Following an artificial increase in centriole number without tetraploidization due to transient overexpression of the kinase PLK4, human cells return to a normal centriole number during the proliferation of the population. We examine the mechanisms responsible for this return to normal centriole number at the population level in human retinal pigment epithelial cells. We find that the return to normal centriole number in the population of induced cells cannot be explained by limited duplication of centrioles, instability of extra centrioles, or by grossly asymmetric segregation of extra centrioles in mitosis. However, cells with extra centrioles display heterogenous phenotypes including extended cell cycle arrest, longer interphase durations, and death, which overall result in a proliferative disadvantage relative to normal cells in the population. Although about half of cells with extra centrioles in a population were able to divide, the extent of the disadvantages conferred by other fates is sufficient to account for the observed rate of return to normal centriole number. These results suggest that only under conditions of either positive selection for cells with extra centrioles, continuous generation of such centrioles, or alleviation of the disadvantageous growth phenotypes, would they be maintained in a population.

    View details for DOI 10.1091/mbc.E19-04-0195

    View details for PubMedID 32966175

  • Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding. PLoS biology Mirvis, M., Siemers, K. A., Nelson, W. J., Stearns, T. P. 2019; 17 (7): e3000381


    The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly is critically important, as misregulation or delay of cilia loss leads to cell cycle defects. The physical means by which cilia are lost are poorly understood but are thought to involve resorption of ciliary components into the cell body. To investigate cilium loss in mammalian cells, we used live-cell imaging to comprehensively characterize individual events. The predominant mode of cilium loss was rapid deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of Gradual resorption was followed by rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently of calcium. Together, these results suggest that mammalian ciliary loss involves a tunable decision between deciliation and resorption.

    View details for DOI 10.1371/journal.pbio.3000381

    View details for PubMedID 31314751

  • Regulation of cilia abundance in multiciliated cells ELIFE Nanjundappa, R., Kong, D., Shim, K., Stearns, T., Brody, S. L., Loncarek, J., Mahjoub, M. R. 2019; 8
  • Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Weiss, L. E., Milenkovic, L., Yoon, J., Stearns, T., Moerner, W. E. 2019; 116 (12): 5550-5557
  • Pocket similarity identifies selective estrogen receptor modulators as microtubule modulators at the taxane site NATURE COMMUNICATIONS Lo, Y., Cormier, O., Liu, T., Nettles, K. W., Katzenellenbogen, J. A., Stearns, T., Altman, R. B. 2019; 10
  • Motional dynamics of single Patched1 molecules in cilia are controlled by Hedgehog and cholesterol. Proceedings of the National Academy of Sciences of the United States of America Weiss, L. E., Milenkovic, L., Yoon, J., Stearns, T., Moerner, W. E. 2019


    The Hedgehog-signaling pathway is an important target in cancer research and regenerative medicine; yet, on the cellular level, many steps are still poorly understood. Extensive studies of the bulk behavior of the key proteins in the pathway established that during signal transduction they dynamically localize in primary cilia, antenna-like solitary organelles present on most cells. The secreted Hedgehog ligand Sonic Hedgehog (SHH) binds to its receptor Patched1 (PTCH1) in primary cilia, causing its inactivation and delocalization from cilia. At the same time, the transmembrane protein Smoothened (SMO) is released of its inhibition by PTCH1 and accumulates in cilia. We used advanced, single molecule-based microscopy to investigate these processes in live cells. As previously observed for SMO, PTCH1 molecules in cilia predominantly move by diffusion and less frequently by directional transport, and spend a fraction of time confined. After treatment with SHH we observed two major changes in the motional dynamics of PTCH1 in cilia. First, PTCH1 molecules spend more time as confined, and less time freely diffusing. This result could be mimicked by a depletion of cholesterol from cells. Second, after treatment with SHH, but not after cholesterol depletion, the molecules that remain in the diffusive state showed a significant increase in the diffusion coefficient. Therefore, PTCH1 inactivation by SHH changes the diffusive motion of PTCH1, possibly by modifying the membrane microenvironment in which PTCH1 resides.

    View details for PubMedID 30819883

  • Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy BIOPHYSICAL JOURNAL Yoon, J., Comerci, C. J., Weiss, L. E., Milenkovic, L., Stearns, T., Moernert, W. E. 2019; 116 (2): 319-329
  • Regulation of cilia abundance in multiciliated cells. eLife Nanjundappa, R. n., Kong, D. n., Shim, K. n., Stearns, T. n., Brody, S. L., Loncarek, J. n., Mahjoub, M. R. 2019; 8


    Multiciliated cells (MCC) contain hundreds of motile cilia used to propel fluid over their surface. To template these cilia, each MCC produces between 100-600 centrioles by a process termed centriole amplification. Yet, how MCC regulate the precise number of centrioles and cilia remains unknown. Airway progenitor cells contain two parental centrioles (PC) and form structures called deuterosomes that nucleate centrioles during amplification. Using an ex vivo airway culture model, we show that ablation of PC does not perturb deuterosome formation and centriole amplification. In contrast, loss of PC caused an increase in deuterosome and centriole abundance, highlighting the presence of a compensatory mechanism. Quantification of centriole abundance in vitro and in vivo identified a linear relationship between surface area and centriole number. By manipulating cell size, we discovered that centriole number scales with surface area. Our results demonstrate that a cell-intrinsic surface area-dependent mechanism controls centriole and cilia abundance in multiciliated cells.

    View details for PubMedID 31025935

  • Pocket similarity identifies selective estrogen receptor modulators as microtubule modulators at the taxane site. Nature communications Lo, Y. C., Cormier, O. n., Liu, T. n., Nettles, K. W., Katzenellenbogen, J. A., Stearns, T. n., Altman, R. B. 2019; 10 (1): 1033


    Taxanes are a family of natural products with a broad spectrum of anticancer activity. This activity is mediated by interaction with the taxane site of beta-tubulin, leading to microtubule stabilization and cell death. Although widely used in the treatment of breast cancer and other malignancies, existing taxane-based therapies including paclitaxel and the second-generation docetaxel are currently limited by severe adverse effects and dose-limiting toxicity. To discover taxane site modulators, we employ a computational binding site similarity screen of > 14,000 drug-like pockets from PDB, revealing an unexpected similarity between the estrogen receptor and the beta-tubulin taxane binding pocket. Evaluation of nine selective estrogen receptor modulators (SERMs) via cellular and biochemical assays confirms taxane site interaction, microtubule stabilization, and cell proliferation inhibition. Our study demonstrates that SERMs can modulate microtubule assembly and raises the possibility of an estrogen receptor-independent mechanism for inhibiting cell proliferation.

    View details for PubMedID 30833575

  • Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy. Biophysical journal Yoon, J., Comerci, C. J., Weiss, L. E., Milenkovic, L., Stearns, T., Moerner, W. E. 2018


    Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of 250nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (2-6 mum long and 200-300nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10-25nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

    View details for PubMedID 30598282

  • Cyclin-dependent kinase control of motile ciliogenesis ELIFE Vladar, E. K., Stratton, M. B., Saal, M. L., Salazar-De Simone, G., Wang, X., Wolgemuth, D., Stearns, T., Axelrod, J. D. 2018; 7


    Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication.

    View details for PubMedID 30152757

  • Cilium structure, assembly, and disassembly regulated by the cytoskeleton. The Biochemical journal Mirvis, M., Stearns, T., James Nelson, W. 2018; 475 (14): 2329–53


    The cilium, once considered a vestigial structure, is a conserved, microtubule-based organelle critical for transducing extracellular chemical and mechanical signals that control cell polarity, differentiation, and proliferation. The cilium undergoes cycles of assembly and disassembly that are controlled by complex inter-relationships with the cytoskeleton. Microtubules form the core of the cilium, the axoneme, and are regulated by post-translational modifications, associated proteins, and microtubule dynamics. Although actin and septin cytoskeletons are not major components of the axoneme, they also regulate cilium organization and assembly state. Here, we discuss recent advances on how these different cytoskeletal systems- affect cilium function, structure, and organization.

    View details for PubMedID 30064990

  • Cilium structure, assembly, and disassembly regulated by the cytoskeleton BIOCHEMICAL JOURNAL Mirvis, M., Stearns, T., Nelson, W. 2018; 475: 2329-2353
  • Quantifying Nanoscale Morphological Features of the Primary Cilium Membrane using Super-Resolution Fluorescence Microscopy Yoon, J., Weiss, L., Milenkovic, L., Stearns, T., Moerner, W. E. CELL PRESS. 2018: 268A
  • The ABCs of Centriole Architecture: The Form and Function of Triplet Microtubules. Cold Spring Harbor symposia on quantitative biology Wang, J. T., Stearns, T. n. 2018


    The centriole is a defining feature of many eukaryotic cells. It nucleates a cilium, organizes microtubules as part of the centrosome, and is duplicated in coordination with the cell cycle. Centrioles have a remarkable structure, consisting of microtubules arranged in a barrel with ninefold radial symmetry. At their base, or proximal end, centrioles have unique triplet microtubules, formed from three microtubules linked to each other. This microtubule organization is not found anywhere else in the cell, is conserved in all major branches of the eukaryotic tree, and likely was present in the last eukaryotic common ancestor. At their tip, or distal end, centrioles have doublet microtubules, which template the cilium. Here, we consider the structures of the compound microtubules in centrioles and discuss potential mechanisms for their formation and their function. We propose that triplet microtubules are required for the structural integrity of centrioles, allowing the centriole to serve as the essential nucleator of the cilium.

    View details for PubMedID 29540555

  • Mitosis sans Mitosis: The Mitotic Oscillator in Differentiation DEVELOPMENTAL CELL Stratton, M., Stearns, T. 2017; 43 (4): 385–86


    Differentiation and proliferation are usually considered to be antagonistic partners in development. However, in a recent issue of Science, Al Jord et al. (2017) show that key regulators of the mitotic cycle are redeployed in differentiating multiciliated cells to promote ciliogenesis without mitotic progression.

    View details for PubMedID 29161589

  • Centriole triplet microtubules are required for stable centriole formation and inheritance in human cell ELIFE Wang, J. T., Kong, D., Hoerner, C. R., Loncarek, J., Stearns, T. 2017; 6
  • Using Yeast to Determine the Functional Consequences of Mutations in the Human p53 Tumor Suppressor Gene: An Introductory Course-Based Undergraduate Research Experience in Molecular and Cell Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Hekmat-Scafe, D. S., Brownell, S. E., Seawell, P. C., Malladi, S., Imam, J. F., Singla, V., Bradon, N., Cyert, M. S., Stearns, T. 2017; 45 (2): 161-178


    The opportunity to engage in scientific research is an important, but often neglected, component of undergraduate training in biology. We describe the curriculum for an innovative, course-based undergraduate research experience (CURE) appropriate for a large, introductory cell and molecular biology laboratory class that leverages students' high level of interest in cancer. The course is highly collaborative and emphasizes the analysis and interpretation of original scientific data. During the course, students work in teams to characterize a collection of mutations in the human p53 tumor suppressor gene via expression and analysis in yeast. Initially, student pairs use both qualitative and quantitative assays to assess the ability of their p53 mutant to activate expression of reporter genes, and they localize their mutation within the p53 structure. Through facilitated discussion, students suggest possible molecular explanations for the transactivation defects displayed by their p53 mutants and propose experiments to test these hypotheses that they execute during the second part of the course. They use a western blot to determine whether mutant p53 levels are reduced, a DNA-binding assay to test whether recognition of any of three p53 target sequences is compromised, and fluorescence microscopy to assay nuclear localization. Students studying the same p53 mutant periodically convene to discuss and interpret their combined data. The course culminates in a poster session during which students present their findings to peers, instructors, and the greater biosciences community. Based on our experience, we provide recommendations for the development of similar large introductory lab courses. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(2):161-178, 2017.

    View details for DOI 10.1002/bmb.21024

    View details for Web of Science ID 000398045000010

  • Centriole triplet microtubules are required for stable centriole formation and inheritance in human cells. eLife Wang, J. T., Kong, D. n., Hoerner, C. R., Loncarek, J. n., Stearns, T. n. 2017; 6


    Centrioles are composed of long-lived microtubules arranged in nine triplets. However, the contribution of triplet microtubules to mammalian centriole formation and stability is unknown. Little is known of the mechanism of triplet microtubule formation, but experiments in unicellular eukaryotes indicate that delta-tubulin and epsilon-tubulin, two less-studied tubulin family members, are required. Here, we report that centrioles in delta-tubulin and epsilon-tubulin null mutant human cells lack triplet microtubules and fail to undergo centriole maturation. These aberrant centrioles are formed de novo each cell cycle, but are unstable and do not persist to the next cell cycle, leading to a futile cycle of centriole formation and--- disintegration. Disintegration can be suppressed by paclitaxel treatment. Delta-tubulin and epsilon-tubulin physically interact, indicating that these tubulins act together to maintain triplet microtubules and that these are necessary for inheritance of centrioles from one cell cycle to the next.

    View details for PubMedID 28906251

  • The ABCs of Centriole Architecture: The Form and Function of Triplet Microtubules Wang, J. T., Stearns, T., Stewart, D., Stillman, B. COLD SPRING HARBOR LABORATORY PRESS. 2017: 145-155
  • A Conversation with Tim Stearns Witkowski, J., Stearns, T., Stewart, D., Stillman, B. COLD SPRING HARBOR LABORATORY PRESS. 2017: 409-412

    View details for DOI 10.1101/sqb.2017.82.035360

    View details for Web of Science ID 000484900900049

    View details for PubMedID 29802152

  • Sperm Centrosomes: Kiss Your Asterless Goodbye, for Fertility's Sake. Current biology Schatten, G., Stearns, T. 2015; 25 (24): R1178-81


    Centrosomes are reduced to their cores in sperm. Emerging molecular explanations for centrosome construction have now helped to elucidate the mechanism of their destruction in sperm. Since centrosome inaccuracies cause aneuploidies responsible for cancers, birth defects and infertility, this new insight into centrosome behavior has broad implications.

    View details for DOI 10.1016/j.cub.2015.11.015

    View details for PubMedID 26702655

  • MDM1 is a microtubule-binding protein that negatively regulates centriole duplication. Molecular biology of the cell Van de Mark, D., Kong, D., Loncarek, J., Stearns, T. 2015; 26 (21): 3788-3802


    Mouse double-minute 1 (Mdm1) was originally identified as a gene amplified in transformed mouse cells and more recently as being highly up-regulated during differentiation of multiciliated epithelial cells, a specialized cell type having hundreds of centrioles and motile cilia. Here we show that the MDM1 protein localizes to centrioles of dividing cells and differentiating multiciliated cells. 3D-SIM microscopy showed that MDM1 is closely associated with the centriole barrel, likely residing in the centriole lumen. Overexpression of MDM1 suppressed centriole duplication, whereas depletion of MDM1 resulted in an increase in granular material that likely represents early intermediates in centriole formation. We show that MDM1 binds microtubules in vivo and in vitro. We identified a repeat motif in MDM1 that is required for efficient microtubule binding and found that these repeats are also present in CCSAP, another microtubule-binding protein. We propose that MDM1 is a negative regulator of centriole duplication and that its function is mediated through microtubule binding.

    View details for DOI 10.1091/mbc.E15-04-0235

    View details for PubMedID 26337392

    View details for PubMedCentralID PMC4626064

  • Zeta-Tubulin Is a Member of a Conserved Tubulin Module and Is a Component of the Centriolar Basal Foot in Multiciliated Cells CURRENT BIOLOGY Turk, E., Wills, A. A., Kwon, T., Sedzinski, J., Wallingford, J. B., Stearns, T. 2015; 25 (16): 2177-2183


    There are six members of the tubulin superfamily in eukaryotes [1]. Alpha- and beta-tubulin form a heterodimer that polymerizes to form microtubules, and gamma-tubulin nucleates microtubules as a component of the gamma-tubulin ring complex. Alpha-, beta-, and gamma-tubulin are conserved in all eukaryotes. In contrast, delta- and epsilon-tubulin are conserved in many, but not all, eukaryotes and are associated with centrioles, although their molecular function is unclear [2-7]. Zeta-tubulin is the sixth and final member of the tubulin superfamily and is largely uncharacterized. We find that zeta-, epsilon-, and delta-tubulin form an evolutionarily co-conserved module, the ZED module, that has been lost at several junctions in eukaryotic evolution and that zeta- and delta-tubulin are evolutionarily interchangeable. Humans lack zeta-tubulin but have delta-tubulin. In Xenopus multiciliated cells, zeta-tubulin is a component of the basal foot, a centriolar appendage that connects centrioles to the apical cytoskeleton, and co-localizes there with epsilon-tubulin. Depletion of zeta-tubulin results in disorganization of centriole distribution and polarity in multiciliated cells. In contrast with multiciliated cells, zeta-tubulin in cycling cells does not localize to centrioles and is associated with the TRiC/CCT cytoplasmic chaperone complex. We conclude that zeta-tubulin facilitates interactions between the centrioles and the apical cytoskeleton as a component of the basal foot in differentiated cells and propose that the ZED tubulins are important for centriole functionalization and orientation of centrioles with respect to cellular polarity axes.

    View details for DOI 10.1016/j.cub.2015.06.063

    View details for Web of Science ID 000359882200031

  • A High-Enrollment Course-Based Undergraduate Research Experience Improves Student Conceptions of Scientific Thinking and Ability to Interpret Data CBE-LIFE SCIENCES EDUCATION Brownell, S. E., Hekmat-Scafe, D. S., Singla, V., Seawell, P. C., Imam, J. F., Eddy, S. L., Stearns, T., Cyert, M. S. 2015; 14 (2)


    We present an innovative course-based undergraduate research experience curriculum focused on the characterization of single point mutations in p53, a tumor suppressor gene that is mutated in more than 50% of human cancers. This course is required of all introductory biology students, so all biology majors engage in a research project as part of their training. Using a set of open-ended written prompts, we found that the course shifts student conceptions of what it means to think like a scientist from novice to more expert-like. Students at the end of the course identified experimental repetition, data analysis, and collaboration as important elements of thinking like a scientist. Course exams revealed that students showed gains in their ability to analyze and interpret data. These data indicate that this course-embedded research experience has a positive impact on the development of students' conceptions and practice of scientific thinking.

    View details for DOI 10.1187/cbe.14-05-0092

    View details for Web of Science ID 000355555900011

    View details for PubMedID 26033869

    View details for PubMedCentralID PMC4477737

  • Observing planar cell polarity in multiciliated mouse airway epithelial cells. Methods in cell biology Vladar, E. K., Lee, Y. L., Stearns, T., Axelrod, J. D. 2015; 127: 37-54


    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type.

    View details for DOI 10.1016/bs.mcb.2015.01.016

    View details for PubMedID 25837385

  • Cell biology. Centrioles, in absentia. Science (New York, N.Y.) Stearns, T. n. 2015; 348 (6239): 1091–92

    View details for PubMedID 26045422

  • Probing mammalian centrosome structure using BioID proximity-dependent biotinylation CENTROSOME & CENTRIOLE Firat-Karalar, E. N., Stearns, T. 2015; 129: 153-170


    Understanding the structure and function of the centrosome will require identification of its constituent components and a detailed characterization of the interactions among these components. Here, we describe the application of proximity-dependent biotin identification (BioID) to identify spatial and temporal relationships among centrosome proteins. The BioID method relies on protein fusions to a promiscuous mutant of the Escherichia coli biotin ligase BirA, which biotinylates proteins that are in a ∼10 nm labeling radius of the enzyme. The biotinylated proteins are captured by affinity and are identified by mass spectrometry. Proteins identified in this way are referred to as "proximity interactors." Application of BioID to a set of centrosome proteins demonstrated the utility of this approach in overcoming inherent limitations in probing centrosome structure. These studies also demonstrated the potential of BioID for building large-scale proximity interaction maps among centrosome proteins. In this chapter, we describe the work flow for identification of proximity interactions of centrosome proteins, including materials and methods for the generation and characterization of a BirA*-fusion protein expression plasmid, expression of BirA*-fusion proteins in cells, and purification and identification of proximity partners by mass spectrometry.

    View details for DOI 10.1016/bs.mcb.2015.03.016

    View details for Web of Science ID 000363929300010

    View details for PubMedID 26175438

  • Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function MOLECULAR BIOLOGY OF THE CELL Lee, Y. L., Sante, J., Comerci, C. J., Cyge, B., Menezes, L. F., Li, F., Germino, G. G., Moerner, W. E., Takemaru, K., Stearns, T. 2014; 25 (19): 2919-2933


    Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1(-/-) cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.

    View details for DOI 10.1091/mbc.E14-02-0735

    View details for Web of Science ID 000343124100004

    View details for PubMedCentralID PMC4230582

  • Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function. Molecular biology of the cell Lee, Y. L., Santé, J., Comerci, C. J., Cyge, B., Menezes, L. F., Li, F., Germino, G. G., Moerner, W. E., Takemaru, K., Stearns, T. 2014; 25 (19): 2919-2933


    Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1(-/-) cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1(-/-) cells.

    View details for DOI 10.1091/mbc.E14-02-0735

    View details for PubMedID 25103236

  • Proteomic analysis of mammalian sperm cells identifies new components of the centrosome JOURNAL OF CELL SCIENCE Firat-Karalar, E. N., Sante, J., Elliott, S., Stearns, T. 2014; 127 (19): 4128-4133


    Centrioles are evolutionarily conserved microtubule-based structures at the core of the animal centrosome that are essential for nucleating the axoneme of cilia. We hypothesized that centriole proteins have been under-represented in proteomic studies of the centrosome, because of the larger amount of pericentriolar material making up the centrosome. In this study, we have overcome this problem by determining the centriolar proteome of mammalian sperm cells, which have a pair of centrioles but little pericentriolar material. Mass spectrometry of sperm centrioles identifies known components of centrioles and many previously uncharacterized candidate centriole proteins. Assessment of localization of a subset of these candidates in cultured cells identified CCDC113, CCDC96, C4orf47, CCDC38, C7orf31, CCDC146, CCDC81 and CCDC116 as centrosome-associated proteins. We examined the highly conserved protein CCDC113 further and found that it is a component of centriolar satellites, is in a complex with the satellite proteins HAP1 and PCM1, and functions in primary cilium formation.

    View details for DOI 10.1242/jcs.157008

    View details for Web of Science ID 000343123500004

    View details for PubMedID 25074808

    View details for PubMedCentralID PMC4179487

  • The centriole duplication cycle PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES Firat-Karalar, E. N., Stearns, T. 2014; 369 (1650)


    Centrosomes are the main microtubule-organizing centre of animal cells and are important for many critical cellular and developmental processes from cell polarization to cell division. At the core of the centrosome are centrioles, which recruit pericentriolar material to form the centrosome and act as basal bodies to nucleate formation of cilia and flagella. Defects in centriole structure, function and number are associated with a variety of human diseases, including cancer, brain diseases and ciliopathies. In this review, we discuss recent advances in our understanding of how new centrioles are assembled and how centriole number is controlled. We propose a general model for centriole duplication control in which cooperative binding of duplication factors defines a centriole 'origin of duplication' that initiates duplication, and passage through mitosis effects changes that license the centriole for a new round of duplication in the next cell cycle. We also focus on variations on the general theme in which many centrioles are created in a single cell cycle, including the specialized structures associated with these variations, the deuterosome in animal cells and the blepharoplast in lower plant cells.

    View details for DOI 10.1098/rstb.2013.0460

    View details for Web of Science ID 000339646500010

    View details for PubMedID 25047614

    View details for PubMedCentralID PMC4113104

  • Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication CURRENT BIOLOGY Firat-Karalar, E. N., Rauniyar, N., Yates, J. R., Stearns, T. 2014; 24 (6): 664-670


    The centrosome consists of a pair of centrioles and surrounding pericentriolar material (PCM). Many vertebrate cells also have an array of granules, termed centriolar satellites, that localize around the centrosome and are associated with centrosome and cilium function. Centriole duplication occurs once per cell cycle and is effected by a set of proteins including PLK4, CEP192, CEP152, CEP63, and CPAP. Information on the relationships between these components is limited due to the difficulty in assaying interactions in the context of the centrosome. Here, we used proximity-dependent biotin identification (BioID) to identify proximity interactions among centriole duplication proteins. PLK4, CEP192, and CEP152 BioID identified known physically interacting proteins and a new interaction between CEP152 and CDK5RAP2 consistent with a function of CEP152 in PCM recruitment. BioID for CEP63 and its paralog CCDC67 revealed extensive proximity interactions with centriolar satellite proteins. Focusing on these satellite proteins identified two new regulators of centriole duplication, CCDC14 and KIAA0753. Both proteins colocalize with CEP63 to satellites, bind to CEP63, and identify other satellite proteins by BioID. KIAA0753 positively regulates centriole duplication and CEP63 centrosome localization, whereas CCDC14 negatively regulates both processes. These results suggest that centriolar satellites have a previously unappreciated function in regulating centriole duplication.

    View details for DOI 10.1016/j.cub.2014.01.067

    View details for Web of Science ID 000333233300027

    View details for PubMedID 24613305

  • Centrosome-kinase fusions promote oncogenic signaling and disrupt centrosome function in myeloproliferative neoplasms. PloS one Lee, J. Y., Hong, W., Majeti, R., Stearns, T. 2014; 9 (3)

    View details for DOI 10.1371/journal.pone.0092641

    View details for PubMedID 24658090

  • Journey to the center of the centrosome. Developmental cell Stearns, T. n. 2014; 28 (6): 603–4


    Little is understood about how the centrosome, a complex organelle and signaling hub consisting of hundreds of components, is assembled. In this issue of Developmental Cell, Conduit et al. (2014) shed light on this issue, showing that modification and recruitment of Centrosomin to the centrosome center creates a dynamic pericentriolar matrix.

    View details for PubMedID 24697894

  • Centrosome-kinase fusions promote oncogenic signaling and disrupt centrosome function in myeloproliferative neoplasms. PloS one Lee, J. Y., Hong, W., Majeti, R., Stearns, T. 2014; 9 (3)


    Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype.

    View details for DOI 10.1371/journal.pone.0092641

    View details for PubMedID 24658090

  • Myb promotes centriole amplification and later steps of the multiciliogenesis program DEVELOPMENT Tan, F. E., Vladar, E. K., Ma, L., Fuentealba, L. C., Hoh, R., Espinoza, F. H., Axelrod, J. D., Alvarez-Buylla, A., Stearns, T., Kintner, C., Krasnow, M. A. 2013; 140 (20): 4277-4286


    The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

    View details for DOI 10.1242/dev.094102

    View details for Web of Science ID 000325153200017

    View details for PubMedID 24048590

    View details for PubMedCentralID PMC3787764

  • Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites NATURE Tang, Z., Lin, M. G., Stowe, T. R., Chen, S., Zhu, M., Stearns, T., Franco, B., Zhong, Q. 2013; 502 (7470): 254-?


    The primary cilium is a microtubule-based organelle that functions in sensory and signalling pathways. Defects in ciliogenesis can lead to a group of genetic syndromes known as ciliopathies. However, the regulatory mechanisms of primary ciliogenesis in normal and cancer cells are incompletely understood. Here we demonstrate that autophagic degradation of a ciliopathy protein, OFD1 (oral-facial-digital syndrome 1), at centriolar satellites promotes primary cilium biogenesis. Autophagy is a catabolic pathway in which cytosol, damaged organelles and protein aggregates are engulfed in autophagosomes and delivered to lysosomes for destruction. We show that the population of OFD1 at the centriolar satellites is rapidly degraded by autophagy upon serum starvation. In autophagy-deficient Atg5 or Atg3 null mouse embryonic fibroblasts, OFD1 accumulates at centriolar satellites, leading to fewer and shorter primary cilia and a defective recruitment of BBS4 (Bardet-Biedl syndrome 4) to cilia. These defects are fully rescued by OFD1 partial knockdown that reduces the population of OFD1 at centriolar satellites. More strikingly, OFD1 depletion at centriolar satellites promotes cilia formation in both cycling cells and transformed breast cancer MCF7 cells that normally do not form cilia. This work reveals that removal of OFD1 by autophagy at centriolar satellites represents a general mechanism to promote ciliogenesis in mammalian cells. These findings define a newly recognized role of autophagy in organelle biogenesis.

    View details for DOI 10.1038/nature12606

    View details for Web of Science ID 000325436100055

    View details for PubMedID 24089205

    View details for PubMedCentralID PMC4075283

  • Remembrance of cilia past. Cell Hoerner, C., Stearns, T. 2013; 155 (2): 271-273


    The primary cilium is thought to be disassembled prior to mitosis, freeing the centrosomes to participate in the mitotic spindle. In this issue, Paridaen et al. demonstrate that a remnant of the ciliary membrane remains attached to the mother centriole and is asymmetrically inherited in the developing neocortex.

    View details for DOI 10.1016/j.cell.2013.09.027

    View details for PubMedID 24120128

  • Myb promotes centriole amplification and later steps of the multiciliogenesis program. Development Tan, F. E., Vladar, E. K., Ma, L., Fuentealba, L. C., Hoh, R., Espinoza, F. H., Axelrod, J. D., Alvarez-Buylla, A., Stearns, T., Kintner, C., Krasnow, M. A. 2013; 140 (20): 4277-4286


    The transcriptional control of primary cilium formation and ciliary motility are beginning to be understood, but little is known about the transcriptional programs that control cilium number and other structural and functional specializations. One of the most intriguing ciliary specializations occurs in multiciliated cells (MCCs), which amplify their centrioles to nucleate hundreds of cilia per cell, instead of the usual monocilium. Here we report that the transcription factor MYB, which promotes S phase and drives cycling of a variety of progenitor cells, is expressed in postmitotic epithelial cells of the mouse airways and ependyma destined to become MCCs. MYB is expressed early in multiciliogenesis, as progenitors exit the cell cycle and amplify their centrioles, then switches off as MCCs mature. Conditional inactivation of Myb in the developing airways blocks or delays centriole amplification and expression of FOXJ1, a transcription factor that controls centriole docking and ciliary motility, and airways fail to become fully ciliated. We provide evidence that MYB acts in a conserved pathway downstream of Notch signaling and multicilin, a protein related to the S-phase regulator geminin, and upstream of FOXJ1. MYB can activate endogenous Foxj1 expression and stimulate a cotransfected Foxj1 reporter in heterologous cells, and it can drive the complete multiciliogenesis program in Xenopus embryonic epidermis. We conclude that MYB has an early, crucial and conserved role in multiciliogenesis, and propose that it promotes a novel S-like phase in which centriole amplification occurs uncoupled from DNA synthesis, and then drives later steps of multiciliogenesis through induction of Foxj1.

    View details for DOI 10.1242/dev.094102

    View details for PubMedID 24048590

  • FOP Is a Centriolar Satellite Protein Involved in Ciliogenesis PLOS ONE Lee, J. Y., Stearns, T. 2013; 8 (3)


    Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G1, when FOP protein levels are lowest, and most labeled in G2. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.

    View details for DOI 10.1371/journal.pone.0058589

    View details for PubMedID 23554904

  • The Rilp-like proteins Rilpl1 and Rilpl2 regulate ciliary membrane content. Molecular biology of the cell Schaub, J. R., Stearns, T. 2013; 24 (4): 453-464


    The primary cilium is a microtubule-based structure found in most cell types in mammals. Disruption of cilium function causes a diverse set of human diseases collectively known as ciliopathies. We report that Rab effector-related proteins Rab-interacting lysosomal protein-like 1 (Rilpl1) and Rilpl2 regulate protein localization in the primary cilium. Rilpl2 was initially identified as up-regulated in ciliating mouse tracheal epithelial cells. Rilpl1 and Rilpl2 both localize to the primary cilium and centrosome, Rilpl1 specifically to the distal end of the mother centriole. Live-cell microscopy reveals that Rilpl2 primary cilium localization is dynamic and that it is associated with tubulovesicular structures at the base of the cilium. Depletion of Rilpl1 and Rilpl2 results in accumulation of signaling proteins in the ciliary membrane and prevents proper epithelial cell organization in three-dimensional culture. These data suggest that Rilp-like proteins function in regulation of ciliary membrane protein concentration by promoting protein removal from the primary cilium.

    View details for DOI 10.1091/mbc.E12-08-0598

    View details for PubMedID 23264467

    View details for PubMedCentralID PMC3571868

  • Cell architecture: putting the building blocks together CURRENT OPINION IN CELL BIOLOGY Akhmanova, A., Stearns, T. 2013; 25 (1): 3-5

    View details for DOI 10.1016/

    View details for Web of Science ID 000315541400002

    View details for PubMedID 23279910

  • Transcriptional Program of Ciliated Epithelial Cells Reveals New Cilium and Centrosome Components and Links to Human Disease PLOS ONE Hoh, R. A., Stowe, T. R., Turk, E., Stearns, T. 2012; 7 (12)


    Defects in the centrosome and cilium are associated with a set of human diseases having diverse phenotypes. To further characterize the components that define the function of these organelles we determined the transcriptional profile of multiciliated tracheal epithelial cells. Cultures of mouse tracheal epithelial cells undergoing differentiation in vitro were derived from mice expressing GFP from the ciliated-cell specific FOXJ1 promoter (FOXJ1:GFP). The transcriptional profile of ciliating GFP+ cells from these cultures was defined at an early and a late time point during differentiation and was refined by subtraction of the profile of the non-ciliated GFP- cells. We identified 649 genes upregulated early, when most cells were forming basal bodies, and 73 genes genes upregulated late, when most cells were fully ciliated. Most, but not all, of known centrosome proteins are transcriptionally upregulated early, particularly Plk4, a master regulator of centriole formation. We found that three genes associated with human disease states, Mdm1, Mlf1, and Dyx1c1, are upregulated during ciliogenesis and localize to centrioles and cilia. This transcriptome for mammalian multiciliated epithelial cells identifies new candidate centrosome and cilia proteins, highlights similarities between components of motile and primary cilia, and identifies new links between cilia proteins and human disease.

    View details for DOI 10.1371/journal.pone.0052166

    View details for Web of Science ID 000313872600011

    View details for PubMedID 23300604

    View details for PubMedCentralID PMC3534086

  • Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling CURRENT BIOLOGY Mahjoub, M. R., Stearns, T. 2012; 22 (17): 1628-1634


    The primary cilium is a nexus of cell signaling, and ciliary dysfunction is associated with polycystic kidney disease, retinal degeneration, polydactyly, neural tube defects, and obesity (ciliopathies). Signaling molecules for cilium-associated pathways are concentrated in the cilium, and this is essential for efficient signaling. Cilia are nucleated from centrioles, and aberrant centriole numbers are seen in many cancers and in some ciliopathies. We tested the effect of supernumerary centrioles on cilium function and found that cells with extra centrioles often formed more than one cilium, had reduced ciliary concentration of Smoothened in response to Sonic hedgehog stimulation, and reduced Shh pathway transcriptional activation. This ciliary dilution phenotype was also observed with the serotonin receptor Htr6, fibrocystin PKHD1, and Arl13b. The presence of extra centrioles and cilia disrupted epithelial organization in 3D spheroid culture. Cells mutant for the tuberous sclerosis gene Tsc2 also had extra cilia and diluted ciliary protein. In most cells, extra cilia were clustered and shared the same ciliary pocket, suggesting that the ciliary pocket is the rate-limiting structure for trafficking of ciliary proteins. Thus, extra centrioles and cilia disrupt signaling and may contribute to disease phenotypes.

    View details for DOI 10.1016/j.cub.2012.06.057

    View details for Web of Science ID 000308849900037

    View details for PubMedID 22840514

  • The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium MOLECULAR BIOLOGY OF THE CELL Stowe, T. R., Wilkinson, C. J., Iqbal, A., Stearns, T. 2012; 23 (17): 3322-3335


    Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.

    View details for DOI 10.1091/mbc.E12-02-0134

    View details for Web of Science ID 000312221100009

    View details for PubMedID 22767577

    View details for PubMedCentralID PMC3431927

  • STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein BIOPHYSICAL JOURNAL Lau, L., Lee, Y. L., Sahl, S. J., Stearns, T., Moerner, W. E. 2012; 102 (12): 2926-2935


    Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ∼300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.

    View details for DOI 10.1016/j.bpj.2012.05.015

    View details for Web of Science ID 000305546500027

    View details for PubMedID 22735543

    View details for PubMedCentralID PMC3379620

  • Mechanosensing by the Primary Cilium: Deletion of Kif3A Reduces Bone Formation Due to Loading PLOS ONE Temiyasathit, S., Tang, W. J., Leucht, P., Anderson, C. T., Monica, S. D., Castillo, A. B., Helms, J. A., Stearns, T., Jacobs, C. R. 2012; 7 (3)


    Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Colα1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.

    View details for DOI 10.1371/journal.pone.0033368

    View details for Web of Science ID 000302381500135

    View details for PubMedID 22428034

    View details for PubMedCentralID PMC3299788

  • A crucial requirement for Hedgehog signaling in small cell lung cancer NATURE MEDICINE Park, K., Martelotto, L. G., Peifer, M., Sos, M. L., Karnezis, A. N., Mahjoub, M. R., Bernard, K., Conklin, J. F., Szczepny, A., Yuan, J., Guo, R., Ospina, B., Falzon, J., Bennett, S., Brown, T. J., Markovic, A., Devereux, W. L., Ocasio, C. A., Chen, J. K., Stearns, T., Thomas, R. K., Dorsch, M., Buonamici, S., Watkins, D. N., Peacock, C. D., Sage, J. 2011; 17 (11): 1504-U1506


    Small-cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer for which there is no effective treatment. Using a mouse model in which deletion of Rb1 and Trp53 in the lung epithelium of adult mice induces SCLC, we found that the Hedgehog signaling pathway is activated in SCLC cells independently of the lung microenvironment. Constitutive activation of the Hedgehog signaling molecule Smoothened (Smo) promoted the clonogenicity of human SCLC in vitro and the initiation and progression of mouse SCLC in vivo. Reciprocally, deletion of Smo in Rb1 and Trp53-mutant lung epithelial cells strongly suppressed SCLC initiation and progression in mice. Furthermore, pharmacological blockade of Hedgehog signaling inhibited the growth of mouse and human SCLC, most notably following chemotherapy. These findings show a crucial cell-intrinsic role for Hedgehog signaling in the development and maintenance of SCLC and identify Hedgehog pathway inhibition as a therapeutic strategy to slow the progression of disease and delay cancer recurrence in individuals with SCLC.

    View details for DOI 10.1038/nm.2473

    View details for PubMedID 21983857

  • Curcumin Inhibits Growth of Saccharomyces cerevisiae through Iron Chelation EUKARYOTIC CELL Minear, S., O'Donnell, A. F., Ballew, A., Giaever, G., Nislow, C., Stearns, T., Cyert, M. S. 2011; 10 (11): 1574-1581


    Curcumin, a polyphenol derived from turmeric, is an ancient therapeutic used in India for centuries to treat a wide array of ailments. Interest in curcumin has increased recently, with ongoing clinical trials exploring curcumin as an anticancer therapy and as a protectant against neurodegenerative diseases. In vitro, curcumin chelates metal ions. However, although diverse physiological effects have been documented for this compound, curcumin's mechanism of action on mammalian cells remains unclear. This study uses yeast as a model eukaryotic system to dissect the biological activity of curcumin. We found that yeast mutants lacking genes required for iron and copper homeostasis are hypersensitive to curcumin and that iron supplementation rescues this sensitivity. Curcumin penetrates yeast cells, concentrates in the endoplasmic reticulum (ER) membranes, and reduces the intracellular iron pool. Curcumin-treated, iron-starved cultures are enriched in unbudded cells, suggesting that the G(1) phase of the cell cycle is lengthened. A delay in cell cycle progression could, in part, explain the antitumorigenic properties associated with curcumin. We also demonstrate that curcumin causes a growth lag in cultured human cells that is remediated by the addition of exogenous iron. These findings suggest that curcumin-induced iron starvation is conserved from yeast to humans and underlies curcumin's medicinal properties.

    View details for DOI 10.1128/EC.05163-11

    View details for Web of Science ID 000296723600022

    View details for PubMedID 21908599

    View details for PubMedCentralID PMC3209049

  • The centrosome cycle: Centriole biogenesis, duplication and inherent asymmetries NATURE CELL BIOLOGY Nigg, E. A., Stearns, T. 2011; 13 (10): 1154-1160


    Centrosomes are microtubule-organizing centres of animal cells. They influence the morphology of the microtubule cytoskeleton, function as the base for the primary cilium and serve as a nexus for important signalling pathways. At the core of a typical centrosome are two cylindrical microtubule-based structures termed centrioles, which recruit a matrix of associated pericentriolar material. Cells begin the cell cycle with exactly one centrosome, and the duplication of centrioles is constrained such that it occurs only once per cell cycle and at a specific site in the cell. As a result of this duplication mechanism, the two centrioles differ in age and maturity, and thus have different functions; for example, the older of the two centrioles can initiate the formation of a ciliary axoneme. We discuss spatial aspects of the centrosome duplication cycle, the mechanism of centriole assembly and the possible consequences of the inherent asymmetry of centrioles and centrosomes.

    View details for DOI 10.1038/ncb2345

    View details for Web of Science ID 000295617900003

    View details for PubMedID 21968988

  • STED Super-resolution Microscopy in Drosophila Tissue and in Mammalian Cells. Proceedings of SPIE--the International Society for Optical Engineering Lau, L., Lee, Y. L., Matis, M., Axelrod, J., Stearns, T., Moerner, W. E. 2011; 7910


    Far-field super-resolution microscopy is a rapidly emerging method that is opening up opportunities for biological imaging beyond the optical diffraction limit. We have implemented a Stimulated Emission Depletion (STED) microscope to image single dye, cell, and tissue samples with 50-80 nm resolution. First, we compare the STED performance imaging single molecules of several common dyes and report a novel STED dye. Then we apply STED to image planar cell polarity protein complexes in intact fixed Drosophila tissue for the first time. Finally, we present a preliminary study of the centrosomal protein Cep164 in mammalian cells. Our images suggest that Cep164 is arranged in a nine-fold symmetric pattern around the centriole, consistent with findings suggested by cryoelectron tomography. Our work demonstrates that STED microscopy can be used for superresolution imaging in intact tissue and provides ultrastructural information in biological samples as an alternative to immuno-electron microscopy.

    View details for PubMedID 23447411

  • STED Super-resolution Microscopy in Drosophila Tissue and in Mammalian Cells Conference on Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications III Lau, L., Lee, Y. L., Matis, M., Axelrod, J., Stearns, T., Moerner, W. E. SPIE-INT SOC OPTICAL ENGINEERING. 2011

    View details for DOI 10.1117/12.881221

    View details for Web of Science ID 000297729300032

  • Cep152 interacts with Plk4 and is required for centriole duplication JOURNAL OF CELL BIOLOGY Hatch, E. M., Kulukian, A., Holland, A. J., Cleveland, D. W., Stearns, T. 2010; 191 (4): 721-729


    Centrioles are microtubule-based structures that organize the centrosome and nucleate cilia. Centrioles duplicate once per cell cycle, and duplication requires Plk4, a member of the Polo-like kinase family; however, the mechanism linking Plk4 activity and centriole formation is unknown. In this study, we show in human and frog cells that Plk4 interacts with the centrosome protein Cep152, the orthologue of Drosophila melanogaster Asterless. The interaction requires the N-terminal 217 residues of Cep152 and the crypto Polo-box of Plk4. Cep152 and Plk4 colocalize at the centriole throughout the cell cycle. Overexpression of Cep152 (1-217) mislocalizes Plk4, but both Cep152 and Plk4 are able to localize to the centriole independently of the other. Depletion of Cep152 prevents both normal centriole duplication and Plk4-induced centriole amplification and results in a failure to localize Sas6 to the centriole, an early step in duplication. Cep152 can be phosphorylated by Plk4 in vitro, suggesting that Cep152 acts with Plk4 to initiate centriole formation.

    View details for DOI 10.1083/jcb.201006049

    View details for Web of Science ID 000284737200006

    View details for PubMedID 21059850

    View details for PubMedCentralID PMC2983069

  • Cep120 is asymmetrically localized to the daughter centriole and is essential for centriole assembly JOURNAL OF CELL BIOLOGY Mahjoub, M. R., Xie, Z., Stearns, T. 2010; 191 (2): 331-346


    Centrioles form the core of the centrosome in animal cells and function as basal bodies that nucleate and anchor cilia at the plasma membrane. In this paper, we report that Cep120 (Ccdc100), a protein previously shown to be involved in maintaining the neural progenitor pool in mouse brain, is associated with centriole structure and function. Cep120 is up-regulated sevenfold during differentiation of mouse tracheal epithelial cells (MTECs) and localizes to basal bodies. Cep120 localizes preferentially to the daughter centriole in cycling cells, and this asymmetry between mother and daughter centrioles is relieved coincident with new centriole assembly. Photobleaching recovery analysis identifies two pools of Cep120, differing in their halftime at the centriole. We find that Cep120 is required for centriole duplication in cycling cells, centriole amplification in MTECs, and centriole overduplication in S phase-arrested cells. We propose that Cep120 is required for centriole assembly and that the observed defect in neuronal migration might derive from a defect in this process.

    View details for DOI 10.1083/jcb.201003009

    View details for Web of Science ID 000283391500013

    View details for PubMedID 20956381

    View details for PubMedCentralID PMC2958470

  • The life cycle of centrioles. Cold Spring Harbor symposia on quantitative biology Hatch, E., Stearns, T. 2010; 75: 425-431


    Centrioles organize the centrosome and nucleate the ciliary axoneme, and the centriole life cycle has many parallels to the chromosome cycle. The centriole cycle in animals begins at fertilization with the contribution of two centrioles by the male gamete. In the ensuing cell cycles, the duplication of centrioles is controlled temporally, spatially, and numerically. As a consequence of the duplication mechanism, the two centrioles in a typical interphase cell are of different ages and have different functions. Here, we discuss how new centrioles are assembled, what mechanisms limit centriole number, and the consequences of the inherent asymmetry of centriole duplication and segregation.

    View details for DOI 10.1101/sqb.2010.75.054

    View details for PubMedID 21502410

  • STEM CELLS A fateful age gap NATURE Stearns, T. 2009; 461 (7266): 891-892

    View details for DOI 10.1038/461891a

    View details for Web of Science ID 000270817700029

    View details for PubMedID 19829363

  • Centriole Age Underlies Asynchronous Primary Cilium Growth in Mammalian Cells CURRENT BIOLOGY Anderson, C. T., Stearns, T. 2009; 19 (17): 1498-1502


    Primary cilia are microtubule-based sensory organelles that play important roles in development and disease . They are required for Sonic hedgehog (Shh) and platelet-derived growth factor (PDGF) signaling. Primary cilia grow from the older of the two centrioles of the centrosome, referred to as the mother centriole. In cycling cells, the cilium typically grows in G1 and is lost before mitosis, but the regulation of its growth is poorly understood. Centriole duplication at G1/S results in two centrosomes, one with an older mother centriole and one with a new mother centriole, that are segregated in mitosis. Here we report that primary cilia grow asynchronously in sister cells resulting from a mitotic division and that the sister cell receiving the older mother centriole usually grows a primary cilium first. We also show that the signaling proteins inversin and PDGFRalpha localize asynchronously to sister cell primary cilia and that sister cells respond asymmetrically to Shh. These results suggest that the segregation of differently aged mother centrioles, an asymmetry inherent to every animal cell division, can influence the ability of sister cells to respond to environmental signals, potentially altering the behavior or fate of one or both sister cells.

    View details for DOI 10.1016/j.cub.2009.07.034

    View details for Web of Science ID 000269920500042

    View details for PubMedID 19682908

    View details for PubMedCentralID PMC3312602

  • Polo Kinase and Separase Regulate the Mitotic Licensing of Centriole Duplication in Human Cells DEVELOPMENTAL CELL Tsou, M. B., Wang, W., George, K. A., Uryu, K., Stearns, T., Jallepalli, P. V. 2009; 17 (3): 344-354


    It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.

    View details for DOI 10.1016/j.devcel.2009.07.015

    View details for Web of Science ID 000270017100008

    View details for PubMedID 19758559

    View details for PubMedCentralID PMC2746921

  • Plk1-Dependent Recruitment of gamma-Tubulin Complexes to Mitotic Centrosomes Involves Multiple PCM Components PLOS ONE Haren, L., Stearns, T., Luders, J. 2009; 4 (6)


    The nucleation of microtubules requires protein complexes containing gamma-tubulin, which are present in the cytoplasm and associate with the centrosome and with the mitotic spindle. We have previously shown that these interactions require the gamma-tubulin targeting factor GCP-WD/NEDD1, which has an essential role in spindle formation. The recruitment of additional gamma-tubulin to the centrosomes occurs during centrosome maturation at the G2/M transition and is regulated by the mitotic kinase Plk1. However, the molecular details of this important pathway are unknown and a Plk1 substrate that controls gamma-tubulin recruitment has not been identified. Here we show that Plk1 associates with GCP-WD in mitosis and Plk1 activity contributes to phosphorylation of GCP-WD. Plk1 depletion or inhibition prevents accumulation of GCP-WD at mitotic centrosomes, but GCP-WD mutants that are defective in Plk1-binding and -phosphorylation still accumulate at mitotic centrosomes and recruit gamma-tubulin. Moreover, Plk1 also controls the recruitment of other PCM proteins implicated in centrosomal gamma-tubulin attachment (Cep192/hSPD2, pericentrin, Cep215/Cdk5Rap2). Our results support a model in which Plk1-dependent recruitment of gamma-tubulin to mitotic centrosomes is regulated upstream of GCP-WD, involves multiple PCM proteins and therefore potentially multiple Plk1 substrates.

    View details for DOI 10.1371/journal.pone.0005976

    View details for Web of Science ID 000267237400004

    View details for PubMedID 19543530

  • Exploring the pole: an EMBO conference on centrosomes and spindle pole bodies NATURE CELL BIOLOGY Jaspersen, S. L., Stearns, T. 2008; 10 (12): 1375-1378


    The centrosome and spindle pole body community gathered for its triennial meeting from 12-16 September, 2008 at EMBL in Heidelberg (Germany).

    View details for DOI 10.1038/ncb1208-1375

    View details for Web of Science ID 000261261800003

    View details for PubMedID 19043428

  • Primary cilia: Cellular sensors for the skeleton 37th International Sun Valley Workshop on Skeletal Tissue Biology Anderson, C. T., Castillo, A. B., Brugmann, S. A., Helms, J. A., Jacobs, C. R., Stearns, T. WILEY-BLACKWELL. 2008: 1074–78


    The primary cilium is a solitary, immotile cilium that is present in almost every mammalian cell type. Primary cilia are thought to function as chemosensors, mechanosensors, or both, depending on cell type, and have been linked to several developmental signaling pathways. Primary cilium malfunction has been implicated in several human diseases, the symptoms of which include vision and hearing loss, polydactyly, and polycystic kidneys. Recently, primary cilia have also been implicated in the development and homeostasis of the skeleton. In this review, we discuss the structure and formation of the primary cilium and some of the mechanical and chemical signals to which it could be sensitive, with a focus on skeletal biology. We also raise several unanswered questions regarding the role of primary cilia as mechanosensors and chemosensors and identify potential research avenues to address these questions.

    View details for DOI 10.1002/ar.20754

    View details for Web of Science ID 000259324900004

    View details for PubMedID 18727074

    View details for PubMedCentralID PMC2879613

  • Primary cilia mediate mechanosensing in bone cells by a calcium-independent mechanism PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Malone, A. M., Anderson, C. T., Tummala, P., Kwon, R. Y., Johnston, T. R., Stearns, T., Jacobs, C. R. 2007; 104 (33): 13325-13330


    Primary cilia are sensory organelles that translate extracellular chemical and mechanical cues into cellular responses. Bone is an exquisitely mechanosensitive organ, and its homeostasis depends on the ability of bone cells to sense and respond to mechanical stimuli. One such stimulus is dynamic fluid flow, which triggers biochemical and transcriptional changes in bone cells by an unknown mechanism. Here we report that bone cells possess primary cilia that project from the cell surface and deflect during fluid flow and that these primary cilia are required for osteogenic and bone resorptive responses to dynamic fluid flow. We also show that, unlike in kidney cells, primary cilia in bone translate fluid flow into cellular responses in bone cells independently of Ca(2+) flux and stretch-activated ion channels. These results suggest that primary cilia might regulate homeostasis in diverse tissues by allowing mechanical signals to alter cellular activity via tissue-specific pathways. Our identification of a mechanism for mechanotransduction in bone could lead to therapeutic approaches for combating bone loss due to osteoporosis and disuse.

    View details for DOI 10.1073/pnas.0700636104

    View details for Web of Science ID 000248899600022

    View details for PubMedID 17673554

    View details for PubMedCentralID PMC1939687

  • Molecular characterization of centriole assembly in ciliated epithelial cells JOURNAL OF CELL BIOLOGY Vladar, E. K., Stearns, T. 2007; 178 (1): 31-42


    Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.

    View details for Web of Science ID 000247864300005

    View details for PubMedID 17606865

  • The molecular logic of the centrosome duplication cycle Tsou, B., Stearns, T. FEDERATION AMER SOC EXP BIOL. 2007: A93
  • Opinion - Microtubule-organizing centres: a re-evaluation NATURE REVIEWS MOLECULAR CELL BIOLOGY Luders, J., Stearns, T. 2007; 8 (2): 161-167


    The number, length, distribution and polarity of microtubules are largely controlled by microtubule-organizing centres, which nucleate and anchor microtubule minus ends in a process that requires gamma-tubulin. Here we discuss recent evidence indicating that gamma-tubulin-dependent formation of new microtubules is not restricted to conventional microtubule-organizing centres. These findings suggest that the spatio-temporal control of microtubule nucleation is more complex than previously thought, leading us to a re-evaluation of the concept of the microtubule-organizing center.

    View details for DOI 10.1038/nrm2100

    View details for Web of Science ID 000247564900005

    View details for PubMedID 17245416

  • Primary cilia: Mechanosensory organelles in bone cells. 28th Annual Meeting of the American-Society-for-Bone-and-Mineral-Research Malone, A. M., Anderson, C. T., Temiyasathit, S., Tang, J., Tummala, P., Stearns, T., Jacobs, C. R. WILEY-BLACKWELL. 2006: S39–S39
  • Mechanism limiting centrosome duplication to once per cell cycle NATURE Tsou, M. B., Stearns, T. 2006; 442 (7105): 947-951


    The centrosome organizes the microtubule cytoskeleton and consists of a pair of centrioles surrounded by pericentriolar material. Cells begin the cell cycle with a single centrosome, which duplicates once before mitosis. During duplication, new centrioles grow orthogonally to existing ones and remain engaged (tightly opposed) with those centrioles until late mitosis or early G1 phase, when they become disengaged. The relationship between centriole engagement/disengagement and centriole duplication potential is not understood, and the mechanisms that control these processes are not known. Here we show that centriole disengagement requires the protease separase at anaphase, and that this disengagement licences centriole duplication in the next cell cycle. We describe an in vitro system using Xenopus egg extract and purified centrioles in which both centriole disengagement and centriole growth occur. Centriole disengagement at anaphase is independent of mitotic exit and Cdk2/cyclin E activity, but requires the anaphase-promoting complex and separase. In contrast to disengagement, new centriole growth occurs in interphase, is dependent on Cdk2/cyclin E, and requires previously disengaged centrioles. This suggests that re-duplication of centrioles within a cell cycle is prevented by centriole engagement itself. We propose that the 'once-only' control of centrosome duplication is achieved by temporally separating licensing in anaphase from growth of new centrioles during S phase. The involvement of separase in both centriole disengagement and sister chromatid separation would prevent premature centriole disengagement before anaphase onset, which can lead to multipolar spindles and genomic instability.

    View details for DOI 10.1038/nature04985

    View details for Web of Science ID 000239960500044

    View details for PubMedID 16862117

  • Controlling centrosome number: licenses and blocks CURRENT OPINION IN CELL BIOLOGY Tsou, M. F., Stearns, T. 2006; 18 (1): 74-78


    Centrosomes organize microtubule structures in animal cells. The centrosome duplicates once per cell cycle in most dividing cells via a pathway that relies on a pre-existing centrosome. The molecular mechanism of this 'once and only once' control is not understood, and recent results show that centrosomes can also be assembled by a de novo pathway that bypasses this control. These results require a rethinking of how proper centrosome number is maintained. We propose that the engagement of centrioles with each other normally blocks centrosome re-duplication, and that disengagement of centrioles from each other at the end of mitosis licenses them for duplication in the subsequent cell cycle.

    View details for DOI 10.1016/

    View details for Web of Science ID 000235242700012

    View details for PubMedID 16361091

  • GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation NATURE CELL BIOLOGY Luders, J., Patel, U. K., Stearns, T. 2006; 8 (2): 137-U10


    The gamma-tubulin ring complex (gammaTuRC) is a large multi-protein complex that is required for microtubule nucleation from the centrosome. Here, we show that the GCP-WD protein (originally named NEDD1) is the orthologue of the Drosophila Dgrip71WD protein, and is a subunit of the human gammaTuRC. GCP-WD has the properties of an attachment factor for the gammaTuRC: depletion or inhibition of GCP-WD results in loss of the gammaTuRC from the centrosome, abolishing centrosomal microtubule nucleation, although the gammaTuRC is intact and able to bind to microtubules. GCP-WD depletion also blocks mitotic chromatin-mediated microtubule nucleation, resulting in failure of spindle assembly. Mitotic phosphorylation of GCP-WD is required for association of gamma-tubulin with the spindle, separately from association with the centrosome. Our results indicate that GCP-WD broadly mediates targeting of the gammaTuRC to sites of microtubule nucleation and to the mitotic spindle, which is essential for spindle formation.

    View details for DOI 10.1038/ncb1349

    View details for Web of Science ID 000235059800008

    View details for PubMedID 16378099

  • Insights into microtubule nucleation from the crystal structure of human gamma-tubulin NATURE Aldaz, H., Rice, L. M., Stearns, T., Agard, D. A. 2005; 435 (7041): 523-527


    Microtubules are hollow polymers of alphabeta-tubulin that show GTP-dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires gamma-tubulin, organized as an oligomer within the 2.2-MDa gamma-tubulin ring complex (gamma-TuRC) of higher eukaryotes. Structural insight is lacking regarding gamma-tubulin, its oligomerization and how it promotes microtubule assembly. Here we report the 2.7-A crystal structure of human gamma-tubulin bound to GTP-gammaS (a non-hydrolysable GTP analogue). We observe a 'curved' conformation for gamma-tubulin-GTPgammaS, similar to that seen for GDP-bound, unpolymerized alphabeta-tubulin. Tubulins are thought to represent a distinct class of GTP-binding proteins, and conformational switching in gamma-tubulin might differ from the nucleotide-dependent switching of signalling GTPases. A crystal packing interaction replicates the lateral contacts between alpha- and beta-tubulins in the microtubule, and this association probably forms the basis for gamma-tubulin oligomerization within the gamma-TuRC. Laterally associated gamma-tubulins in the gamma-TuRC might promote microtubule nucleation by providing a template that enhances the intrinsically weak lateral interaction between alphabeta-tubulin heterodimers. Because they are dimeric, alphabeta-tubulins cannot form microtubule-like lateral associations in the curved conformation. The lateral array of gamma-tubulins we observe in the crystal reveals a unique functional property of a monomeric tubulin.

    View details for DOI 10.1038/nature03586

    View details for Web of Science ID 000229337800060

    View details for PubMedID 15917813

  • Mammalian cells lack checkpoints for tetraploidy, aberrant centrosome number, and cytokinesis failure BMC CELL BIOLOGY Wong, C., Stearns, T. 2005; 6


    Mammalian cells have been reported to have a p53-dependent tetraploidy checkpoint that blocks cell cycle progression in G1 in response to failure of cell division. In most cases where the tetraploidy checkpoint has been observed cell division was perturbed by anti-cytoskeleton drug treatments. However, other evidence argues against the existence of a tetraploidy checkpoint. Cells that have failed to divide differ from normal cells in having two nuclei, two centrosomes, a decreased surface to volume ratio, and having undergone an abortive cytokinesis. We tested each of these to determine which, if any, cause a G1 cell cycle arrest.Primary human diploid fibroblasts with intact cell cycle checkpoints were used in all experiments. Synchronized cells exhibited G1 arrest in response to division failure caused by treatment with either cytochalasin or the myosin II inhibitor blebbistatin. The role of tetraploidy, aberrant centrosome number, and increased cell size were tested by cell/cell and cell/cytoplast fusion experiments; none of these conditions resulted in G1 arrest. Instead we found that various drug treatments of the cells resulted in cellular damage, which was the likely cause of the arrest. When cytokinesis was blocked in the absence of damage-inducing drug treatments no G1 arrest was observed.We show that neither tetraploidy, aberrant centrosome number, cell size, nor failure of cytokinesis lead to G1 arrest, suggesting that there is no tetraploidy checkpoint. Rather, certain standard synchronization treatments cause damage that is the likely cause of G1 arrest. Since tetraploid cells can cycle when created with minimal manipulation, previous reports of a tetraploidy checkpoint can probably be explained by side effects of the drug treatments used to observe them.

    View details for DOI 10.1186/1471-2121-6-6

    View details for Web of Science ID 000227559200001

    View details for PubMedID 15713235

    View details for PubMedCentralID PMC554097

  • Using femtosecond laser subcellular surgery to studycell biology Shen, N., Colvin, M., Genin, F., Huser, T., Cortopassi, G. A., Stearns, T., LeDuc, P., Ingber, D. E., Mazur, E. BIOPHYSICAL SOCIETY. 2004: 520A
  • Centrosome number is controlled by a centrosome-intrinsic block to reduplication NATURE CELL BIOLOGY Wong, C., Stearns, T. 2003; 5 (6): 539-544


    The centrosome duplicates once in S phase. To determine whether there is a block in centrosome reduplication, we used a cell fusion assay to compare the duplication potential of unduplicated G1 centrosomes and recently duplicated G2 centrosomes. By fusing cells in different cell cycle stages, we found that G2 centrosomes were unable to reduplicate in a cellular environment that supports centrosome duplication. Furthermore, G2 cytoplasm did not inhibit centrosome duplication in fused cells, indicating that the block to reduplication is intrinsic to the centrosomes rather than the cytoplasm. To test the underlying mechanism, we created mononucleate G1 cells with two centrosomes by fusing cells with enucleated cytoplasts. Both centrosomes duplicated, indicating that the block is not controlled by centrosome:nucleus ratio. We also found that human primary cells have tight control over centrosome number during prolonged S-phase arrest and that this control is partially abrogated in transformed cells. This suggests a link between the control of centrosome duplication and maintenance of genomic stability.

    View details for DOI 10.1038/ncb993

    View details for Web of Science ID 000183202800015

    View details for PubMedID 12766773

  • Centrosome biology: A SAS-sy centriole in the cell cycle CURRENT BIOLOGY Wong, C., Stearns, T. 2003; 13 (9): R351-R352


    A novel protein in Caenorhabditis elegans, SAS-4, is a component of centrioles and is required for centriole duplication. Depletion of SAS-4 results in stunted centrioles and a smaller centrosome, suggesting a link to organelle size control.

    View details for DOI 10.1016/S0960-9822(03)00273-2

    View details for Web of Science ID 000182639900009

    View details for PubMedID 12725749

  • Controlling centrosome number: Evidence for a block to centrosome over-duplication EMBO/EMBL Conference on Centrosomes and Spindle Pole Bodies Wong, C., Stearns, T. WILEY-LISS. 2003: 192–92
  • Centrosome structure and duplication EMBO/EMBL Conference on Centrosomes and Spindle Pole Bodies Stearns, T., Chang, P., Patel, U., Wong, C. WILEY-LISS. 2003: 157–57
  • Epsilon-tubulin is required for centrosome duplication and structure EMBO/EMBL Conference on Centrosomes and Spindle Pole Bodies Chang, P., Stearns, T. WILEY-LISS. 2003: 173–73
  • epsilon-tubulin is required for centriole duplication and microtubule organization NATURE CELL BIOLOGY Chang, P., Giddings, T. H., Winey, M., Stearns, T. 2003; 5 (1): 71-76


    Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified epsilon-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of epsilon-tubulin to the new centrosome can only occur after exit from S phase and that epsilon-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis epsilon-tubulin was cloned and shown to be similar to human epsilon-tubulin in both sequence and localization. Depletion of epsilon-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that epsilon-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that epsilon-tubulin is required for centriole duplication and organization of the pericentriolar material.

    View details for DOI 10.1038/ncb900

    View details for Web of Science ID 000180223700016

    View details for PubMedID 12510196

  • Characterization of delta-tubulin in animal cells 42nd Annual Meeting of the American-Society-for-Cell-Biology Ruster, K. S., Chang, P., Stearns, T. AMER SOC CELL BIOLOGY. 2002: 197A–198A
  • Controlling centrosome number: Evidence for a block to centrosome over-duplication 42nd Annual Meeting of the American-Society-for-Cell-Biology Wong, C., Stearns, T. AMER SOC CELL BIOLOGY. 2002: 50A–50A
  • gamma-tubulin CURRENT BIOLOGY Patel, U., Stearns, T. 2002; 12 (12): R408-R409

    View details for Web of Science ID 000176468000005

    View details for PubMedID 12123586

  • Systematic structure-function analysis of the small GTPase Arf1 in yeast MOLECULAR BIOLOGY OF THE CELL Click, E. S., Stearns, T., Botstein, D. 2002; 13 (5): 1652-1664


    Members of the ADP-ribosylation factor (Arf) family of small GTPases are implicated in vesicle traffic in the secretory pathway, although their precise function remains unclear. We generated a series of 23 clustered charge-to-alanine mutations in the Arf1 protein of Saccharomyces cerevisiae to determine the portions of this protein important for its function in cells. These mutants display a number of phenotypes, including conditional lethality at high or low temperature, defects in glycosylation of invertase, dominant lethality, fluoride sensitivity, and synthetic lethality with the arf2 null mutation. All mutations were mapped onto the available crystal structures for Arf1p: Arf1p bound to GDP, to GTP, and complexed with the regulatory proteins ArfGEF and ArfGAP. From this systematic structure-function analysis we demonstrate that all essential mutations studied map to one hemisphere of the protein and provide strong evidence in support of the proposed ArfGEF contact site on Arf1p but minimal evidence in support of the proposed ArfGAP-binding site. In addition, we describe the isolation of a spatially distant intragenic suppressor of a dominant lethal mutation in the guanine nucleotide-binding region of Arf1p.

    View details for DOI 10.1091/mbc.02-01-0007

    View details for Web of Science ID 000175812900018

    View details for PubMedID 12006660

    View details for PubMedCentralID PMC111134

  • GCP5 and GCP6: Two new members of the human gamma-tubulin complex MOLECULAR BIOLOGY OF THE CELL Murphy, S. M., Preble, A. M., Patel, U. K., O'Connell, K. L., Dias, D. P., Moritz, M., Agard, D., Stults, J. T., Stearns, T. 2001; 12 (11): 3340-3352


    The gamma-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human gamma-tubulin complex and the identification of its subunits. The human gamma-tubulin complex is a ring of ~25 nm, has a subunit structure similar to that reported for gamma-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human gamma-tubulin complex components confirmed the presence of four previously identified components (gamma-tubulin and gamma-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the gamma-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire gamma-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of gamma-tubulin, GCP2, GCP3, and GCP4 within the gamma-tubulin complex. Thus, the gamma-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.

    View details for Web of Science ID 000172357200004

    View details for PubMedID 11694571

    View details for PubMedCentralID PMC60259

  • Pericentrin interacts with GCP 2/3 to direct assembly of soluble gamma tubulin ring complexes onto centrosomes Zimmerman, W., Sillibourne, J., Dictenberg, J., Murphy, S., Stearns, T., Doxsey, S. J. AMER SOC CELL BIOLOGY. 2001: 439A
  • Centrosome duplication: A centriolar pas de deux CELL Stearns, T. 2001; 105 (4): 417-420

    View details for Web of Science ID 000168840700001

    View details for PubMedID 11371338

  • Molecular mechanisms of centrosome duplication Piard-Ruster, K. S., Reynolds-Lacey, K., Chang, P., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 342A–342A
  • Characterization of the human gamma-tubulin complex Patel, U., Murphy, S. M., Preble, A. M., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 187A
  • GIG1, a novel S-cerevisiae gene, encodes a protein that interacts with gamma-tubulin. Marschall, L. G., Chiem, K., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 188A
  • Pericentrin interacts with members of the gamma tubulin complex. Zimmerman, W. C., Murphy, S., Stearns, T., Doxsey, S. J. AMER SOC CELL BIOLOGY. 2000: 201A
  • Genetic analysis of ADP-Ribosylation Factor 1 (ARF1) in yeast Click, E. S., Stearns, T., Botstein, D. AMER SOC CELL BIOLOGY. 2000: 210A
  • Genetic analysis of the role of Cdc28p in spindle pole body duplication Byrnes, M. J., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 343A
  • Defining the human gamma-tubulin complex: identification of two new components as members of the GCP superfamily. Murphy, S. M., Preble, A., Patel, U. A., O'Connell, K., Stults, J., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 361A
  • The DNA-damage checkpoint signal in budding yeast is nuclear-limited. Demeter, J., Lee, S. E., Haber, J. E., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 38A
  • Does the presence of multiple centrosomes lead to aneuploidy? Wong, C. C., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 203A–203A
  • Delta-tubulin and epsilon-tubulin: new tubulins at the centrosome Chang, P., Stearns, T. AMER SOC CELL BIOLOGY. 2000: 552A–552A
  • The DNA damage checkpoint signal in budding yeast is nuclear limited MOLECULAR CELL Demeter, J., Lee, S. E., Haber, J. E., Stearns, T. 2000; 6 (2): 487-492


    The nature of the DNA damage-induced checkpoint signal that causes the arrest of cells prior to mitosis is unknown. To determine if this signal is transmitted through the cytoplasm or is confined to the nucleus, we created binucleate heterokaryon yeast cells in which one nucleus suffered an unrepairable double-strand break, and the second nucleus was undamaged. In most of these binucleate cells, the damaged nucleus arrested prior to spindle elongation, while the undamaged nucleus completed mitosis, even when the strength of the damage signal was increased. The arrest of the damaged nucleus was dependent upon the function of the RAD9 checkpoint gene. Thus, the DNA damage checkpoint causing G2/M arrest is regulated by a signal that is nuclear limited.

    View details for PubMedID 10983994

  • delta-Tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function NATURE CELL BIOLOGY Chang, P., Stearns, T. 2000; 2 (1): 30-35


    The centrosome organizes microtubules, which are made up of alpha-tubulin and beta-tubulin, and contains centrosome-bound gamma-tubulin, which is involved in microtubule nucleation. Here we identify two new human tubulins and show that they are associated with the centrosome. One is a homologue of the Chlamydomonas delta-tubulin Uni3, and the other is a new tubulin, which we have named epsilon-tubulin. Localization of delta-tubulin and epsilon-tubulin to the centrosome is independent of microtubules, and the patterns of localization are distinct from each other and from that of gamma-tubulin. Delta-tubulin is found in association with the centrioles, whereas epsilon-tubulin localizes to the pericentriolar material. epsilon-Tubulin exhibits a cell-cycle-specific pattern of localization, first associating with only the older of the centrosomes in a newly duplicated pair and later associating with both centrosomes. epsilon-Tubulin thus distinguishes the old centrosome from the new at the level of the pericentriolar material, indicating that there may be a centrosomal maturation event that is marked by the recruitment of epsilon-tubulin.

    View details for Web of Science ID 000084843600016

    View details for PubMedID 10620804

  • Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae Cold Spring Harbor Symposium on Quantitative Biology Lee, S. E., Pellicioli, A., Demeter, J., Vaze, M. P., Gasch, A. P., Malkova, A., Brown, P. O., Botstein, D., Stearns, T., Foiani, M., Haber, J. E. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 2000: 303–314

    View details for Web of Science ID 000169676800031

    View details for PubMedID 12760044

  • gamma-Tubulin complexes: size does matter TRENDS IN CELL BIOLOGY Jeng, R., Stearns, T. 1999; 9 (9): 339-342


    gamma-Tubulin is a conserved component of all microtubule-organizing centres and is required for these organelles to nucleate microtubule polymerization. However, the mechanism of nucleation is not known. In addition to its localization to organizing centres, a large pool of gamma-tubulin exists in the cytoplasm in a complex with other proteins. The size of the gamma-tubulin complex and number of associated proteins vary among organisms, and the functional significance of these differences is unknown. Recently, the nature of these gamma-tubulin complexes has been explored in different organisms, and this has led us closer to a molecular understanding of microtubule nucleation.

    View details for Web of Science ID 000082139400002

    View details for PubMedID 10461186

  • Components of an SCE ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle GENES & DEVELOPMENT Freed, E., Lacey, K. R., Huie, P., Lyapina, S. A., Deshaies, R. J., Stearns, T., Jackson, P. K. 1999; 13 (17): 2242-2257


    Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.

    View details for Web of Science ID 000082647200006

    View details for PubMedID 10485847

    View details for PubMedCentralID PMC316987

  • Primer - The centrosome CURRENT BIOLOGY Urbani, L., Stearns, T. 1999; 9 (9): R315-R317

    View details for Web of Science ID 000080232900004

    View details for PubMedID 10322119

  • Cyclin-dependent kinase control of centrosome duplication PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lacey, K. R., Jackson, P. K., Stearns, T. 1999; 96 (6): 2817-2822


    Centrosomes nucleate microtubules and duplicate once per cell cycle. This duplication and subsequent segregation in mitosis results in maintenance of the one centrosome/cell ratio. Centrosome duplication occurs during the G1/S transition in somatic cells and must be coupled to the events of the nuclear cell cycle; failure to coordinate duplication and mitosis results in abnormal numbers of centrosomes and aberrant mitoses. Using both in vivo and in vitro assays, we show that centrosome duplication in Xenopus laevis embryos requires cyclin/cdk2 kinase activity. Injection of the cdk (cyclin-dependent kinase) inhibitor p21 into one blastomere of a dividing embryo blocks centrosome duplication in that blastomere; the related cdk inhibitor p27 has a similar effect. An in vitro system using Xenopus extracts carries out separation of the paired centrioles within the centrosome. This centriole separation activity is dependent on cyclin/cdk2 activity; depletion of either cdk2 or of the two activating cyclins, cyclin A and cyclin E, eliminates centriole separation activity. In addition, centriole separation is inhibited by the mitotic state, suggesting a mechanism of linking the cell cycle to periodic duplication of the centrosome.

    View details for Web of Science ID 000079224500048

    View details for PubMedID 10077594

    View details for PubMedCentralID PMC15852

  • Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin JOURNAL OF CELL BIOLOGY Feierbach, B., Nogales, E., Downing, K. H., Stearns, T. 1999; 144 (1): 113-124


    Tubulin is a heterodimer of alpha- and beta-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287-296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821-832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with alpha-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with alpha-tubulin. Mutations in alpha-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of alpha-tubulin; this domain is distinct from the region of interaction between alpha-tubulin and beta-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in alpha-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast alpha-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional alpha-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester alpha-tubulin from interaction with beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

    View details for Web of Science ID 000078084800011

    View details for PubMedID 9885248

    View details for PubMedCentralID PMC2148126

  • Cytoskeletal dynamics in yeast METHODS IN CELL BIOLOGY, VOL 58 Carminati, J. L., Stearns, T. 1999; 58: 87-105

    View details for Web of Science ID 000165166500006

    View details for PubMedID 9891376

  • Centrosome reduction during mouse spermiogenesis DEVELOPMENTAL BIOLOGY Manandhar, G., Sutovsky, P., Joshi, H. C., Stearns, T., Schatten, G. 1998; 203 (2): 424-434


    The sperm does not contribute the centrosome during murine fertilization. To determine the manner in which a functional centrosome is reduced, we have studied centrosome degeneration during spermiogenesis of mice. The round spermatids display normal centrosomes consisting of a pair of centrioles along with gamma-tubulin containing foci. However, they do not seem to organize microtubules. Elongating spermatids display gamma-tubulin spots in the neck region, while microtubules are organized from the perinuclear ring as the manchette. Electron microscopic studies using immunogold labeling revealed that gamma-tubulin is mainly localized in the centriolar adjunct from which an aster of microtubules emanates. Microtubules repolymerized randomly in the cytoplasm after nocodazole treatment and reversal. gamma-Tubulin dissociates from the neck region and is discarded in the residual bodies during spermiation. The distal centriole degenerates during testicular stage of spermiogenesis, while the proximal centriole is lost during epididymal stage. Loss of centrosomal protein and centrioles in mouse sperm further confirm the maternal inheritance of centrosome during murine fertilization.

    View details for PubMedID 9808791

  • The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components Spc97p and Spc98p JOURNAL OF CELL BIOLOGY Murphy, S. M., Urbani, L., Stearns, T. 1998; 141 (3): 663-674


    gamma-Tubulin is a universal component of microtubule organizing centers where it is believed to play an important role in the nucleation of microtubule polymerization. gamma-Tubulin also exists as part of a cytoplasmic complex whose size and complexity varies in different organisms. To investigate the composition of the cytoplasmic gamma-tubulin complex in mammalian cells, cell lines stably expressing epitope-tagged versions of human gamma-tubulin were made. The epitope-tagged gamma-tubulins expressed in these cells localize to the centrosome and are incorporated into the cytoplasmic gamma-tubulin complex. Immunoprecipitation of this complex identifies at least seven proteins, with calculated molecular weights of 48, 71, 76, 100, 101, 128, and 211 kD. We have identified the 100- and 101-kD components of the gamma-tubulin complex as homologues of the yeast spindle pole body proteins Spc97p and Spc98p, and named the corresponding human proteins hGCP2 and hGCP3. Sequence analysis revealed that these proteins are not only related to their respective homologues, but are also related to each other. GCP2 and GCP3 colocalize with gamma-tubulin at the centrosome, cosediment with gamma-tubulin in sucrose gradients, and coimmunoprecipitate with gamma-tubulin, indicating that they are part of the gamma-tubulin complex. The conservation of a complex involving gamma-tubulin, GCP2, and GCP3 from yeast to mammals suggests that structurally diverse microtubule organizing centers such as the yeast spindle pole body and the animal centrosome share a common molecular mechanism for microtubule nucleation.

    View details for Web of Science ID 000073499300009

    View details for PubMedID 9566967

    View details for PubMedCentralID PMC2132743

  • Parallel analysis of genetic selections using whole genome oligonucleotide arrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Cho, R. J., Fromont-Racine, M., Wodicka, L., Feierbach, B., Stearns, T., Legrain, P., Lockhart, D. J., Davis, R. W. 1998; 95 (7): 3752-3757


    Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.

    View details for Web of Science ID 000072848500079

    View details for PubMedID 9520439

    View details for PubMedCentralID PMC19909

  • Expression of amino- and carboxyl-terminal gamma- and alpha-tubulin mutants in cultured epithelial cells JOURNAL OF BIOLOGICAL CHEMISTRY Leask, A., Stearns, T. 1998; 273 (5): 2661-2668


    Three distinct tubulin proteins are essential for microtubule function: alpha-, beta-, and gamma-tubulin. After translation, alpha- and beta-tubulin proteins combine into a soluble, 7 S heterodimer that is multimerized to form the microtubule filament. Conversely, gamma-tubulin combines with several proteins into a soluble, 25 S multi-protein particle, the gammasome that is essential for nucleating microtubule filaments at the centrosome. The proteins that assist tubulins in executing their specific functions are largely unknown. As an initial approach to address this issue, we first decided to identify domains of mammalian alpha- and gamma-tubulin necessary for their function by creating mutant mammalian alpha- and gamma-tubulin (both deletion and hybrid mutants) and assaying their behavior in stably transfected Chinese hamster ovary epithelial cells. First, we demonstrated that addition of a carboxyl-terminal epitope tag had no effect on the subcellular localization of either alpha- and gamma-tubulin. Second, we found that both the amino and carboxyl termini of gamma-tubulin were essential for its incorporation into the gammasome. Third, we found that the amino and carboxyl termini of alpha-tubulin were necessary for incorporation of the alpha-beta-tubulin heterodimer into the microtubule filament network. In general, alpha-tubulin sequences could not replace those of gamma-tubulin and vice versa. Taken together, these results suggest that the amino and carboxyl termini of alpha- and gamma-tubulin and perhaps regions throughout these proteins were necessary for their specific functions.

    View details for Web of Science ID 000071736600025

    View details for PubMedID 9446570

  • Nucleation and capture of large cell surface-associated microtubule arrays that are not located near centrosomes in certain cochlear epithelial cells JOURNAL OF ANATOMY Tucker, J. B., Mogensen, M. M., Henderson, C. G., Doxsey, S. J., Wright, M., Stearns, T. 1998; 192: 119-130


    This report deals with the as yet undetermined issue of whether cell-surface associated microtubules in certain cochlear epithelial cells are centrosomally nucleated and subsequently migrate to microtubule-capturing sites located at the surface regions in question. Alternatively, the cells may possess additional nucleating sites which are noncentrosomal and surface-associated. These alternative possibilities have been investigated for highly polarised epithelial cells called supporting cells in the mouse and guinea pig organ of Corti using antibodies to pericentrin and gamma-tubulin. There is substantial evidence that both proteins are essential components of microtubule-nucleating sites in cells generally. Each mature supporting cell possesses a large microtubule array that is remotely located with respect to its centrosome (more than 10 microns away). The antibodies bind to a cell's centrosome. No binding has been detected at 2 other microtubule-organising centres that are associated with the ends of the centrosomally-remote microtubule array while it is being constructed. Such arrays include thousands of microtubules in some of the cell types that have been examined. If all a cell's microtubules are nucleated by its centrosome then the findings reported above imply that microtubules escape from the centrosomal nucleating site and migrate to a new location. Furthermore capture of the plus and minus ends of the errant microtubules is taking place because both ends of a centrosomally-remote microtubule array are attached to sites that are precisely positioned at certain cell surface locations. Minus ends are locating targets with an exactitude comparable to that which has been demonstrated for plus ends in certain cell types. These cells apparently operate a single control centre strategy for microtubule nucleation that is complemented by precise positioning of plus and minus end-capturing sites at the cell surface.

    View details for Web of Science ID 000072900100012

    View details for PubMedID 9568567

    View details for PubMedCentralID PMC1467745

  • Cytoskeleton: Anatomy of an organizing center CURRENT BIOLOGY Marschall, L. G., Stearns, T. 1997; 7 (12): R754-R756


    One component of the yeast spindle pole body, Spc42p, has been found to form a crystalline array within one of the central layers of the structure; the Spc42p crystal might provide a scaffold around which the spindle pole body is assembled, and could be involved in regulating the size of the spindle pole body.

    View details for Web of Science ID A1997YL44000009

    View details for PubMedID 9382821

  • The cell center at 100 CELL Stearns, T., Winey, M. 1997; 91 (3): 303-309

    View details for Web of Science ID A1997YD94100004

    View details for PubMedID 9363939

  • Motoring to the finish: Kinesin and dynein work together to orient the yeast mitotic spindle JOURNAL OF CELL BIOLOGY Stearns, T. 1997; 138 (5): 957-960

    View details for Web of Science ID A1997XW46300002

    View details for PubMedID 9281575

    View details for PubMedCentralID PMC2136760

  • Tubulin subunits exist in an activated conformational state generated and maintained by protein cofactors JOURNAL OF CELL BIOLOGY Tian, G. L., Lewis, S. A., Feierbach, B., Stearns, T., Rommelaere, H., Ampe, C., Cowan, N. J. 1997; 138 (4): 821-832


    The production of native alpha/beta tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on beta-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from alpha-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of alpha-tubulin folding in vitro. This enabled an integrated study of alpha- and beta-tubulin folding: we find that the pathways leading to the formation of native alpha- and beta-tubulin converge in that the folding of the alpha subunit requires the participation of cofactor complexes containing the beta subunit and vice versa. We also show that sequestration of native alpha-or beta-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining alpha-and beta-tubulin polypeptides in an activated conformational state required for the formation of native alpha/beta heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.

    View details for Web of Science ID A1997XU36000008

    View details for PubMedID 9265649

  • Microtubules orient the mitotic spindle in yeast through dynein-dependent interactions with the cell cortex JOURNAL OF CELL BIOLOGY Carminati, J. L., Stearns, T. 1997; 138 (3): 629-641


    Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)-tubulin fusion protein to observe microtubules in living yeast cells. GFP-tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.

    View details for Web of Science ID A1997XR47800013

    View details for PubMedID 9245791

  • Synaptically coupled central nervous system neurons lack centrosomal gamma-tubulin NEUROSCIENCE LETTERS Leask, A., Obrietan, K., Stearns, T. 1997; 229 (1): 17-20


    In cycling cells, microtubule assembly is initiated at the centrosome and requires the centrosomal protein gamma-tubulin. Previously, it was reported that gamma-tubulin is present at the centrosome of cervical ganglion cells undergoing axonal growth, but not in the axons or dendrites. We find that although gamma-tubulin is present at the centrosomes of neurons just beginning to extend processes, it is not associated with centrosomes in hypothalamic and cortical neurons on which functional synaptic connections have formed. In contrast, another centrosomal protein, pericentrin, is associated with the centrosome at all stages. These results suggest that centrosomal microtubule nucleation is required for early stages of neurogenesis to supply sufficient microtubule polymer to support rapid axonal growth, but is not required for maintenance of axonal microtubules in synaptically coupled neurons.

    View details for Web of Science ID A1997XK37000005

    View details for PubMedID 9224791

  • Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin JOURNAL OF CELL BIOLOGY Vogel, J. M., Stearns, T., Rieder, C. L., Palazzo, R. E. 1997; 137 (1): 193-202


    Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

    View details for Web of Science ID A1997WU02800017

    View details for PubMedID 9105047

    View details for PubMedCentralID PMC2139867

  • Centrosomal deployment of gamma-tubulin and pericentrin: Evidence for a microtubule-nucleating domain and a minus-end docking domain in certain mouse epithelial cells CELL MOTILITY AND THE CYTOSKELETON Mogensen, M. M., Mackie, J. B., Doxsey, S. J., Stearns, T., Tucker, J. B. 1997; 36 (3): 276-290


    This report provides evidence for two functionally and spatially distinct centrosomal domains in certain mouse cochlear epithelial cells. The vast majority of microtubules elongate from sites associated with the apical cell surface in these cells rather than from pericentriolar material surrounding the immediate environs of their apically situate centrioles. The distribution of gamma-tubulin and pericentrin at cell apices has been examined while microtubule nucleation is progressing because these centrosomal proteins are believed to be essential for microtubule nucleation. Antibodies to both proteins bind to pericentriolar regions but no binding has been detected at the apical cell surface-associated sites where the ends of thousands of recently nucleated microtubules are concentrated. Sparse transient microtubule populations can be detected between pericentriolar regions and surface sites while microtubule assembly advances. A procedure apparently operates in which the pericentriolar region functions as a microtubule-nucleating domain and the cell surface-associated sites operate as docking domains which capture the minus ends of microtubules that migrate to them shortly after nucleation. Docking domains may include some components of the pericentriolar material that have been relocated at the cell apex. A docking element hypothesis for centrosomal control of minus end positioning and dynamics in animal cells generally is proposed. This investigation has also shown that the concentration of gamma-tubulin and pericentrin around centrioles differs spatially and quantitatively in ways that are characteristic for the four cell types studied. Some of these characteristics can be related to differences in control of microtubule number and positioning.

    View details for Web of Science ID A1997WL72400008

    View details for PubMedID 9067623

  • Analysis of Tub4p, a yeast gamma-tubulin-like protein: Implications for microtubule-organizing center function JOURNAL OF CELL BIOLOGY Marschall, L. G., Jeng, R. L., Mulholland, J., Stearns, T. 1996; 134 (2): 443-454


    gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

    View details for Web of Science ID A1996UX94600015

    View details for PubMedID 8707828

  • Cytoskeleton: Microtubule nucleation takes shape CURRENT BIOLOGY Murphy, S. M., Stearns, T. 1996; 6 (6): 642-644


    The centrosomal protein gamma-tubulin is part of a ring-shaped complex that can induce microtubule polymerization. This complex may explain how the centrosome nucleates microtubule polymerization, and thereby organizes the microtubule cytoskeleton.

    View details for Web of Science ID A1996UR92800012

    View details for PubMedID 8793282

  • RECRUITMENT OF MATERNAL GAMMA-TUBULIN TO THE BOVINE SPERM CENTROSOME Navara, C. S., Zoran, S. S., Salisbury, J. L., Simerly, C., Stearns, T., Schatten, G. AMER SOC CELL BIOLOGY. 1995: 227–227


    Green fluorescent protein allows gene expression and protein localization to be observed in living cells.

    View details for Web of Science ID A1995QM82300014

    View details for PubMedID 7780736

  • MUTATIONAL ANALYSIS OF SACCHAROMYCES-CEREVISIAE ARF1 JOURNAL OF BIOLOGICAL CHEMISTRY Kahn, R. A., Clark, J., RULKA, C., Stearns, T., Zhang, C. J., Randazzo, P. A., Terui, T., Cavenagh, M. 1995; 270 (1): 143-150


    Wild type and eight point mutants of Saccharomyces cerevisiae ARF1 were expressed in yeast and bacteria to determine the roles of specific residues in in vivo and in vitro activities. Mutations at either Gly2 or Asp26 resulted in recessive loss of function. It was concluded that N-myristoylation is required for Arf action in cells but not for either nucleotide exchange or cofactor activities in vitro. Asp26 (homologous to Gly12 of p21ras) was essential for the binding of the activating nucleotide, guanosine 5'-3-O-(thio)triphosphate. This is in marked contrast to results obtained after mutagenesis of the homologous residue in p21ras or Gs alpha, and suggests a fundamental difference in the guanine nucleotide binding site of Arf with respect to these other GTP-binding proteins. Two dominant alleles were also identified, one activating dominant ([Q71L]Arf1) and the other ([N126I]) a negative dominant. A conditional allele, [W66R]Arf1, was characterized and shown to have approximately 300-fold lower specific activity in an in vitro Arf assay. Two high-copy suppressors of this conditional phenotype were cloned and sequenced. One of these suppressors, SFS4, was found to be identical to PBS2/HOG4, recently shown to encode a microtubule-associated protein kinase kinase in yeast.

    View details for Web of Science ID A1995QA28700026

    View details for PubMedID 7814365

  • THE FORM AND THE SUBSTANCE NATURE MEDICINE Stearns, T. 1995; 1 (1): 19-20

    View details for Web of Science ID A1995QX55700017

    View details for PubMedID 7584942



    The centrosome nucleates microtubule polymerization, affecting microtubule number, polarity, and structure. We use an in vitro system based on extracts of Xenopus eggs to examine the role of gamma-tubulin in centrosome assembly and function. gamma-Tubulin is present in the cytoplasm of frog eggs and vertebrate somatic cells in a large approximately 25S complex. The egg extracts assemble centrosomes around sperm centrioles. Formation of a centrosome in the extract requires both the gamma-tubulin complex and ATP and can take place in the absence of microtubules. gamma-Tubulin is not present on the sperm prior to incubation in extract, but is recruited from the cytoplasm during centrosome assembly. The gamma-tubulin complex also binds to microtubules, likely the minus end, independent of the centrosome. These results suggest that gamma-tubulin is an essential component of the link between the centrosome and the microtubule, probably playing a direct role in microtubule nucleation.

    View details for Web of Science ID A1994MZ28500006

    View details for PubMedID 8124706

  • SPECIFICITY DOMAINS DISTINGUISH THE RAS-RELATED GTPASES YPT1 AND SEC4 NATURE Dunn, B., Stearns, T., Botstein, D. 1993; 362 (6420): 563-565


    The essential Ras-related GTPases Ypt1 and Sec4 act at distinct stages of the secretion pathway in the yeast Saccharomyces cerevisiae: Ypt1 is required for vesicular transport from the endoplasmic reticulum to the Golgi apparatus, whereas Sec4 is required for fusion of secretory vesicles to the plasma membrane. Here we use chimaeras of the two proteins to identify a 9-residue segment of Ypt1 that, when substituted for the analogous segment of Sec4, allows the chimaera to perform the minimal functions of both proteins in vivo. This segment corresponds to loop L7 of the p21ras crystal structure. Substitution of a 24-residue Ypt1 segment, including the residues just mentioned, together with 12 residues of Ypt1 corresponding to the 'effector region' of p21ras (loop L2; refs 7,8), transforms Sec4 into a fully functional Ypt1 protein without residual Sec4 function.

    View details for Web of Science ID A1993KW45300061

    View details for PubMedID 8464499

  • Spindle positioning and cell polarity. Current biology Hyman, A. A., Stearns, T. 1992; 2 (9): 469-471

    View details for PubMedID 15335895

  • At the heart of the organizing center. Current biology Cande, W. Z., Stearns, T. 1991; 1 (4): 254-256

    View details for PubMedID 15336136

  • GAMMA-TUBULIN IS A HIGHLY CONSERVED COMPONENT OF THE CENTROSOME CELL Stearns, T., Evans, L., Kirschner, M. 1991; 65 (5): 825-836


    We have cloned and characterized gamma-tubulin genes from both X. laevis and S. pombe, and partial genes from maize, diatom, and a budding yeast. The proteins encoded by these genes are very similar to each other and to the original Aspergillus protein, indicating that gamma-tubulins are an ubiquitous and highly conserved subfamily of the tubulin family. A null mutation of the S. pombe gene is lethal. gamma-tubulin is a minor protein, present at less than 1% the level of alpha- and beta-tubulin, and is limited to the centrosome. In particular, gamma-tubulin is associated with the pericentriolar material, the microtubule-nucleating material of the centrosome. gamma-Tubulin remains associated with the centrosome when microtubules are depolymerized, suggesting that it is an integral component that might play a role in microtubule organization.

    View details for Web of Science ID A1991FP51600013

    View details for PubMedID 1840506



    ADP ribosylation factor (ARF) is a ubiquitous 21-kDa GTP-binding protein in eucaryotes. ARF was first identified in animal cells as the protein factor required for the efficient ADP-ribosylation of the mammalian G protein Gs by cholera toxin in vitro. A gene (ARF1) encoding a protein homologous to mammalian ARF was recently cloned from Saccharomyces cerevisiae (Sewell and Kahn, Proc. Natl. Acad. Sci. USA, 85:4620-4624, 1988). We have found a second gene encoding ARF in S. cerevisiae, ARF2. The two ARF genes are within 28 centimorgans of each other on chromosome IV, and the proteins encoded by them are 96% identical. Disruption of ARF1 causes slow growth, cold sensitivity, and sensitivity to normally sublethal concentrations of fluoride ion in the medium. Disruption of ARF2 causes no detectable phenotype. Disruption of both genes is lethal; thus, ARF is essential for mitotic growth. The ARF1 and ARF2 proteins are functionally homologous, and the phenotypic differences between mutations in the two genes can be accounted for by the level of expression; ARF1 produces approximately 90% of total ARF. Among revertants of the fluoride sensitivity of an arf1 null mutation were ARF1-ARF2 fusion genes created by a gene conversion event in which the deleted ARF1 sequences were repaired by recombination with ARF2.

    View details for Web of Science ID A1990EJ60200063

    View details for PubMedID 2123295



    ADP-ribosylation factor (ARF) is a ubiquitous, highly conserved 21-kDa GTP-binding protein, first identified in animal cells as the cofactor required for the in vitro ADP-ribosylation of the stimulatory regulatory subunit of adenylate cyclase, Gs, by cholera toxin. As the relevance of this activity to in vivo function is unknown, we have taken advantage of the conserved nature of ARF to study its function in Saccharomyces cerevisiae. Yeast cells bearing an arf1 null mutation display a number of phenotypes suggesting a defect in the secretory pathway. Secreted invertase is only partially glycosylated, and there is a small internal accumulation of invertase. Genetic experiments revealed interactions between ARF1 and other genes known to be involved in the secretory pathway, including YPT1, which encodes a different GTP-binding protein. In accord with these genetic results, immunofluorescence and immunoelectron microscopy show that ARF protein is localized to the Golgi apparatus in mammalian cells, in particular to the cytosolic surface of predominantly cis-Golgi membranes. Together, these results indicate that ARF functions in intracellular protein transport to or within the Golgi apparatus, a role not predicted by the previous in vitro biochemical studies.

    View details for Web of Science ID A1990CM07700080

    View details for PubMedID 2105501

  • The cytoskeleton of Saccharomyces cerevisiae CURRENT OPINION IN CELL BIOLOGY BARNES, G., Drubin, D. G., Stearns, T. 1990; 2 (1): 109-115


    Three new genes affecting microtubule function in Saccharomyces cerevisiae were isolated by screening for mutants displaying supersensitivity to the antimicrotubule drug benomyl. Such mutants fall into six complementation groups: TUB1, TUB2 and TUB3, the three tubulin genes of yeast, and three new genes, which we have named CIN1, CIN2 and CIN4. Mutations in each of the CIN genes were also independently isolated by screening for mutants with increased rates of chromosome loss. Strains bearing mutations in the CIN genes are approximately tenfold more sensitive than wild type to both benomyl and to the related antimicrotubule drug, nocodazole. This phenotype is recessive for all alleles isolated. The CIN1, CIN2 and CIN4 genes were cloned by complementation of the benomyl-supersensitive phenotype. Null mutants of each of the genes are viable, and have phenotypes similar to those of the point mutants. Genetic evidence for the involvement of the CIN gene products in microtubule function comes from the observation that some tubulin mutations are suppressed by cin mutations, while other tubulin mutations are lethal in combination with cin mutations. Additional genetic experiments with cin mutants suggest that the three genes act together in the same pathway or structure to affect microtubule function.

    View details for Web of Science ID A1990CM17600005

    View details for PubMedID 2407611

  • The cytoskeleton of Saccharomyces cerevisiae. Current opinion in cell biology BARNES, G., Drubin, D. G., Stearns, T. 1990; 2 (1): 109-115

    View details for PubMedID 2183834



    By using a multiply marked supernumerary chromosome III as an indicator, we isolated mutants of Saccharomyces cerevisiae that display increased rates of chromosome loss. In addition to mutations in the tubulin-encoding TUB genes, we found mutations in the CIN1, CIN2, and CIN4 genes. These genes have been defined independently by mutations causing benomyl supersensitivity and are distinct from other known yeast genes that affect chromosome segregation. Detailed phenotypic characterization of cin mutants revealed several other phenotypes similar to those of tub mutants. Null alleles of these genes caused cold sensitivity for viability. At 11 degrees C, cin mutants arrest at the mitosis stage of their cell cycle because of loss of most microtubule structure. cin1, cin2, and cin4 mutations also cause defects in two other microtubule-mediated processes, nuclear migration and nuclear fusion (karyogamy). Overproduction of the CIN1 gene product was found to cause the same phenotype as loss of function, supersensitivity to benomyl. Our findings suggest that the CIN1, CIN2, and CIN4 proteins contribute to microtubule stability either by regulating the activity of a yeast microtubule component or as structural components of microtubules.

    View details for Web of Science ID A1990CE81900025

    View details for PubMedID 2403635



    The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.

    View details for Web of Science ID A1990MC41900023

    View details for PubMedID 2199782


    View details for Web of Science ID A1990CG91300001

    View details for PubMedID 2403845



    The hypothesis that DNA topoisomerase II facilitates the separation of replicated sister chromatids was tested by examining the consequences of chromosome segregation in the absence of topoisomerase II activity. We observed a substantial elevation in the rate of nondisjunction in top2/top2 cells incubated at the restrictive temperature for one generation time. In contrast, only a minor increase in the amount of chromosome breakage was observed by either physical or genetic assays. These results suggest that aneuploidy is a major cause of the nonviability observed when top2 cells undergo mitosis at the restrictive temperature. In related experiments, we determined that topoisomerase II must act specifically during mitosis. This latter observation is consistent with the hypothesis that the mitotic spindle is necessary to allow topoisomerase II to complete the untangling of sister chromatids.

    View details for Web of Science ID A1989R643200020

    View details for PubMedID 2538717

  • FLUORESCENCE MICROSCOPY METHODS FOR YEAST METHODS IN CELL BIOLOGY Pringle, J. R., Preston, R. A., Adams, A. E., Stearns, T., Drubin, D. G., Haarer, B. K., Jones, E. W. 1989; 31: 357-435

    View details for Web of Science ID A1989AV40500019

    View details for PubMedID 2476649



    Mutations in genes of Saccharomyces cerevisiae that code for proteins that interact with beta-tubulin were sought by screening for unlinked mutations that fail to complement mutations in the single beta-tubulin-encoding gene (TUB2). Among the first three noncomplementing mutations examined, two are linked to TUB2 while one is unlinked. The unlinked mutation was shown to be a conditional-lethal allele of the major alpha-tubulin-encoding gene (TUB1) and represents the first such mutation in that gene. The tub1-1 mutation itself causes a cold-sensitive cell-cycle arrest, and confers supersensitivity to the antimicrotubule drug benomyl. These phenotypes occur in the presence of a wild-type copy of the minor alpha-tubulin-encoding gene, TUB3; the combination of tub1-1 and a tub3 null mutation is inviable in haploids. Through further application of this method, new mutations in TUB2 and TUB3 were isolated as unlinked noncomplementers of tub1-1. The noncomplementation between tub1 and tub2 mutations is gene specific and allele specific, suggesting that the phenotype is due to an interaction at the protein level. We conclude that isolation of unlinked noncomplementing mutations is likely to be a generally useful method for isolating mutations in interacting gene products.

    View details for Web of Science ID A1988N663300004

    View details for PubMedID 3294100


    View details for Web of Science ID A1988AP53900009

    View details for PubMedID 3151179