Lucy Erin O'Brien

All Publications

• Basal stem cell progeny establish their apical surface in a junctional niche during turnover of an adult barrier epithelium. Nature cell biology Galenza, A., Moreno-Roman, P., Su, Y. H., Acosta-Alvarez, L., Debec, A., Guichet, A., Knapp, J. M., Kizilyaprak, C., Humbel, B. M., Kolotuev, I., O'Brien, L. E. 2023

  Abstract

  Barrier epithelial organs face the constant challenge of sealing the interior body from the external environment while simultaneously replacing the cells that contact this environment. New replacement cells-the progeny of basal stem cells-are born without barrier-forming structures such as a specialized apical membrane and occluding junctions. Here, we investigate how new progeny acquire barrier structures as they integrate into the intestinal epithelium of adult Drosophila. We find they gestate their future apical membrane in a sublumenal niche created by a transitional occluding junction that envelops the differentiating cell and enables it to form a deep, microvilli-lined apical pit. The transitional junction seals the pit from the intestinal lumen until differentiation-driven, basal-to-apical remodelling of the niche opens the pit and integrates the now-mature cell into the barrier. By coordinating junctional remodelling with terminal differentiation, stem cell progeny integrate into a functional, adult epithelium without jeopardizing barrier integrity.

  View details for DOI 10.1038/s41556-023-01116-w

  View details for PubMedID 36997641

  View details for PubMedCentralID 5742542

• Bellymount enables longitudinal, intravital imaging of abdominal organs and the gut microbiota in adult Drosophila. PLoS biology Koyama, L. A., Aranda-Díaz, A. n., Su, Y. H., Balachandra, S. n., Martin, J. L., Ludington, W. B., Huang, K. C., O'Brien, L. E. 2020; 18 (1): e3000567

  Abstract

  Cell- and tissue-level processes often occur across days or weeks, but few imaging methods can capture such long timescales. Here, we describe Bellymount, a simple, noninvasive method for longitudinal imaging of the Drosophila abdomen at subcellular resolution. Bellymounted animals remain live and intact, so the same individual can be imaged serially to yield vivid time series of multiday processes. This feature opens the door to longitudinal studies of Drosophila internal organs in their native context. Exploiting Bellymount's capabilities, we track intestinal stem cell lineages and gut microbial colonization in single animals, revealing spatiotemporal dynamics undetectable by previously available methods.

  View details for DOI 10.1371/journal.pbio.3000567

  View details for PubMedID 31986129

• Disruption of EGF Feedback by Intestinal Tumors and Neighboring Cells in Drosophila. Current biology : CB Ngo, S. n., Liang, J. n., Su, Y. H., O'Brien, L. E. 2020

  Abstract

  In healthy adult organs, robust feedback mechanisms control cell turnover to enforce homeostatic equilibrium between cell division and death [1, 2]. Nascent tumors must subvert these mechanisms to achieve cancerous overgrowth [3-7]. Elucidating the nature of this subversion can reveal how cancers become established and may suggest strategies to prevent tumor progression. In adult Drosophila intestine, a well-studied model of homeostatic cell turnover, the linchpin of cell equilibrium is feedback control of the epidermal growth factor (EGF) protease Rhomboid (Rho). Expression of Rho in apoptotic cells enables them to secrete EGFs, which stimulate nearby stem cells to undergo replacement divisions [8]. As in mammals, loss of adenomatous polyposis coli (APC) causes Drosophila intestinal stem cells to form adenomas [9]. Here, we demonstrate that Drosophila APC-/- tumors trigger widespread Rho expression in non-apoptotic cells, resulting in chronic EGF signaling. Initially, nascent APC-/- tumors induce rho in neighboring wild-type cells via acute, non-autonomous activation of Jun N-terminal kinase (JNK). During later growth and multilayering, APC-/- tumors induce rho in tumor cells by autonomous downregulation of E-cadherin (E-cad) and consequent activity of p120-catenin. This sequential dysregulation of tumor non-autonomous and -autonomous EGF signaling converts tissue-level feedback into feed-forward activation that drives cancerous overgrowth. Because Rho, EGF receptor (EGFR), and E-cad are associated with colorectal cancer in humans [10-17], our findings may shed light on how human colorectal tumors progress.

  View details for DOI 10.1016/j.cub.2020.01.082

  View details for PubMedID 32243854

• Long-term live imaging of the Drosophila adult midgut reveals real-time dynamics of division, differentiation, and loss. eLife Martin, J. L., Sanders, E. N., Moreno-Roman, P., Jaramillo Koyama, L. A., Balachandra, S., Du, X., O'Brien, L. E. 2018; 7

  Abstract

  Organ renewal is governed by the dynamics of cell division, differentiation, and loss. To study these dynamics in real time, we present a platform for extended live imaging of the adult Drosophila midgut, a premier genetic model for stem cell-based organs. A window cut into a living animal allows the midgut to be imaged while intact and physiologically functioning. This approach prolongs imaging sessions to 12-16 hours and yields movies that document cell and tissue dynamics at vivid spatiotemporal resolution. Applying a pipeline for movie processing and analysis, we uncover new, intriguing cell behaviors: that mitotic stem cells dynamically re-orient, that daughter cells use slow kinetics of Notch activation to reach a fate-specifying threshold, and that enterocytes extrude via ratcheted constriction of a junctional ring. By enabling real-time study of midgut phenomena that were previously inaccessible, our platform opens a new realm for dynamic understanding of adult organ renewal.

  View details for PubMedID 30427308

• Feedback regulation of steady-state epithelial turnover and organ size NATURE Liang, J., Balachandra, S., Ngo, S., O'Brien, L. 2017; 548 (7669): 588-+

  Abstract

  Epithelial organs undergo steady-state turnover throughout adult life, with old cells being continually replaced by the progeny of stem cell divisions. To avoid hyperplasia or atrophy, organ turnover demands strict equilibration of cell production and loss. However, the mechanistic basis of this equilibrium is unknown. Here we show that robustly precise turnover of the adult Drosophila intestine arises through a coupling mechanism in which enterocyte apoptosis breaks feedback inhibition of stem cell division. Healthy enterocytes inhibit stem cell division through E-cadherin, which prevents secretion of mitogenic epidermal growth factors (EGFs) by repressing transcription of the EGF maturation factor rhomboid. Individual apoptotic enterocytes promote divisions by loss of E-cadherin, which releases cadherin-associated β-catenin (Armadillo in Drosophila) and p120-catenin to induce rhomboid. Induction of rhomboid in the dying enterocyte triggers activation of the EGF receptor (Egfr) in stem cells within a discrete radius. When we blocked apoptosis, E-cadherin-controlled feedback suppressed divisions, and the organ retained the same number of cells. When we disrupted feedback, apoptosis and divisions were uncoupled, and the organ developed either hyperplasia or atrophy. Together, our results show that robust cellular balance hinges on the obligate coupling of divisions to apoptosis, which limits the proliferative potential of a stem cell to the precise time and place at which a replacement cell is needed. In this way, localized cell-cell communication gives rise to tissue-level homeostatic equilibrium and constant organ size.

  View details for PubMedID 28847000

  View details for PubMedCentralID PMC5742542

• Beyond the niche: tissue-level coordination of stem cell dynamics. Annual review of cell and developmental biology O'Brien, L. E., Bilder, D. 2013; 29: 107-136

  Abstract

  Adult animals rely on populations of stem cells to ensure organ function throughout their lifetime. Stem cells are governed by signals from stem cell niches, and much is known about how single niches promote stemness and direct stem cell behavior. However, most organs contain a multitude of stem cell-niche units, which are often distributed across the entire expanse of the tissue. Beyond the biology of individual stem cell-niche interactions, the next challenge is to uncover the tissue-level processes that orchestrate spatial control of stem-based renewal, repair, and remodeling throughout a whole organ. Here we examine what is known about higher order mechanisms for interniche coordination in epithelial organs, whose simple geometry offers a promising entry point for understanding the regulation of niche number, distribution, and activity. We also consider the potential existence of stem cell territories and how tissue architecture may influence niche coordination.

  View details for DOI 10.1146/annurev-cellbio-101512-122319

  View details for PubMedID 23937350

• Tissue Homeostasis and Non-Homeostasis: From Cell Life Cycles to Organ States. Annual review of cell and developmental biology O'Brien, L. E. 2022

  Abstract

  Although tissue homeostasis-the steady state-implies stability, our organs are in a state of continual, large-scale cellular flux. This flux underpins an organ's ability to homeostatically renew, to non-homeostatically resize upon altered functional demand, and to return to homeostasis after resizing or injury-in other words, to be dynamic. Here, I examine the basic unit of organ-scale cell dynamics: the cellular life cycle of birth, differentiation, and death. Focusing on epithelial organs, I discuss how spatial patterns and temporal kinetics of life cycle stages depend upon lineage organization and tissue architecture. I review how signaling between stages coordinates life cycle dynamics to enforce homeostasis, and I highlight how particular stages are transiently unbalanced to drive organ resizing or repair. Finally, I offer that considering organs as a collective of not cells but rather cell life cycles provides a powerful vantage for deciphering homeostatic and non-homeostatic tissue states. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 38 is October 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-cellbio-120420-114855

  View details for PubMedID 35850152

• Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ. Journal of cell science Kim, A. A., Nguyen, A., Marchetti, M., Du, X., Montell, D. J., Pruitt, B. L., O'Brien, L. E. 2022

  Abstract

  Cytosolic calcium is a highly dynamic, tightly regulated, and broadly conserved cellular signal. Calcium dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. Here we examine calcium dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem cell daughters adopt strikingly distinct patterns of calcium oscillations after differentiation: Enteroendocrine cells exhibit single-cell calcium oscillations, while enterocytes exhibit rhythmic, long-range calcium waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), whose oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates calcium oscillations in all cell types--even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. Our findings establish that cells adopt fate-specific modes of calcium dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently.

  View details for DOI 10.1242/jcs.260249

  View details for PubMedID 35722729

• Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly. Science (New York, N.Y.) Li, H., Janssens, J., De Waegeneer, M., Kolluru, S. S., Davie, K., Gardeux, V., Saelens, W., David, F. P., Brbic, M., Spanier, K., Leskovec, J., McLaughlin, C. N., Xie, Q., Jones, R. C., Brueckner, K., Shim, J., Tattikota, S. G., Schnorrer, F., Rust, K., Nystul, T. G., Carvalho-Santos, Z., Ribeiro, C., Pal, S., Mahadevaraju, S., Przytycka, T. M., Allen, A. M., Goodwin, S. F., Berry, C. W., Fuller, M. T., White-Cooper, H., Matunis, E. L., DiNardo, S., Galenza, A., O'Brien, L. E., Dow, J. A., FCA Consortium, Jasper, H., Oliver, B., Perrimon, N., Deplancke, B., Quake, S. R., Luo, L., Aerts, S., Agarwal, D., Ahmed-Braimah, Y., Arbeitman, M., Ariss, M. M., Augsburger, J., Ayush, K., Baker, C. C., Banisch, T., Birker, K., Bodmer, R., Bolival, B., Brantley, S. E., Brill, J. A., Brown, N. C., Buehner, N. A., Cai, X. T., Cardoso-Figueiredo, R., Casares, F., Chang, A., Clandinin, T. R., Crasta, S., Desplan, C., Detweiler, A. M., Dhakan, D. B., Dona, E., Engert, S., Floc'hlay, S., George, N., Gonzalez-Segarra, A. J., Groves, A. K., Gumbin, S., Guo, Y., Harris, D. E., Heifetz, Y., Holtz, S. L., Horns, F., Hudry, B., Hung, R., Jan, Y. N., Jaszczak, J. S., Jefferis, G. S., Karkanias, J., Karr, T. L., Katheder, N. S., Kezos, J., Kim, A. A., Kim, S. K., Kockel, L., Konstantinides, N., Kornberg, T. B., Krause, H. M., Labott, A. T., Laturney, M., Lehmann, R., Leinwand, S., Li, J., Li, J. S., Li, K., Li, K., Li, L., Li, T., Litovchenko, M., Liu, H., Liu, Y., Lu, T., Manning, J., Mase, A., Matera-Vatnick, M., Matias, N. R., McDonough-Goldstein, C. E., McGeever, A., McLachlan, A. D., Moreno-Roman, P., Neff, N., Neville, M., Ngo, S., Nielsen, T., O'Brien, C. E., Osumi-Sutherland, D., Ozel, M. N., Papatheodorou, I., Petkovic, M., Pilgrim, C., Pisco, A. O., Reisenman, C., Sanders, E. N., Dos Santos, G., Scott, K., Sherlekar, A., Shiu, P., Sims, D., Sit, R. V., Slaidina, M., Smith, H. E., Sterne, G., Su, Y., Sutton, D., Tamayo, M., Tan, M., Tastekin, I., Treiber, C., Vacek, D., Vogler, G., Waddell, S., Wang, W., Wilson, R. I., Wolfner, M. F., Wong, Y. E., Xie, A., Xu, J., Yamamoto, S., Yan, J., Yao, Z., Yoda, K., Zhu, R., Zinzen, R. P. 2022; 375 (6584): eabk2432

  Abstract

  For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

  View details for DOI 10.1126/science.abk2432

  View details for PubMedID 35239393

• The nature of cell division forces in epithelial monolayers. The Journal of cell biology Gupta, V. K., Nam, S., Yim, D., Camuglia, J., Martin, J. L., Sanders, E. N., O'Brien, L. E., Martin, A. C., Kim, T., Chaudhuri, O. 2021; 220 (8)

  Abstract

  Epithelial cells undergo striking morphological changes during division to ensure proper segregation of genetic and cytoplasmic materials. These morphological changes occur despite dividing cells being mechanically restricted by neighboring cells, indicating the need for extracellular force generation. Beyond driving cell division itself, forces associated with division have been implicated in tissue-scale processes, including development, tissue growth, migration, and epidermal stratification. While forces generated by mitotic rounding are well understood, forces generated after rounding remain unknown. Here, we identify two distinct stages of division force generation that follow rounding: (1) Protrusive forces along the division axis that drive division elongation, and (2) outward forces that facilitate postdivision spreading. Cytokinetic ring contraction of the dividing cell, but not activity of neighboring cells, generates extracellular forces that propel division elongation and contribute to chromosome segregation. Forces from division elongation are observed in epithelia across many model organisms. Thus, division elongation forces represent a universal mechanism that powers cell division in confining epithelia.

  View details for DOI 10.1083/jcb.202011106

  View details for PubMedID 34132746

• Microfluidics for mechanobiology of model organisms. Methods in cell biology Kim, A. A., Nekimken, A. L., Fechner, S., O'Brien, L. E., Pruitt, B. L. 2018; 146: 217–59

  Abstract

  Mechanical stimuli play a critical role in organ development, tissue homeostasis, and disease. Understanding how mechanical signals are processed in multicellular model systems is critical for connecting cellular processes to tissue- and organism-level responses. However, progress in the field that studies these phenomena, mechanobiology, has been limited by lack of appropriate experimental techniques for applying repeatable mechanical stimuli to intact organs and model organisms. Microfluidic platforms, a subgroup of microsystems that use liquid flow for manipulation of objects, are a promising tool for studying mechanobiology of small model organisms due to their size scale and ease of customization. In this work, we describe design considerations involved in developing a microfluidic device for studying mechanobiology. Then, focusing on worms, fruit flies, and zebrafish, we review current microfluidic platforms for mechanobiology of multicellular model organisms and their tissues and highlight research opportunities in this developing field.

  View details for PubMedID 30037463

• A Model for Adult Organ Resizing Demonstrates Stem Cell Scaling through a Tunable Commitment Rate. Biophysical journal Du, X. n., O'Brien, L. E., Riedel-Kruse, I. H. 2017; 113 (1): 174–84

  Abstract

  Many adult organs grow or shrink to accommodate different physiological demands. Often, as total cell number changes, stem cell number changes proportionally in a phenomenon called "stem cell scaling". The cellular behaviors that give rise to scaling are unknown. Here we study two complementary theoretical models of the adult Drosophila midgut, a stem cell-based organ with known resizing dynamics. First, we derive a differential equations model of midgut resizing and show that the in vivo kinetics of growth can be recapitulated if the rate of fate commitment depends on the tissue's stem cell proportion. Second, we develop a 2D simulation of the midgut and find that proportion-dependent commitment rate and stem cell scaling can arise phenomenologically from the stem cells' exploration of physical tissue space during its lifetime. Together, these models provide a biophysical understanding of how stem cell scaling is maintained during organ growth and shrinkage.

  View details for PubMedID 28700915

• Regional specificity in the Drosophila midgut: setting boundaries with stem cells. Cell stem cell O'Brien, L. E. 2013; 13 (4): 375-376

  Abstract

  Many organs consist of distinct subregions with specialized physiological roles, but how regional boundaries are upheld during cellular renewal is largely unknown. Recently, Buchon et al. (2013) and Marianes and Spradling (2013) showed that subregions of the Drosophila midgut are maintained by patterned transcription factors and compartmentalized stem cell progeny.

  View details for DOI 10.1016/j.stem.2013.09.008

  View details for PubMedID 24094316

• Altered Modes of Stem Cell Division Drive Adaptive Intestinal Growth CELL O'Brien, L. E., Soliman, S. S., Li, X., Bilder, D. 2011; 147 (3): 603-614

  Abstract

  Throughout life, adult organs continually adapt to variable environmental factors. Adaptive mechanisms must fundamentally differ from homeostatic maintenance, but little is known about how physiological factors elicit tissue remodeling. Here, we show that specialized stem cell responses underlie the adaptive resizing of a mature organ. In the adult Drosophila midgut, intestinal stem cells interpret a nutrient cue to "break homeostasis" and drive growth when food is abundant. Activated in part by niche production of insulin, stem cells direct a growth program through two altered modes of behavior: accelerated division rates and predominance of symmetric division fates. Together, these altered modes produce a net increase in total intestinal cells, which is reversed upon withdrawal of food. Thus, tissue renewal programs are not committed to maintain cellular equilibrium; stem cells can remodel organs in response to physiological triggers.

  View details for DOI 10.1016/j.cell.2011.08.048

  View details for Web of Science ID 000296573700015

  View details for PubMedID 22036568

  View details for PubMedCentralID PMC3246009

• STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis MOLECULAR BIOLOGY OF THE CELL Kim, M., O'Brien, L. E., Kwon, S., Mostov, K. E. 2010; 21 (22): 3926-3933

  Abstract

  Tubule formation in vitro using Madin-Darby canine kidney (MDCK) epithelial cells consists mainly of two processes. First, the cells undergo a partial epithelial-mesenchymal transition (pEMT), losing polarity and migrating. Second, the cells redifferentiate, forming cords and then tubules with continuous lumens. We have shown previously that extracellular signal-regulated kinase activation is required for pEMT. However, the mechanism of how the pEMT phase is turned off and the redifferentiation phase is initiated is largely unknown. To address the central question of the sequential control of these two phases, we used MDCK cells grown as cysts and treated with hepatocyte growth factor to model tubulogenesis. We show that signal transducer and activator of transcription (STAT)1 controls the sequential progression from the pEMT phase to the redifferentiation phase. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 allows redifferentiation to occur even when cells are otherwise prevented from progressing beyond the pEMT phase by exogenous activation of Raf. Moreover, tyrosine phosphorylation defective STAT1 partially restored cord formation in such cells, suggesting that STAT1 functions in part as nonnuclear protein mediating signal transduction in this process. Constitutively active or inactive forms of STAT1 did not promote lumen maturation, suggesting this requires a distinct signal.

  View details for DOI 10.1091/mbc.E10-02-0112

  View details for Web of Science ID 000284216800043

  View details for PubMedID 20861313

• Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures METHODS IN ENZYMOLOGY, VOL 406, REGULATORS AND EFFECTORS OF SMALL GTPASES: RHO FAMILY O'Brien, L. E., Yu, W., Tang, K., Jou, T. S., Zegers, M. M., MOSTOV, K. E. 2006; 406: 676-691

  Abstract

  Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three-dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo. However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin-Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.

  View details for DOI 10.1016/S0076-6879(06)06053-8

  View details for Web of Science ID 000235750600053

  View details for PubMedID 16472697

• beta 1-integrin orients epithelial polarity via Rac1 and laminin MOLECULAR BIOLOGY OF THE CELL Yu, W., Datta, A., Leroy, P., O'Brien, L. E., Mak, G., Jou, T. S., Matlin, K. S., MOSTOV, K. E., Zegers, M. M. 2005; 16 (2): 433-445

  Abstract

  Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.

  View details for DOI 10.1091/mcb.E04-05-0435

  View details for Web of Science ID 000226563600001

  View details for PubMedID 15574881

• Formation of multicellular epithelial structures. Novartis Foundation symposium Mostov, K., Brakeman, P., Datta, A., Gassama, A., Katz, L., Kim, M., Leroy, P., Levin, M., Liu, K., Martin, F., O'Brien, L. E., Verges, M., Su, T., Tang, K., Tanimizu, N., Yamaji, T., Yu, W. 2005; 269: 193-200

  Abstract

  The kidney is primarily comprised of highly polarized epithelial cells. Much has been learned recently about the mechanisms of epithelial polarization. However, in most experimental systems the orientation of this polarity is determined by external cues, such as growth of epithelial cells on a filter support. When Madin-Darby canine kidney (MDCK) cells are grown instead in a three-dimensional (3D) collagen gel, the cells form hollow cysts lined by a monolayer of epithelial cells, with their apical surfaces all facing the central lumen. We have found that expression of a dominant-negative (DN) form of the small GTPase Rac1 causes an inversion of epithelial polarity, such that the apical surface of the cells instead faces the periphery of the cyst. This indicates that the establishment of polarity and the orientation of polarity can be experimentally separated by growing cells in a 3D collagen gel, where there is no filter support to provide an external cue for orientation. DN Rac1 causes a defect in the assembly of laminin into its normal basement membrane network, and addition of a high concentration of exogenous laminin rescues the inversion of polarity caused by DN Rac1.

  View details for PubMedID 16355541

• ERK and MMPs sequentially regulate distinct stages of epithelial tubule development DEVELOPMENTAL CELL O'Brien, L. E., Tang, K., Kats, E. S., Schutz-Geschwender, A., Lipschutz, J. H., Mostov, K. E. 2004; 7 (1): 21-32

  Abstract

  Epithelial cells undergo tubulogenesis in response to morphogens such as hepatocyte growth factor (HGF). To organize into tubules, cells must execute a complex series of morphogenetic events; however, the mechanisms that underlie the timing and sequence of these events are poorly understood. Here, we show that downstream effectors of HGF coordinately regulate successive stages of tubulogenesis. Activation of extracellular-regulated kinase (ERK) is necessary and sufficient for the initial stage, during which cells depolarize and migrate. ERK becomes dispensable for the latter stage, during which cells repolarize and differentiate. Conversely, the activity of matrix metalloproteases (MMPs) is essential for the late stage but not the initial stage. Thus, ERK and MMPs define two regulatory subprograms that act in sequence. By inducing these reciprocal signals, HGF directs the morphogenetic progression of tubule development.

  View details for Web of Science ID 000222696300007

  View details for PubMedID 15239951

• Epithelial polarity and tubulogenesis in vitro TRENDS IN CELL BIOLOGY Zegers, M. M., O'Brien, L. E., Yu, W., Datta, A., MOSTOV, K. E. 2003; 13 (4): 169-176

  Abstract

  The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.

  View details for DOI 10.1016/S0962-8924(03)00036-9

  View details for Web of Science ID 000182394700004

  View details for PubMedID 12667754

• Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures MOLECULAR BIOLOGY OF THE CELL Yu, W., O'Brien, L. E., Wang, F., Bourne, H., MOSTOV, K. E., Zegers, M. M. 2003; 14 (2): 748-763

  Abstract

  Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.

  View details for Web of Science ID 000182184300030

  View details for PubMedID 12589067

• Opinion - Building epithelial architecture: insights from three-dimensional culture models NATURE REVIEWS MOLECULAR CELL BIOLOGY O'Brien, L. E., Zegers, M. M., Mostov, K. E. 2002; 3 (7): 531-537

  Abstract

  How do individual cells organize into multicellular tissues? Here, we propose that the morphogenetic behaviour of epithelial cells is guided by two distinct elements: an intrinsic differentiation programme that drives formation of a lumen-enclosing monolayer, and a growth factor-induced, transient de-differentiation that allows this monolayer to be remodelled.

  View details for DOI 10.1038/nrm859

  View details for Web of Science ID 000176563500018

  View details for PubMedID 12094219

• Analysis of membrane traffic in polarized epithelial cells. Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.] Lipschutz, J. H., O'Brien, L. E., Altschuler, Y., Avrahami, D., Nguyen, Y., Tang, K., MOSTOV, K. E. 2001; Chapter 15: Unit 15 5-?

  Abstract

  Spatial asymmetry is fundamental to the structure and function of most eukaryotic cells. A basic aspect of this polarity is that the cell's plasma membrane is divided into discrete domains. The best studied and simplest example of this occurs in epithelial cells, which line exposed body surfaces. Epithelial cells use two pathways to send proteins to the cell surface. Newly made proteins can travel directly from the trans-Golgi network (TGN) to either the apical or basolateral surface. Alternatively, proteins can be sent to the basolateral surface and then endocytosed and transcytosed to the apical surface. Epithelial cells grown on porous filters adopt a typical polarized morphology; transfected epithelial cells can be used to biosynthetically characterize the trafficking patterns of a given protein. These cells can also be used to study delivery to a particular surface and to localize the protein by immunofluorescence.

  View details for DOI 10.1002/0471143030.cb1505s12

  View details for PubMedID 18228332

• Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly NATURE CELL BIOLOGY O'Brien, L. E., Jou, T. S., Pollack, A. L., Zhang, Q. H., Hansen, S. H., Yurchenco, P., Mostov, K. E. 2001; 3 (9): 831-838

  Abstract

  Cellular polarization involves the generation of asymmetry along an intracellular axis. In a multicellular tissue, the asymmetry of individual cells must conform to the overlying architecture of the tissue. However, the mechanisms that couple cellular polarization to tissue morphogenesis are poorly understood. Here, we report that orientation of apical polarity in developing Madin-Darby canine kidney (MDCK) epithelial cysts requires the small GTPase Rac1 and the basement membrane component laminin. Dominant-negative Rac1 alters the supramolecular assembly of endogenous MDCK laminin and causes a striking inversion of apical polarity. Exogenous laminin is recruited to the surface of these cysts and rescues apical polarity. These findings implicate Rac1-mediated laminin assembly in apical pole orientation. By linking apical orientation to generation of the basement membrane, epithelial cells ensure the coordination of polarity with tissue architecture.

  View details for Web of Science ID 000170979600017

  View details for PubMedID 11533663

• Exocyst is involved in cystogenesis and tubulogenesis and acts by modulating synthesis and delivery of basolateral plasma membrane and secretory proteins MOLECULAR BIOLOGY OF THE CELL Lipschutz, J. H., Guo, W., O'Brien, L. E., Nguyen, Y. H., Novick, P., Mostov, K. E. 2000; 11 (12): 4259-4275

  Abstract

  Epithelial cyst and tubule formation are critical processes that involve transient, highly choreographed changes in cell polarity. Factors controlling these changes in polarity are largely unknown. One candidate factor is the highly conserved eight-member protein complex called the exocyst. We show that during tubulogenesis in an in vitro model system the exocyst relocalized along growing tubules consistent with changes in cell polarity. In yeast, the exocyst subunit Sec10p is a crucial component linking polarized exocytic vesicles with the rest of the exocyst complex and, ultimately, the plasma membrane. When the exocyst subunit human Sec10 was exogenously expressed in epithelial Madin-Darby canine kidney cells, there was a selective increase in the synthesis and delivery of apical and basolateral secretory proteins and a basolateral plasma membrane protein, but not an apical plasma membrane protein. Overexpression of human Sec10 resulted in more efficient and rapid cyst formation and increased tubule formation upon stimulation with hepatocyte growth factor. We conclude that the exocyst plays a central role in the development of epithelial cysts and tubules.

  View details for Web of Science ID 000165955000016

  View details for PubMedID 11102522