Honors & Awards
Reed-Hodgson Professor, Human Biology (2004-present)
Member, American Academy of Arts and Sciences (2006-present)
Member, National Academy of Sciences (2008-present)
Member, Institute of Medicine (2011-present)
Ph. D., Mass Inst Tech, Microbiology (1980)
B. A., Brandeis Univ., Physics (1974)
Postdoctoral, Indiana University, Developmental Genetics (1983)
Current Research and Scholarly Interests
A central focus of our work concerns the mechanisms that regulate stem cell behavior. The central characteristic of adult stem cells is their long-term capacity to divide as relatively undifferentiated precursors while also producing daughter cells that initiate differentiation. Understanding the mechanisms that regulate stem cell specification and the choice between stem cell self-renewal and differentiation is crucial for realizing the potential of stem cells for regenerative medicine. We are using the Drosophila male germ line as a powerful genetic system to identify both the cell autonomous determinants and the extrinsic cell-cell interactions that govern stem cell specification, self-renewal, and differentiation. One of the great advantages of this system is that stem cells can be studied in situ, in the context of their normal support cells. Our results indicate that signals from surrounding somatic support cells specify asymmetric division of male germ line stem cells by inducing one daughter cell to self-renew stem cell identity while directing the other daughter cell to differentiate. A second focus of our work concerns how the developmental program directs cellular differentiation. Fundamental cellular functions like the cell cycle, the cytoskeleton, and the general transcription machinery are remodelled during development to give rise to specialized cell types. We investigate the mechanisms that regulate and mediate cellular differentiation during male gametogenesis in Drosophila. Our current work focuses on three areas. 1) We are investigating the mechanisms that regulate the unique program of gene expression that takes place in primary spermatocytes in preparation for the dramatic morphogenetic events of spermatid differentiation. We have discovered that both progression of the meiotic cell cycle and expression of spermatid differentiation genes are regulated by tissue specific versions of the general PolII transcription machinery. In addition, our work implicates components upstream of the Rb pathway in the control of terminal differentiation. 2) We are exploring the mechanisms that regulate remodeling of sub-cellular organelles. Our studies revealed the first known protein mediator of mitochondrial fusion, required for formation of specialized mitochondrial structures in spermatids. Our current work indicates that human homologs of the Drosophila mitofusin protein regulate mitochondrial morphology in human cells and may play a role in differentiation of heart and skeletal muscle. 3) We are dissecting the mechanisms that remodel the actin cytoskeleton and lead to localized assembly and constriction of the acto-myosin contractile machinery during cytokinesis. We have identified mutations in over 20 new genes that block different stages of contractile ring assembly and function during male meiosis. To investigate the underlying molecular mechanisms that regulate and mediate cytokinesis, we are cloning selected of these genes. Our initial results indicate that shared mechanisms involving addition of new membrane are required for both cleavage furrow constriction during cytokinesis and polarized cell elongation during later terminal differentiation.
- Cell and Developmental Biology
HUMBIO 3A (Win)
- The Biology of Stem Cells
HUMBIO 157 (Spr)
Independent Studies (20)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Directed Reading in Developmental Biology
DBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Genetics
GENE 299 (Aut, Win, Spr, Sum)
- Directed Reading in Stem Cell Biology and Regenerative Medicine
STEMREM 299 (Aut, Win, Spr)
- Directed Reading/Special Projects
HUMBIO 199 (Aut, Win, Spr)
- Graduate Research
CBIO 399 (Win, Spr)
- Graduate Research
DBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
GENE 399 (Aut, Win, Spr, Sum)
- Graduate Research
STEMREM 399 (Aut, Win, Spr)
HUMBIO 194 (Aut, Win, Spr)
- Medical Scholars Research
DBIO 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
GENE 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
STEMREM 370 (Aut, Win, Spr)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Aut, Win, Spr)
- Research in Human Biology
HUMBIO 193 (Aut, Win)
- Supervised Study
GENE 260 (Aut, Win, Spr, Sum)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Undergraduate Research
DBIO 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
GENE 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
STEMREM 199 (Aut, Win, Spr)
- Directed Reading in Cancer Biology
Prior Year Courses
- Cell and Developmental Biology
HUMBIO 3A (Win)
- Cell and Developmental Biology
HUMBIO 3A (Win)
- Evolution of Life in Neotropical Forests of Ecuador
OSPGEN 75 (Sum)
- The Biology of Stem Cells
HUMBIO 157 (Spr)
- Cell and Developmental Biology
HUMBIO 3A (Win)
- Cell and Developmental Biology
Graduate and Fellowship Programs
Developmental phosphoproteomics identifies the kinase CK2 as a driver of Hedgehog signaling and a therapeutic target in medulloblastoma.
2018; 11 (547)
A major limitation of targeted cancer therapy is the rapid emergence of drug resistance, which often arises through mutations at or downstream of the drug target or through intrinsic resistance of subpopulations of tumor cells. Medulloblastoma (MB), the most common pediatric brain tumor, is no exception, and MBs that are driven by sonic hedgehog (SHH) signaling are particularly aggressive and drug-resistant. To find new drug targets and therapeutics for MB that may be less susceptible to common resistance mechanisms, we used a developmental phosphoproteomics approach in murine granule neuron precursors (GNPs), the developmental cell of origin of MB. The protein kinase CK2 emerged as a driver of hundreds of phosphorylation events during the proliferative, MB-like stage of GNP growth, including the phosphorylation of three of the eight proteins commonly amplified in MB. CK2 was critical to the stabilization and activity of the transcription factor GLI2, a late downstream effector in SHH signaling. CK2 inhibitors decreased the viability of primary SHH-type MB patient cells in culture and blocked the growth of murine MB tumors that were resistant to currently available Hh inhibitors, thereby extending the survival of tumor-bearing mice. Because of structural interactions, one CK2 inhibitor (CX-4945) inhibited both wild-type and mutant CK2, indicating that this drug may avoid at least one common mode of acquired resistance. These findings suggest that CK2 inhibitors may be effective for treating patients with MB and show how phosphoproteomics may be used to gain insight into developmental biology and pathology.
View details for DOI 10.1126/scisignal.aau5147
View details for PubMedID 30206138
The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline
The switch from mitosis to meiosis is the key event marking onset of differentiation in the germline stem cell lineage. In Drosophila, the translational repressor Bgcn is required for spermatogonia to stop mitosis and transition to meiotic prophase and the spermatocyte state. Here we show that the mammalian Bgcn homolog YTHDC2 facilitates a clean switch from mitosis to meiosis in mouse germ cells, revealing a conserved role for YTHDC2 in this critical cell fate transition. YTHDC2-deficient male germ cells enter meiosis but have a mixed identity, maintaining expression of Cyclin A2 and failing to properly express many meiotic markers. Instead of continuing through meiotic prophase, the cells attempt an abnormal mitotic-like division and die. YTHDC2 binds multiple transcripts including Ccna2 and other mitotic transcripts, binds specific piRNA precursors, and interacts with RNA granule components, suggesting that proper progression of germ cells through meiosis is licensed by YTHDC2 through post-transcriptional regulation.
View details for DOI 10.7554/eLife.26116
View details for Web of Science ID 000424298600001
View details for PubMedID 29087293
View details for PubMedCentralID PMC5703642
Blocking promiscuous activation at cryptic promoters directs cell type-specific gene expression
2017; 356 (6339): 717-721
To selectively express cell type-specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. Here, we show in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. Our results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation.
View details for DOI 10.1126/science.aal3096
View details for Web of Science ID 000401508400039
View details for PubMedID 28522526
Cell type-specific translational repression of Cyclin B during meiosis in males
2015; 142 (19): 3394-3402
The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I.
View details for DOI 10.1242/dev.122341
View details for Web of Science ID 000365025500014
View details for PubMedID 26443637
View details for PubMedCentralID PMC4631753
Somatic cell lineage is required for differentiation and not maintenance of germline stem cells in Drosophila testes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (45): 18477-18481
Adult stem cells are believed to be maintained by a specialized microenvironment, the niche, which provides short-range signals that either instruct stem cells to self-renew or inhibit execution of preprogrammed differentiation pathways. In Drosophila testes, somatic cyst stem cells (CySCs) and the apical hub form the niche for neighboring germline stem cells (GSCs), with CySCs as the proposed source of instructive self-renewal signals [Leatherman JL, Dinardo S (2010) Nat Cell Biol 12(8):806-811]. In contrast to this model, we show that early germ cells with GSC characteristics can be maintained over time after ablation of CySCs and their cyst cell progeny. Without CySCs and cyst cells, early germ cells away from the hub failed to initiate differentiation. Our results suggest that CySCs do not have a necessary instructive role in specifying GSC self-renewal and that the differentiated progeny of CySCs provide an environment necessary to trigger GSC differentiation. This work highlights the complex interaction between different stem cell populations in the same niche and how the state of one stem cell population can influence the fate of the other.
View details for DOI 10.1073/pnas.1215516109
View details for Web of Science ID 000311156700055
View details for PubMedID 23091022
A Self-Limiting Switch Based on Translational Control Regulates the Transition from Proliferation to Differentiation in an Adult Stem Cell Lineage
CELL STEM CELL
2012; 11 (5): 689-700
In adult stem cell lineages, progenitor cells commonly undergo mitotic transit amplifying (TA) divisions before terminal differentiation, allowing production of many differentiated progeny per stem cell division. Mechanisms that limit TA divisions and trigger the switch to differentiation may protect against cancer by preventing accumulation of oncogenic mutations in the proliferating population. Here we show that the switch from TA proliferation to differentiation in the Drosophila male germline stem cell lineage is mediated by translational control. The TRIM-NHL tumor suppressor homolog Mei-P26 facilitates accumulation of the differentiation regulator Bam in TA cells. In turn, Bam and its partner Bgcn bind the mei-P26 3' untranslated region and repress translation of mei-P26 in late TA cells. Thus, germ cells progress through distinct, sequential regulatory states, from Mei-P26 on/Bam off to Bam on/Mei-P26 off. TRIM-NHL homologs across species facilitate the switch from proliferation to differentiation, suggesting a conserved developmentally programmed tumor suppressor mechanism.
View details for DOI 10.1016/j.stem.2012.08.012
View details for Web of Science ID 000311471900014
View details for PubMedID 23122292
Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (52): 22311-22316
A key feature of many adult stem cell lineages is that stem cell daughters destined for differentiation undergo several transit amplifying (TA) divisions before initiating terminal differentiation, allowing few and infrequently dividing stem cells to produce many differentiated progeny. Although the number of progenitor divisions profoundly affects tissue (re)generation, and failure to control these divisions may contribute to cancer, the mechanisms that limit TA proliferation are not well understood. Here, we use a model stem cell lineage, the Drosophila male germ line, to investigate the mechanism that counts the number of TA divisions. The Drosophila Bag of Marbles (Bam) protein is required for male germ cells to cease spermatogonial TA divisions and initiate spermatocyte differentiation [McKearin DM, et al. (1990) Genes Dev 4:2242-2251]. Contrary to models involving dilution of a differentiation repressor, our results suggest that the switch from proliferation to terminal differentiation is triggered by accumulation of Bam protein to a critical threshold in TA cells and that the number of TA divisions is set by the timing of Bam accumulation with respect to the rate of cell cycle progression.
View details for DOI 10.1073/pnas.0912454106
View details for Web of Science ID 000273178700050
View details for PubMedID 20018708
Asymmetric inheritance of mother versus daughter centrosome in stem cell division
2007; 315 (5811): 518-521
Adult stem cells often divide asymmetrically to produce one self-renewed stem cell and one differentiating cell, thus maintaining both populations. The asymmetric outcome of stem cell divisions can be specified by an oriented spindle and local self-renewal signals from the stem cell niche. Here we show that developmentally programmed asymmetric behavior and inheritance of mother and daughter centrosomes underlies the stereotyped spindle orientation and asymmetric outcome of stem cell divisions in the Drosophila male germ line. The mother centrosome remains anchored near the niche while the daughter centrosome migrates to the opposite side of the cell before spindle formation.
View details for DOI 10.1126/science.1134910
View details for Web of Science ID 000243726600048
View details for PubMedID 17255513
Tissue-specific TAFs counteract polycomb to turn on terminal differentiation
2005; 310 (5749): 869-872
Polycomb transcriptional silencing machinery is implicated in the maintenance of precursor fates, but how this repression is reversed to allow cell differentiation is unknown. Here we show that testis-specific TAF (TBP-associated factor) homologs required for terminal differentiation of male germ cells may activate target gene expression in part by counteracting repression by Polycomb. Chromatin immunoprecipitation revealed that testis TAFs bind to target promoters, reduce Polycomb binding, and promote local accumulation of H3K4me3, a mark of Trithorax action. Testis TAFs also promoted relocalization of Polycomb Repression Complex 1 components to the nucleolus in spermatocytes, implicating subnuclear architecture in the regulation of terminal differentiation.
View details for DOI 10.1126/science.1118101
View details for Web of Science ID 000233121800050
View details for PubMedID 16272126
Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome
2003; 301 (5639): 1547-1550
Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. Here, we show that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation.
View details for Web of Science ID 000185255300054
View details for PubMedID 12970569
Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue
2001; 294 (5551): 2542-2545
Stem cells generate many differentiated, short-lived cell types, such as blood, skin, and sperm, throughout adult life. Stem cells maintain a long-term capacity to divide, producing daughter cells that either self-renew or initiate differentiation. Although the surrounding microenvironment or "niche" influences stem cell fate decisions, few signals that emanate from the niche to specify stem cell self-renewal have been identified. Here we demonstrate that the apical hub cells in the Drosophila testis act as a cellular niche that supports stem cell self-renewal. Hub cells express the ligand Unpaired (Upd), which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in adjacent germ cells to specify self-renewal and continual maintenance of the germ line stem cell population.
View details for Web of Science ID 000172927700057
View details for PubMedID 11752574
Somatic support cells restrict germline stem cell self-renewal and promote differentiation
2000; 407 (6805): 750-754
Stem cells maintain populations of highly differentiated, short-lived cell-types, including blood, skin and sperm, throughout adult life. Understanding the mechanisms that regulate stem cell behaviour is crucial for realizing their potential in regenerative medicine. A fundamental characteristic of stem cells is their capacity for asymmetric division: daughter cells either retain stem cell identity or initiate differentiation. However, stem cells are also capable of symmetric division where both daughters remain stem cells, indicating that mechanisms must exist to balance self-renewal capacity with differentiation. Here we present evidence that support cells surrounding the stem cells restrict self-renewal and control stem cell number by ensuring asymmetric division. Loss of function of the Drosophila Epidermal growth factor receptor in somatic cells disrupted the balance of self-renewal versus differentiation in the male germline, increasing the number of germline stem cells. We propose that activation of this receptor specifies normal behaviour of somatic support cells; in turn, the somatic cells play a guardian role, providing information that prevents self-renewal of stem cell identity by the germ cell they enclose.
View details for Web of Science ID 000089773900044
View details for PubMedID 11048722
Developmentally regulated mitochondrial fusion mediated by a conserved, novel, predicted GTPase
1997; 90 (1): 121-129
The Drosophila melanogaster fuzzy onions (fzo) gene encodes the first known protein mediator of mitochondrial fusion. During Drosophila spermatogenesis, mitochondria in early postmeiotic spermatids aggregate, fuse, and elongate beside the growing flagellar axoneme. fzo mutant males are defective in this developmentally regulated mitochondrial fusion and are sterile. fzo encodes a large, novel, predicted transmembrane GTPase that becomes detectable on spermatid mitochondria late in meiosis II, just prior to fusion, and disappears soon after fusion is complete. Missense mutations that alter conserved residues required for GTP binding in other GTPases inhibit the fusogenic activity of Fzo in vivo but do not affect its localization. Fzo has homologs of unknown function in mammals, nematodes, and yeast.
View details for Web of Science ID A1997XL36200014
View details for PubMedID 9230308
- Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila BMC CELL BIOLOGY 2016; 18
Differentiation in Stem Cell Lineages and in Life: Explorations in the Male Germ Line Stem Cell Lineage
ESSAYS ON DEVELOPMENTAL BIOLOGY, PT A
2016; 116: 375-390
I have been privileged to work on cellular differentiation during a great surge of discovery that has revealed the molecular mechanisms and genetic regulatory circuitry that control embryonic development and adult tissue maintenance and repair. Studying the regulation of proliferation and differentiation in the male germ line stem cell lineage has allowed us investigate how the developmental program imposes layers of additional controls on fundamental cellular processes like cell cycle progression and gene expression to give rise to the huge variety of specialized cell types in our bodies. We are beginning to understand how local signals from somatic support cells specify self-renewal versus differentiation in the stem cell niche at the apical tip of the testis. We are discovering the molecular events that block cell proliferation and initiate terminal differentiation at the switch from mitosis to meiosis-a signature event of the germ cell program. Our work is beginning to reveal how the developmental program that sets up the dramatic new cell type-specific transcription program that prepares germ cells for meiotic division and spermatid differentiation is turned on when cells become spermatocytes. I have had the privilege of working with incredible students, postdocs, and colleagues who have discovered, brainstormed, challenged, and refined our science and our ideas of how developmental pathways and cellular mechanisms work together to drive differentiation.
View details for DOI 10.1016/bs.ctdb.2015.11.041
View details for Web of Science ID 000376939100021
View details for PubMedID 26970629
- Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage PLOS GENETICS 2015; 11 (12)
- Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila PLOS GENETICS 2015; 11 (11)
GOLPH3 Is Essential for Contractile Ring Formation and Rab11 Localization to the Cleavage Site during Cytokinesis in Drosophila melanogaster
2014; 10 (5)
The highly conserved Golgi phosphoprotein 3 (GOLPH3) protein has been described as a Phosphatidylinositol 4-phosphate [PI(4)P] effector at the Golgi. GOLPH3 is also known as a potent oncogene, commonly amplified in several human tumors. However, the molecular pathways through which the oncoprotein GOLPH3 acts in malignant transformation are largely unknown. GOLPH3 has never been involved in cytokinesis. Here, we characterize the Drosophila melanogaster homologue of human GOLPH3 during cell division. We show that GOLPH3 accumulates at the cleavage furrow and is required for successful cytokinesis in Drosophila spermatocytes and larval neuroblasts. In premeiotic spermatocytes GOLPH3 protein is required for maintaining the organization of Golgi stacks. In dividing spermatocytes GOLPH3 is essential for both contractile ring and central spindle formation during cytokinesis. Wild type function of GOLPH3 enables maintenance of centralspindlin and Rho1 at cell equator and stabilization of Myosin II and Septin rings. We demonstrate that the molecular mechanism underlying GOLPH3 function in cytokinesis is strictly dependent on the ability of this protein to interact with PI(4)P. Mutations that abolish PI(4)P binding impair recruitment of GOLPH3 to both the Golgi and the cleavage furrow. Moreover telophase cells from mutants with defective GOLPH3-PI(4)P interaction fail to accumulate PI(4)P-and Rab11-associated secretory organelles at the cleavage site. Finally, we show that GOLPH3 protein interacts with components of both cytokinesis and membrane trafficking machineries in Drosophila cells. Based on these results we propose that GOLPH3 acts as a key molecule to coordinate phosphoinositide signaling with actomyosin dynamics and vesicle trafficking during cytokinesis. Because cytokinesis failures have been associated with premalignant disease and cancer, our studies suggest novel insight into molecular circuits involving the oncogene GOLPH3 in cytokinesis.
View details for DOI 10.1371/journal.pgen.1004305
View details for Web of Science ID 000337145100011
View details for PubMedID 24786584
Escargot Restricts Niche Cell to Stem Cell Conversion in the Drosophila Testis
2014; 7 (3): 722-734
Stem cells reside within specialized microenvironments, or niches, that control many aspects of stem cell behavior. Somatic hub cells in the Drosophila testis regulate the behavior of cyst stem cells (CySCs) and germline stem cells (GSCs) and are a primary component of the testis stem cell niche. The shutoff (shof) mutation, characterized by premature loss of GSCs and CySCs, was mapped to a locus encoding the evolutionarily conserved transcription factor Escargot (Esg). Hub cells depleted of Esg acquire CySC characteristics and differentiate as cyst cells, resulting in complete loss of hub cells and eventually CySCs and GSCs, similar to the shof mutant phenotype. We identified Esg-interacting proteins and demonstrate an interaction between Esg and the corepressor C-terminal binding protein (CtBP), which was also required for maintenance of hub cell fate. Our results indicate that niche cells can acquire stem cell properties upon removal of a single transcription factor in vivo.
View details for DOI 10.1016/j.celrep.2014.04.025
View details for Web of Science ID 000335560900015
View details for PubMedID 24794442
The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells
2014; 141 (1): 73-82
Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. Here, we demonstrate that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia.
View details for DOI 10.1242/dev.101931
View details for Web of Science ID 000328944500009
View details for PubMedID 24346697
- The Histone Variant His2Av is Required for Adult Stem Cell Maintenance in the Drosophila Testis. PLoS genetics 2013; 9 (11)
The polyubiquitin gene Ubi-p63E is essential for male meiotic cell cycle progression and germ cell differentiation in Drosophila.
2013; 140 (17): 3522-3531
The ubiquitin proteasome system (UPS) regulates many biological pathways by post-translationally ubiquitylating proteins for degradation. Although maintaining a dynamic balance between free ubiquitin and ubiquitylated proteins is key to UPS function, the mechanisms that regulate ubiquitin homeostasis in different tissues through development are not clear. Here we show, via analysis of the magellan (magn) complementation group, that loss of function of the Drosophila polyubiquitin Ubi-p63E results specifically in meiotic arrest sterility in males. Ubi-p63E contributes predominantly to maintaining the free ubiquitin pool in testes. The function of Ubi-p63E is required cell-autonomously for proper meiotic chromatin condensation, cell cycle progression and spermatid differentiation. magn mutant germ cells develop normally to the spermatocyte stage but arrest at the G2/M transition of meiosis I, with lack of protein expression of the key meiotic cell cycle regulators Boule and Cyclin B. Loss of Ubi-p63E function did not strongly affect the spermatocyte transcription program regulated by the testis TBP-associated factor (tTAF) or meiosis arrest complex (tMAC) genes. Knocking down proteasome function specifically in spermatocytes caused a different meiotic arrest phenotype, suggesting that the magn phenotype might not result from general defects in protein degradation. Our results suggest a conserved role of polyubiquitin genes in male meiosis and a potential mechanism leading to meiosis I maturation arrest.
View details for DOI 10.1242/dev.098947
View details for PubMedID 23884444
Three levels of regulation lead to protamine and Mst77F expression in Drosophila
2013; 377 (1): 33-45
Differentiation from a haploid round spermatid to a highly streamlined, motile sperm requires temporal and spatial regulation of the expression of numerous proteins. One form of regulation is the storage of translationally repressed mRNAs. In Drosophila spermatocytes, the transcription of many of these translationally delayed mRNAs during spermiogenesis is in turn directly or indirectly regulated by testis-specific homologs of TATA-box-binding-protein-associated factors (tTAFs). Here we present evidence that expression of Mst77F, which is a specialized linker histone-like component of sperm chromatin, and of protamine B (ProtB), which contributes to formation of condensed sperm chromatin, is regulated at three levels. Transcription of Mst77F is guided by a short, promoter-proximal region, while expression of the Mst77F protein is regulated at two levels, early by translational repression via sequences mainly in the 5' part of the ORF and later by either protein stabilization or translational activation, dependent on sequences in the ORF. The protB gene is a direct target of tTAFs, with very short upstream regulatory regions of protB (-105 to +94 bp) sufficient for both cell-type-specific transcription and repression of translation in spermatocytes. In addition, efficient accumulation of the ProtB protein in late elongating spermatids depends on sequences in the ORF. We present evidence that spermatocytes provide the transacting mechanisms for translational repression of these mRNAs, while spermatids contain the machinery to activate or stabilize protamine accumulation for sperm chromatin components. Thus, the proper spatiotemporal expression pattern of major sperm chromatin components depends on cell-type-specific mechanisms of transcriptional and translational control.
View details for DOI 10.1016/j.ydbio.2013.02.018
View details for Web of Science ID 000318757300004
View details for PubMedID 23466740
The transcriptional regulator lola is required for stem cell maintenance and germ cell differentiation in the Drosophila testis
2013; 373 (2): 310-321
Stem cell behavior is regulated by extrinsic signals from specialized microenvironments, or niches, and intrinsic factors required for execution of context-appropriate responses to niche signals. Here we show that function of the transcriptional regulator longitudinals lacking (lola) is required cell autonomously for germline stem cell and somatic cyst stem cell maintenance in the Drosophila testis. In addition, lola is also required for proper execution of key developmental transitions during male germ cell differentiation, including the switch from transit amplifying progenitor to spermatocyte growth and differentiation, as well as meiotic cell cycle progression and spermiogenesis. Different lola isoforms, each having unique C-termini and zinc finger domains, may control different aspects of proliferation and differentiation in the male germline and somatic cyst stem cell lineages.
View details for DOI 10.1016/j.ydbio.2012.11.004
View details for Web of Science ID 000313381200008
View details for PubMedID 23159836
- Polycomb Group Genes Psc and Su(z)2 Maintain Somatic Stem Cell Identity and Activity in Drosophila PLOS ONE 2012; 7 (12)
Mutations in Cog7 affect Golgi structure, meiotic cytokinesis and sperm development during Drosophila spermatogenesis
JOURNAL OF CELL SCIENCE
2012; 125 (22): 5441-5452
The conserved oligomeric Golgi (COG) complex plays essential roles in Golgi function, vesicle trafficking and glycosylation. Deletions in the human COG7 gene are associated with a rare multisystemic congenital disorder of glycosylation that causes mortality within the first year of life. In this paper, we characterise the Drosophila orthologue of COG7 (Cog7). Loss-of-function Cog7 mutants are viable but male sterile. The Cog7 gene product is enriched in the Golgi stacks and in Golgi-derived structures throughout spermatogenesis. Mutations in the Cog7 gene disrupt Golgi architecture and reduce the number of Golgi stacks in primary spermatocytes. During spermiogenesis, loss of the Cog7 protein impairs the assembly of the Golgi-derived acroblast in spermatids and affects axoneme architecture. Similar to the Cog5 homologue, four way stop (Fws), Cog7 enables furrow ingression during cytokinesis. We show that the recruitment of the small GTPase Rab11 and the phosphatidylinositol transfer protein Giotto (Gio) to the cleavage site requires a functioning wild-type Cog7 gene. In addition, Gio coimmunoprecipitates with Cog7 and with Rab11 in the testes. Our results altogether implicate Cog7 as an upstream component in a gio-Rab11 pathway controlling membrane addition during cytokinesis.
View details for DOI 10.1242/jcs.108878
View details for Web of Science ID 000314511900018
View details for PubMedID 22946051
What Drosophila Spermatocytes Tell Us About the Mechanisms Underlying Cytokinesis
2012; 69 (11): 869-881
Cytokinesis separates the genomic material and organelles of a dividing cell equitably into two physically distinct daughter cells at the end of cell division. This highly choreographed process involves coordinated reorganization and regulated action of the actin and microtubule cytoskeletal systems, an assortment of motor proteins, and membrane trafficking components. Due to their large size, the ease with which exquisite cytological analysis may be performed on them, and the availability of numerous mutants and other genetic tools, Drosophila spermatocytes have provided an excellent system for exploring the mechanistic basis for the temporally programmed and precise spatially localized events of cytokinesis. Mutants defective in male meiotic cytokinesis can be easily identified in forward genetic screens by the production of multinucleate spermatids. In addition, the weak spindle assembly checkpoint in spermatocytes, which causes only a small delay of anaphase onset in the presence of unattached chromosomes, allows investigation of whether gene products required for spindle assembly and chromosome segregation are also involved in cytokinesis. Perhaps due to the large size of spermatocytes and the requirement for two rapid-fire rounds of division without intervening S or growth phases during meiosis, male meiotic mutants have also revealed much about molecular mechanisms underlying new membrane addition during cytokinesis. Finally, cell type-specific differences in the events that set up and complete cytokinesis are emerging from comparison of spermatocytes with cells undergoing mitosis either elsewhere in the organism or in tissue culture.
View details for DOI 10.1002/cm.21063
View details for Web of Science ID 000313747400002
View details for PubMedID 22927345
A Novel Human Polycomb Binding Site Acts As a Functional Polycomb Response Element in Drosophila
2012; 7 (5)
Polycomb group (PcG) proteins are key chromatin regulators implicated in multiple processes including embryonic development, tissue homeostasis, genomic imprinting, X-chromosome inactivation, and germ cell differentiation. The PcG proteins recognize target genomic loci through cis DNA sequences known as Polycomb Response Elements (PREs), which are well characterized in Drosophila. However, mammalian PREs have been elusive until two groups reported putative mammalian PREs recently. Consistent with the existence of mammalian PREs, here we report the identification and characterization of a potential PRE from human T cells. The putative human PRE has enriched binding of PcG proteins, and such binding is dependent on a key PcG component SUZ12. We demonstrate that the putative human PRE carries both genetic and molecular features of Drosophila PRE in transgenic flies, implying that not only the trans PcG proteins but also certain features of the cis PREs are conserved between mammals and Drosophila.
View details for DOI 10.1371/journal.pone.0036365
View details for Web of Science ID 000305343400023
View details for PubMedID 22570707
The receptor tyrosine phosphatase Lar regulates adhesion between Drosophila male germline stem cells and the niche
2012; 139 (8): 1381-1390
The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. Here we show that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions.
View details for DOI 10.1242/dev.070052
View details for Web of Science ID 000301945100003
View details for PubMedID 22378638
Polycomb group genes Psc and Su(z)2 maintain somatic stem cell identity and activity in Drosophila.
2012; 7 (12)
Adult stem cells are essential for the proper function of many tissues, yet the mechanisms that maintain the proper identity and regulate proliferative capacity in stem cell lineages are not well understood. Polycomb group (PcG) proteins are transcriptional repressors that have recently emerged as important regulators of stem cell maintenance and differentiation. Here we describe the role of Polycomb Repressive Complex 1 (PRC1) genes Posterior sex combs (Psc) and Suppressor of zeste two (Su(z)2) in restricting the proliferation and maintaining the identity of the Cyst Stem Cell (CySC) lineage in the Drosophila testis. In contrast, Psc and Su(z)2 seem to be dispensable for both germline stem cell (GSC) maintenance and germ cell development. We show that loss of Psc and Su(z)2 function in the CySC lineage results in the formation of aggregates of mutant cells that proliferate abnormally, and display abnormal somatic identity correlated with derepression of the Hox gene Abdominal-B. Furthermore, we show that tumorigenesis in the CySC lineage interferes non-cell autonomously with maintenance of GSCs most likely by displacing them from their niche.
View details for DOI 10.1371/journal.pone.0052892
View details for PubMedID 23285219
Germline Stem Cells
COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY
2011; 3 (11)
Sperm and egg production requires a robust stem cell system that balances self-renewal with differentiation. Self-renewal at the expense of differentiation can cause tumorigenesis, whereas differentiation at the expense of self-renewal can cause germ cell depletion and infertility. In most organisms, and sometimes in both sexes, germline stem cells (GSCs) often reside in a defined anatomical niche. Factors within the niche regulate a balance between GSC self-renewal and differentiation. Asymmetric division of the germline stem cell to form daughter cells with alternative fates is common. The exception to both these tendencies is the mammalian testis where there does not appear to be an obvious anatomical niche and where GSC homeostasis is likely accomplished by a stochastic balance of self-renewal and differentiation and not by regulated asymmetric cell division. Despite these apparent differences, GSCs in all organisms share many common mechanisms, although not necessarily molecules, to guarantee survival of the germline.
View details for DOI 10.1101/cshperspect.a002642
View details for Web of Science ID 000296695500001
View details for PubMedID 21791699
- Role of Survivin in cytokinesis revealed by a separation-of-function allele MOLECULAR BIOLOGY OF THE CELL 2011; 22 (20): 3779-3790
Sequential changes at differentiation gene promoters as they become active in a stem cell lineage
2011; 138 (12): 2441-2450
Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here, we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC), a tissue-specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription.
View details for DOI 10.1242/dev.056572
View details for Web of Science ID 000290909400005
View details for PubMedID 21610025
View details for PubMedCentralID PMC3100706
E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells
2010; 5 (8)
Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.
View details for DOI 10.1371/journal.pone.0012473
View details for Web of Science ID 000281405300011
View details for PubMedID 20824213
The Drosophila SUN protein Spag4 cooperates with the coiled-coil protein Yuri Gagarin to maintain association of the basal body and spermatid nucleus
JOURNAL OF CELL SCIENCE
2010; 123 (16): 2763-2772
Maintaining the proximity of centrosomes to nuclei is important in several cellular contexts, and LINC complexes formed by SUN and KASH proteins are crucial in this process. Here, we characterize the presumed Drosophila ortholog of the mammalian SUN protein, sperm-associated antigen 4 (Spag4, previously named Giacomo), and demonstrate that Spag4 is required for centriole and nuclear attachment during spermatogenesis. Production of spag4 mRNA is limited to the testis, and Spag4 protein shows a dynamic pattern of association with the germline nuclei, including a concentration of protein at the site of attachment of the single spermatid centriole. In the absence of Spag4, nuclei and centrioles or basal bodies (BBs) dissociate from each other after meiosis. This role of Spag4 in centriolar attachment does not involve either of the two KASH proteins of the Drosophila genome (Klarsicht and MSP-300), but does require the coiled-coil protein Yuri Gagarin. Yuri shows an identical pattern of localization at the nuclear surface to Spag4 during spermatogenesis, and epistasis studies show that the activities of Yuri and dynein-dynactin are downstream of spag4 in this centriole attachment pathway. The later defects in spermatogenesis seen for yuri and spag4 mutants are similar, suggesting they could be secondary to initial disruption of events at the nuclear surface.
View details for DOI 10.1242/jcs.066589
View details for Web of Science ID 000280622700009
View details for PubMedID 20647369
Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila
MOLECULAR BIOLOGY OF THE CELL
2010; 21 (9): 1546-1555
During spermiogenesis, Drosophila melanogaster spermatids coordinate their elongation in interconnected cysts that become highly polarized, with nuclei localizing to one end and sperm tail growth occurring at the other. Remarkably little is known about the signals that drive spermatid polarity and elongation. Here we identify phosphoinositides as critical regulators of these processes. Reduction of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) by low-level expression of the PIP(2) phosphatase SigD or mutation of the PIP(2) biosynthetic enzyme Skittles (Sktl) results in dramatic defects in spermatid cysts, which become bipolar and fail to fully elongate. Defects in polarity are evident from the earliest stages of elongation, indicating that phosphoinositides are required for establishment of polarity. Sktl and PIP(2) localize to the growing end of the cysts together with the exocyst complex. Strikingly, the exocyst becomes completely delocalized when PIP(2) levels are reduced, and overexpression of Sktl restores exocyst localization and spermatid cyst polarity. Moreover, the exocyst is required for polarity, as partial loss of function of the exocyst subunit Sec8 results in bipolar cysts. Our data are consistent with a mechanism in which localized synthesis of PIP(2) recruits the exocyst to promote targeted membrane delivery and polarization of the elongating cysts.
View details for DOI 10.1091/mbc.E09-07-0582
View details for Web of Science ID 000277179600011
View details for PubMedID 20237161
TRAPPII is required for cleavage furrow ingression and localization of Rab11 in dividing male meiotic cells of Drosophila
JOURNAL OF CELL SCIENCE
2009; 122 (24): 4526-4534
Although membrane addition is crucial for cytokinesis in many animal cell types, the specific mechanisms supporting cleavage furrow ingression are not yet understood. Mutations in the gene brunelleschi (bru), which encodes the Drosophila ortholog of the yeast Trs120p subunit of TRAPPII, cause failure of furrow ingression in male meiotic cells. In non-dividing cells, Brunelleschi protein fused to GFP is dispersed throughout the cytoplasm and enriched at Golgi organelles, similarly to another Drosophila TRAPPII subunit, dBet3. Localization of the membrane-trafficking GTPase Rab11 to the cleavage furrow requires wild-type function of bru, and genetic interactions between bru and Rab11 increase the failure of meiotic cytokinesis and cause synthetic lethality. bru also genetically interacts with four wheel drive (fwd), which encodes a PI4Kbeta, such that double mutants exhibit enhanced failure of male meiotic cytokinesis. These results suggest that Bru cooperates with Rab11 and PI4Kbeta to regulate the efficiency of membrane addition to the cleavage furrow, thus promoting cytokinesis in Drosophila male meiotic cells.
View details for DOI 10.1242/jcs.054536
View details for Web of Science ID 000272329300013
View details for PubMedID 19934220
- Regulation of self-renewal, proliferation and differentiation in an adult stem cell lineage 16th Annual Conference of the International-Society-of-Development-Biologists ELSEVIER SCIENCE BV. 2009: S3–S3
Molecular Evolution of the Testis TAFs of Drosophila
MOLECULAR BIOLOGY AND EVOLUTION
2009; 26 (5): 1103-1116
The basal transcription machinery is responsible for initiating transcription at core promoters. During metazoan evolution, its components have expanded in number and diversified to increase the complexity of transcriptional regulation in tissues and developmental stages. To explore the evolutionary events and forces underlying this diversification, we analyzed the evolution of the Drosophila testis TAFs (TBP-associated factors), paralogs of TAFs from the basal transcription factor TFIID that are essential for normal transcription during spermatogenesis of a large set of specific genes involved in terminal differentiation of male gametes. There are five testis-specific TAFs in Drosophila, each expressed only in primary spermatocytes and each a paralog of a different generally expressed TFIID subunit. An examination of the presence of paralogs across taxa as well as molecular clock dating indicates that all five testis TAFs likely arose within a span of approximately 38 My 63-250 Ma by independent duplication events from their generally expressed paralogs. Furthermore, the evolution of the testis TAFs has been rapid, with apparent further accelerations in multiple Drosophila lineages. Analysis of between-species divergence and intraspecies polymorphism indicates that the major forces of evolution on these genes have been reduced purifying selection, pervasive positive selection, and coevolution. Other genes that exhibit similar patterns of evolution in the Drosophila lineages are also characterized by enriched expression in the testis, suggesting that the pervasive positive selection acting on the tTAFs is likely to be related to their expression in the testis.
View details for DOI 10.1093/molbev/msp030
View details for Web of Science ID 000265274000014
View details for PubMedID 19244474
Centrosome misorientation reduces stem cell division during ageing
2008; 456 (7222): 599-U40
Asymmetric division of adult stem cells generates one self-renewing stem cell and one differentiating cell, thereby maintaining tissue homeostasis. A decline in stem cell function has been proposed to contribute to tissue ageing, although the underlying mechanism is poorly understood. Here we show that changes in the stem cell orientation with respect to the niche during ageing contribute to the decline in spermatogenesis in the male germ line of Drosophila. Throughout the cell cycle, centrosomes in germline stem cells (GSCs) are oriented within their niche and this ensures asymmetric division. We found that GSCs containing misoriented centrosomes accumulate with age and that these GSCs are arrested or delayed in the cell cycle. The cell cycle arrest is transient, and GSCs appear to re-enter the cell cycle on correction of centrosome orientation. On the basis of these findings, we propose that cell cycle arrest associated with centrosome misorientation functions as a mechanism to ensure asymmetric stem cell division, and that the inability of stem cells to maintain correct orientation during ageing contributes to the decline in spermatogenesis. We also show that some of the misoriented GSCs probably originate from dedifferentiation of spermatogonia.
View details for DOI 10.1038/nature07386
View details for Web of Science ID 000261340000032
View details for PubMedID 18923395
A role for very-long-chain fatty acids in furrow ingression during cytokinesis in Drosophila spermatocytes
2008; 18 (18): 1426-1431
Cell shape and membrane remodeling rely on regulated interactions between the lipid bilayer and cytoskeletal arrays at the cell cortex. During cytokinesis, animal cells build an actomyosin ring anchored to the plasma membrane at the equatorial cortex. Ring constriction coupled to plasma-membrane ingression separates the two daughter cells. Plasma-membrane lipids influence membrane biophysical properties such as membrane curvature and elasticity and play an active role in cell function, and specialized membrane domains are emerging as important factors in regulating assembly and rearrangement of the cytoskeleton. Here, we show that mutations in the gene bond, which encodes a Drosophila member of the family of Elovl proteins that mediate elongation of very-long-chain fatty acids, block or dramatically slow cleavage-furrow ingression during early telophase in dividing spermatocytes. In bond mutant cells at late stages of division, the contractile ring frequently detaches from the cortex and constricts or collapses to one side of the cell, and the cleavage furrow regresses. Our findings implicate very-long-chain fatty acids or their derivative complex lipids in allowing supple membrane deformation and the stable connection of cortical contractile components to the plasma membrane during cell division.
View details for DOI 10.1016/j.cub.2008.08.061
View details for Web of Science ID 000259523600030
View details for PubMedID 18804373
Moesin and its activating kinase Slik are required for cortical stability and microtubule organization in mitotic cells
JOURNAL OF CELL BIOLOGY
2008; 180 (4): 739-746
Cell division requires cell shape changes involving the localized reorganization of cortical actin, which must be tightly linked with chromosome segregation operated by the mitotic spindle. How this multistep process is coordinated remains poorly understood. In this study, we show that the actin/membrane linker moesin, the single ERM (ezrin, radixin, and moesin) protein in Drosophila melanogaster, is required to maintain cortical stability during mitosis. Mitosis onset is characterized by a burst of moesin activation mediated by a Slik kinase-dependent phosphorylation. Activated moesin homogenously localizes at the cortex in prometaphase and is progressively restricted at the equator in later stages. Lack of moesin or inhibition of its activation destabilized the cortex throughout mitosis, resulting in severe cortical deformations and abnormal distribution of actomyosin regulators. Inhibiting moesin activation also impaired microtubule organization and precluded stable positioning of the mitotic spindle. We propose that the spatiotemporal control of moesin activation at the mitotic cortex provides localized cues to coordinate cortical contractility and microtubule interactions during cell division.
View details for DOI 10.1083/jcb.200709161
View details for Web of Science ID 000253494000024
View details for PubMedID 18283112
Asymmetric centrosome behavior and the mechanisms of stem cell division
JOURNAL OF CELL BIOLOGY
2008; 180 (2): 261-266
The ability of dividing cells to produce daughters with different fates is an important developmental mechanism conserved from bacteria to fungi, plants, and metazoan animals. Asymmetric outcomes of a cell division can be specified by two general mechanisms: asymmetric segregation of intrinsic fate determinants or asymmetric placement of daughter cells into microenvironments that provide extrinsic signals that direct cells to different states. For both, spindle orientation must be coordinated with the localization of intrinsic determinants or source of extrinsic signals to achieve the proper asymmetric outcome. Recent work on spindle orientation in Drosophila melanogaster male germline stem cells and neuroblasts has brought into sharp focus the key role of differential centrosome behavior in developmentally programmed asymmetric division (for reviews see Cabernard, C., and C.Q. Doe. 2007. Curr. Biol. 17:R465-R467; Gonzalez, C. 2007. Nat. Rev. Genet. 8:462-472). These findings provide new insights and suggest intriguing new models for how cells coordinate spindle orientation with their cellular microenvironment to regulate and direct cell fate decisions within tissues.
View details for Web of Science ID 000252746900004
View details for PubMedID 18209101
Regulation of Self-renewal and Differentiation in Adult Stem Cell Lineages: Lessons from the Drosophila Male Germ Line
73rd Cold Spring Harbor Symposium on Quantitative Biology
COLD SPRING HARBOR LABORATORY PRESS. 2008: 137–145
The ability to identify stem cells and trace their descendants in vivo has yielded insights into how self-renewal, proliferation, and differentiation are regulated in adult stem cell lineages. Analysis of male germ-line stem cells in Drosophila has revealed the importance of local signals from the microenvironment, the stem cell niche, in controlling stem cell behavior. Germ-line stem cells physically attach to the niche via localized adherens junctions that provide a polarity cue for orientation of centrosomes in interphase and the spindle in mitosis. As a result, stem cells divide asymmetrically: One daughter inherits attachment to the niche and remains within its embrace, whereas the other is displaced away and initiates differentiation. Strikingly, much as leukemia inhibitory factor (LIF) and transforming growth factor-beta (TGF-beta) signaling maintain mouse embryonic stem (ES) cells, maintenance of stem cell state in the Drosophila male germ line is regulated by cytokine-like signals from hub cells that activate the transcription factor STAT (signal transducer and activator of transcription) and TGF-beta class signals from surrounding support cells that repress expression of a key differentiation factor. Surprisingly, transit-amplifying cells can revert to the stem cell state if they reoccupy the niche. Upon cessation of mitosis and the switch to terminal differentiation, germ cells express cell-type- and stage-specific transcription machinery components that drive expression of terminal differentiation genes, in part by removing Polycomb transcriptional silencing machinery.
View details for Web of Science ID 000267135700017
View details for PubMedID 19329574
- Centrosome misorientation reduces stem cell division during ageing. Nature 2008; 456: 599-604
The Drosophila homolog of the Exo84 exocyst subunit promotes apical epithelial identity
JOURNAL OF CELL SCIENCE
2007; 120 (17): 3099-3110
The polarized architecture of epithelial tissues involves a dynamic balance between apical and basolateral membrane domains. Here we show that epithelial polarity in the Drosophila embryo requires the exocyst complex subunit homolog Exo84. Exo84 activity is essential for the apical localization of the Crumbs transmembrane protein, a key determinant of epithelial apical identity. Adherens junction proteins become mislocalized at the cell surface in Exo84 mutants in a pattern characteristic of defects in apical, but not basolateral, components. Loss of Crumbs from the cell surface precedes the disruption of Bazooka and Armadillo localization in Exo84 mutants. Moreover, Exo84 mutants display defects in apical cuticle secretion that are similar to crumbs mutants and are suppressed by a reduction in the basolateral proteins Dlg and Lgl. In Exo84 mutants at advanced stages of epithelial degeneration, apical and adherens junction proteins accumulate in an expanded recycling endosome compartment. These results suggest that epithelial polarity in the Drosophila embryo is actively maintained by exocyst-dependent apical localization of the Crumbs transmembrane protein.
View details for DOI 10.1242/jcs.004770
View details for Web of Science ID 000249559000012
View details for PubMedID 17698923
Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog
2007; 134 (15): 2863-2869
Translational control is crucial for proper timing of developmental events that take place in the absence of transcription, as in meiotic activation in oocytes, early embryogenesis in many organisms, and spermatogenesis. Here we show that a novel form of the translation initiation complex component eIF4G in Drosophila, eIF4G2, is required specifically for male germ cells to undergo meiotic division and proper spermatid differentiation. Flies mutant for eIF4G2 are viable and female fertile but male sterile. Spermatocytes form, but the germ cells in mutant males skip the major events of the meiotic divisions and form aberrant spermatids with large nuclei. Consistent with the failure to undergo the meiotic divisions, function of eIF4G2 is required post-transcriptionally for normal accumulation of the core cell cycle regulatory proteins Twine and CycB in mature spermatocytes. Loss of eIF4G2 function also causes widespread defects in spermatid differentiation. Although differentiation markers Dj and Fzo are expressed in late-stage eIF4G2 mutant germ cells, several key steps of spermatid differentiation fail, including formation of a compact mitochondrial derivative and full elongation. Our results suggest that an alternate form of the translation initiation machinery may be required for regulation and execution of key steps in male germ cell differentiation.
View details for DOI 10.1242/dev.003764
View details for Web of Science ID 000248381600015
View details for PubMedID 17611220
Antagonistic roles of Rac and Rho in organizing the germ cell microenvironment
2007; 17 (14): 1253-1258
The capacity of stem cells to self renew and the ability of stem cell daughters to differentiate into highly specialized cells depend on external cues provided by their cellular microenvironments [1-3]. However, how microenvironments are shaped is poorly understood. In testes of Drosophila melanogaster, germ cells are enclosed by somatic support cells. This physical interrelationship depends on signaling from germ cells to the Epidermal growth factor receptor (Egfr) on somatic support cells . We show that germ cells signal via the Egf class ligand Spitz (Spi) and provide evidence that the Egfr associates with and acts through the guanine nucleotide exchange factor Vav to regulate activities of Rac1. Reducing activity of the Egfr, Vav, or Rac1 from somatic support cells enhanced the germ cell enclosure defects of a conditional spi allele. Conversely, reducing activity of Rho1 from somatic support cells suppressed the germ cell enclosure defects of the conditional spi allele. We propose that a differential in Rac and Rho activities across somatic support cells guides their growth around the germ cells. Our novel findings reveal how signals from one cell type regulate cell-shape changes in another to establish a critical partnership required for proper differentiation of a stem cell lineage.
View details for DOI 10.1016/j.cub.2007.06.048
View details for Web of Science ID 000248202600031
View details for PubMedID 17629483
Male and female Drosophila germline stem cells: Two versions of immortality
2007; 316 (5823): 402-404
Drosophila male and female germline stem cells (GSCs) are sustained by niches and regulatory pathways whose common principles serve as models for understanding mammalian stem cells. Despite striking cellular and genetic similarities that suggest a common evolutionary origin, however, male and female GSCs also display important differences. Comparing these two stem cells and their niches in detail is likely to reveal how a common heritage has been adapted to the differing requirements of male and female gamete production.
View details for DOI 10.1126/science.1140861
View details for Web of Science ID 000245813400041
View details for PubMedID 17446390
Phosphorylation of histone H4 Ser1 regulates sporulation in yeast and is conserved in fly and mouse spermatogenesis
GENES & DEVELOPMENT
2006; 20 (18): 2580-2592
Sporulation in Saccharomyces cerevisiae is a highly regulated process wherein a diploid cell gives rise to four haploid gametes. In this study we show that histone H4 Ser1 is phosphorylated (H4 S1ph) during sporulation, starting from mid-sporulation and persisting to germination, and is temporally distinct from earlier meiosis-linked H3 S10ph involved in chromosome condensation. A histone H4 S1A substitution mutant forms aberrant spores and has reduced sporulation efficiency. Deletion of sporulation-specific yeast Sps1, a member of the Ste20 family of kinases, nearly abolishes the sporulation-associated H4 S1ph modification. H4 S1ph may promote chromatin compaction, since deletion of SPS1 increases accessibility to antibody immunoprecipitation; furthermore, either deletion of Sps1 or an H4 S1A substitution results in increased DNA volume in nuclei within spores. We find H4 S1ph present during Drosophila melanogaster and mouse spermatogenesis, and similar to yeast, this modification extends late into sperm differentiation relative to H3 S10ph. Thus, H4 S1ph may be an evolutionarily ancient histone modification to mark the genome for gamete-associated packaging.
View details for DOI 10.1101/gad.1457006
View details for Web of Science ID 000240526700010
View details for PubMedID 16980586
Stem cells and cancer: Two faces of eve
2006; 124 (6): 1111-1115
Recent evidence suggests that a subset of cancer cells within some tumors, the so-called cancer stem cells, may drive the growth and metastasis of these tumors. Understanding the pathways that regulate proliferation, self-renewal, survival, and differentiation of malignant and normal stem cells may shed light on mechanisms that lead to cancer and suggest better modes of treatment.
View details for DOI 10.1016/j.cell.2006.03.011
View details for Web of Science ID 000237241400007
View details for PubMedID 16564000
Histone modifications in spermatogenesis
31st Annual Meeting of the American-Society-of-Andrology
AMER SOC ANDROLOGY, INC. 2006: 34–34
View details for Web of Science ID 000235688200004
The class IPITP giotto is required for Drosophila cytokinesis
2006; 16 (2): 195-201
Phosphatidylinositol transfer proteins (PITPs) are highly conserved polypeptides that bind phosphatidylinositol or phosphatidylcholine monomers, facilitating their transfer from one membrane compartment to another . Although PITPs have been implicated in a variety of cellular functions, including lipid-mediated signaling and membrane trafficking, the precise biological roles of most PITPs remain to be elucidated . Here we show for the first time that a class I PITP is involved in cytokinesis. We found that giotto (gio), a Drosophila gene that encodes a class I PITP, serves an essential function required for both mitotic and meiotic cytokinesis. Neuroblasts and spermatocytes from gio mutants both assemble regular actomyosin rings. However, these rings fail to constrict to completion, leading to cytokinesis failures. Moreover, gio mutations cause an abnormal accumulation of Golgi-derived vesicles at the equator of spermatocyte telophases, suggesting that Gio is implicated in membrane-vesicle fusion. Consistent with these results, we found that Gio is enriched at the cleavage furrow, the ER, and the spindle envelope. We propose that Gio mediates transfer of lipid monomers from the ER to the equatorial membrane, causing a specific local enrichment in phosphatidylinositol. This change in membrane composition would ultimately facilitate vesicle fusion, allowing membrane addition to the furrow and/or targeted delivery of proteins required for cytokinesis.
View details for DOI 10.1016/j.cub.2005.12.011
View details for Web of Science ID 000235105900025
View details for PubMedID 16431372
Asymmetric stem cell division and function of the niche in the Drosophila male germ line
INTERNATIONAL JOURNAL OF HEMATOLOGY
2005; 82 (5): 377-380
The balance between stem cell and differentiating cell populations is critical for the long-term maintenance of tissue renewal for cell types derived from adult stem cell lineages such as blood, skin, intestinal epithelium, and sperm. To keep this balance, stem cells have the potential to divide asymmetrically, producing one daughter cell that maintains stem cell identity and one daughter cell that initiates differentiation. In many adult stem cell systems, the maintenance, proliferation, and number of stem cells appear to be controlled by the microenvironment, or niche. The Drosophila male and female germ line provide excellent model systems in which to study asymmetric stem cell divisions within the stem cell niche. In addition to signals from the niche that specify stem cell self-renewal, the stem cells themselves have elaborate cellular mechanisms to ensure the asymmetric outcome of cell division.
View details for DOI 10.1532/IJH97.05097
View details for Web of Science ID 000234398000002
View details for PubMedID 16533738
Signaling in stem cell niches: lessons from the Drosophila germline
JOURNAL OF CELL SCIENCE
2005; 118 (4): 665-672
Stem cells are cells that, upon division, can produce new stem cells as well as daughter cells that initiate differentiation along a specific lineage. Studies using the Drosophila germline as a model system have demonstrated that signaling from the stem cell niche plays a crucial role in controlling stem cell behavior. Surrounding support cells secrete growth factors that activate signaling within adjacent stem cells to specify stem cell self-renewal and block differentiation. In addition, cell-cell adhesion between stem cells and surrounding support cells is important for holding stem cells close to self-renewal signals. Furthermore, a combination of localized signaling and autonomously acting proteins might polarize stem cells in such a way as to ensure asymmetric stem cell divisions. Recent results describing stem cell niches in other adult stem cells, including hematopoietic and neural stem cells, have demonstrated that the features characteristic of stem cell niches in Drosophila gonads might be conserved.
View details for DOI 10.1242/jcs.01680
View details for Web of Science ID 000227741500004
View details for PubMedID 15701923
Belle is a Drosophila DEAD-box protein required for viability and in the germ line
2005; 277 (1): 92-101
DEAD-box proteins are ATP-dependent RNA helicases that function in various stages of RNA processing and in RNP remodeling. Here, we report identification and characterization of the Drosophila protein Belle (Bel), which belongs to a highly conserved subfamily of DEAD-box proteins including yeast Ded1p, Xenopus An3, mouse PL10, human DDX3/DBX, and human DBY. Mutations in DBY are a frequent cause of male infertility in humans. Bel can substitute in vivo for Ded1p, an essential yeast translation factor, suggesting a requirement for Bel in translation initiation. Consistent with an essential cellular function, strong loss of function mutations in bel are recessive lethal with a larval growth defect phenotype. Hypomorphic bel mutants are male-sterile. Bel is also closely related to the Drosophila DEAD-box protein Vasa (Vas), a germ line-specific translational regulator. We find that Bel and Vas colocalize in nuage and at the oocyte posterior during oogenesis, and that bel function is required for female fertility. However, unlike Vas, Bel is not specifically enriched in embryonic pole cells. We conclude that the DEAD-box protein Bel has evolutionarily conserved roles in fertility and development.
View details for DOI 10.1016/j.ydbio.2004.09.009
View details for Web of Science ID 000225741200008
View details for PubMedID 15572142
Testis-specific TAF homologs collaborate to control a tissue-specific transcription program
2004; 131 (21): 5297-5308
Alternate forms of the PolII transcription initiation machinery have been proposed to play a role in selective activation of cell-type-specific gene expression programs during cellular differentiation. The cannonball (can) gene of Drosophila encodes a homolog of a TBP-associated factor (dTAF5) protein expressed only in spermatocytes, where it is required for normal transcription of genes required for spermatid differentiation. We show that Drosophila primary spermatocytes also express four additional tissue-specific TAFs: nht (homolog of dTAF4), mia (homolog of dTAF6), sa (homolog of dTAF8) and rye (homolog of dTAF12). Mutations in nht, mia and sa have similar effects in primary spermatocytes on transcription of several target genes involved in spermatid differentiation, and cause the same phenotypes as mutations in can, blocking both meiotic cell cycle progression and spermatid differentiation. The nht, mia, sa and rye proteins contain histone fold domain dimerization motifs. The nht and rye proteins interact structurally when co-expressed in bacteria, similarly to their generally expressed homologs TAF4 and TAF12, which heterodimerize. Strikingly, the structural interaction is tissue specific: nht did not interact with dTAF12 and dTAF4 did not interact with rye in a bacterial co-expression assay. We propose that the products of the five Drosophila genes encoding testis TAF homologs collaborate in an alternative TAF-containing protein complex to regulate a testis-specific gene expression program in primary spermatocytes required for terminal differentiation of male germ cells.
View details for Web of Science ID 000225422200009
View details for PubMedID 15456720
Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila
2004; 572 (1-3): 141-146
A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.
View details for DOI 10.1016/j.febslet.2004.07.025
View details for Web of Science ID 000223519300026
View details for PubMedID 15304338
A misexpression screen reveals effects of bag-of-marbles and TGF beta class signaling on the Drosophila male germ-line stem cell lineage
2004; 167 (2): 707-723
Male gametes are produced throughout reproductive life by a classic stem cell mechanism. However, little is known about the molecular mechanisms for lineage production that maintain male germ-line stem cell (GSC) populations, regulate mitotic amplification divisions, and ensure germ cell differentiation. Here we utilize the Drosophila system to identify genes that cause defects in the male GSC lineage when forcibly expressed. We conducted a gain-of-function screen using a collection of 2050 EP lines and found 55 EP lines that caused defects at early stages of spermatogenesis upon forced expression either in germ cells or in surrounding somatic support cells. Most strikingly, our analysis of forced expression indicated that repression of bag-of-marbles (bam) expression in male GSC is important for male GSC survival, while activity of the TGF beta signal transduction pathway may play a permissive role in maintenance of GSCs in Drosophila testes. In addition, forced activation of the TGF beta signal transduction pathway in germ cells inhibits the transition from the spermatogonial mitotic amplification program to spermatocyte differentiation.
View details for Web of Science ID 000222650500014
View details for PubMedID 15238523
Genetic dissection of meiotic cytokinesis in Drosophila males
MOLECULAR BIOLOGY OF THE CELL
2004; 15 (5): 2509-2522
We have used Drosophila male meiosis as a model system for genetic dissection of the cytokinesis mechanism. Drosophila mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. We have screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, we analyzed meiotic divisions in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. Our results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, our genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis.
View details for Web of Science ID 000221189300039
View details for PubMedID 15004238
Regulation of transcription of meiotic cell cycle and terminal differentiation genes by the testis-specific Zn-finger protein matotopetli
2004; 131 (8): 1691-1702
A robust developmentally regulated and cell type specific transcriptional programme is activated in primary spermatocytes in preparation for differentiation of the male gametes during spermatogenesis. Work in Drosophila is beginning to reveal the genetic networks that regulate this gene expression. The Drosophila aly-class meiotic arrest loci are essential for activation of transcription of many differentiation-specific genes, as well as several genes important for meiotic cell cycle progression, thus linking meiotic cell cycle progression to cellular differentiation during spermatogenesis. The three previously described aly-class proteins (aly, comr and achi/vis) form a complex and are associated with chromatin in primary spermatocytes. We identify, clone and characterize a new aly-class meiotic arrest gene, matotopetli (topi), which encodes a testis-specific Zn-finger protein that physically interacts with Comr. The topi mutant phenotype is most like achi/vis in that topi function is not required for the nuclear localization of Aly or Comr, but is required for their accumulation on chromatin. Most target genes in the transcriptional programme depend on both topi and achi/vis; however, a small subset of target genes are differentially sensitive to loss of topi or achi/vis, suggesting that these aly-class predicted DNA binding proteins can act independently in some contexts.
View details for DOI 10.1242/dev.01032
View details for Web of Science ID 000221155900003
View details for PubMedID 15084455
Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state
2003; 130 (26): 6625-6634
Gap junctions coordinate processes ranging from muscle contraction to ovarian follicle development. Here we show that the gap junction protein Zero population growth (Zpg) is required for germ cell differentiation in the Drosophila ovary. In the absence of Zpg the stem cell daughter destined to differentiate dies. The zpg phenotype is novel, and we used this phenotype to genetically dissect the process of stem cell maintenance and differentiation. Our findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual.
View details for DOI 10.1242/dev.00853
View details for Web of Science ID 000188254100021
View details for PubMedID 14660550
Mitofusin-1 protein is a generally expressed mediator of mitochondrial fusion in mammalian cells
JOURNAL OF CELL SCIENCE
2003; 116 (13): 2763-2774
Mitochondrial fusion may regulate mitochondrial morphogenesis and underlie complementation between mitochondrial genomes in mammalian cells. The nuclear encoded mitochondrial proteins Mfn1 and Mfn2 are human homologues of the only known protein mediators of mitochondrial fusion, the Drosophila Fzo GTPase and Saccharomyces cerevisiae yFzo1p. Although the Mfn1 and Mfn2 genes were broadly expressed, the two genes showed different levels of mRNA expression in different tissues. Two Mfn1 transcripts were detected at similar levels in a variety of human tissues and were dramatically elevated in heart, while Mfn2 mRNA was abundantly expressed in heart and muscle tissue but present only at low levels in many other tissues. Human Mfn1 protein localized to mitochondria and participated in a high molecular weight, detergent extractable protein complex. Forced expression of Mfn1 in cultured cells caused formation of characteristic networks of mitochondria. Introduction of a point mutation in the conserved G1 region of the predicted GTPase domain (Mfn1K88T) dramatically decreased formation of mitochondrial networks upon Mfn1 overexpression, suggesting that network formation required completion of the Mfn1 GTPase cycle. Conversely, a protein variant carrying a point mutation in the G2 motif of the Mfn1 GTPase domain acted as a dominant negative: overexpression of Mfn1T109A resulted in fragmentation of mitochondria. We propose that Mfn1T109A interferes with fusion activity of endogenous Mfn1 protein, possibly by binding necessary cofactors, so to allow unopposed mitochondrial fission. Thus, Mfn1 appears to be a key player in mediating mitochondrial fusion and morphology in mammalian cells.
View details for DOI 10.1242/jcs.00479
View details for Web of Science ID 000184096800015
View details for PubMedID 12759376
The Drosophila Cog5 homologue is required for cytokinesis, cell elongation, and assembly of specialized golgi architecture during spermatogenesis
MOLECULAR BIOLOGY OF THE CELL
2003; 14 (1): 190-200
The multisubunit conserved oligomeric Golgi (COG) complex has been shown previously to be involved in Golgi function in yeast and mammalian tissue culture cells. Despite this broad conservation, several subunits, including Cog5, were not essential for growth and showed only mild effects on secretion when mutated in yeast, raising questions about what functions these COG complex subunits play in the life of the cell. Here, we show that function of the gene four way stop (fws), which encodes the Drosophila Cog5 homologue, is necessary for dramatic changes in cellular and subcellular morphology during spermatogenesis. Loss-of-function mutations in fws caused failure of cleavage furrow ingression in dividing spermatocytes and failure of cell elongation in differentiating spermatids and disrupted the formation and/or stability of the Golgi-based spermatid acroblast. Consistent with the lack of a growth defect in yeast lacking Cog5, animals lacking fws function were viable, although males were sterile. Fws protein localized to Golgi structures throughout spermatogenesis. We propose that Fws may directly or indirectly facilitate efficient vesicle traffic through the Golgi to support rapid and extensive increases in cell surface area during spermatocyte cytokinesis and polarized elongation of differentiating spermatids. Our study suggests that Drosophila spermatogenesis can be an effective sensitized genetic system to uncover in vivo functions for proteins involved in Golgi architecture and/or vesicle transport.
View details for DOI 10.1091/mbc.E02-06-0343
View details for Web of Science ID 000180497300016
View details for PubMedID 12529436
Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis
JOURNAL OF CELL BIOLOGY
2002; 159 (6): 931-938
We find that Bax, a proapoptotic member of the Bcl-2 family, translocates to discrete foci on mitochondria during the initial stages of apoptosis, which subsequently become mitochondrial scission sites. A dominant negative mutant of Drp1, Drp1K38A, inhibits apoptotic scission of mitochondria, but does not inhibit Bax translocation or coalescence into foci. However, Drp1K38A causes the accumulation of mitochondrial fission intermediates that are associated with clusters of Bax. Surprisingly, Drp1 and Mfn2, but not other proteins implicated in the regulation of mitochondrial morphology, colocalize with Bax in these foci. We suggest that Bax participates in apoptotic fragmentation of mitochondria.
View details for DOI 10.1083/jcb.200209124
View details for Web of Science ID 000180150200003
View details for PubMedID 12499352
View details for PubMedCentralID PMC2173996
Signaling from germ cells mediated by the rhomboid homolog stet organizes encapsulation by somatic support cells
2002; 129 (19): 4523-4534
Germ cells normally differentiate in the context of encapsulating somatic cells. However, the mechanisms that set up the special relationship between germ cells and somatic support cells and the signals that mediate the crucial communications between the two cell types are poorly understood. We show that interactions between germ cells and somatic support cells in Drosophila depend on wild-type function of the stet gene. In males, stet acts in germ cells to allow their encapsulation by somatic cyst cells and is required for germ cell differentiation. In females, stet function allows inner sheath cells to enclose early germ cells correctly at the tip of the germarium. stet encodes a homolog of rhomboid, a component of the epidermal growth factor receptor signaling pathway involved in ligand activation in the signaling cell. The stet mutant phenotype suggests that stet facilitates signaling from germ cells to the epidermal growth factor receptor on somatic cells, resulting in the encapsulation of germ cells by somatic support cells. The micro-environment provided by the surrounding somatic cells may, in turn, regulate differentiation of the germ cells they enclose.
View details for Web of Science ID 000178640100013
View details for PubMedID 12223409
Differential expression of the Drosophila mitofusin genes fuzzy onions (fzo) and dmfn
MECHANISMS OF DEVELOPMENT
2002; 116 (1-2): 213-216
Mitofusins comprise a family of evolutionarily conserved, nuclear encoded mitochondrial guanosine triphoshatases that control mitochondrial fusion and morphology. The fuzzy onions (fzo) and Drosophila mitofusin (dmfn) genes, which encode the only Mitofusin homologs in Drosophila are differentially expressed during development. Dmfn-mRNA was widely expressed during embryogenesis accumulating in the mesoderm and endoderm during gut development, during oogenesis with transcripts maternally deposited into the early embryo and in the male germ line, where dmfn-mRNA was expressed in spermatogonia, spermatocytes and early spermatids. In contrast, expression of the fzo was tightly restricted to the male germ line, with mRNA accumulation in spermatocytes and early spermatids. In addition, expression of dmfn and fzo in the same cell type, primary spermatocytes, was under control of different regulatory mechanisms.
View details for Web of Science ID 000177333000027
View details for PubMedID 12128227
A germline-specific gap junction protein required for survival of differentiating early germ cells
2002; 129 (10): 2529-2539
Germ cells require intimate associations and signals from the surrounding somatic cells throughout gametogenesis. The zero population growth (zpg) locus of Drosophila encodes a germline-specific gap junction protein, Innexin 4, that is required for survival of differentiating early germ cells during gametogenesis in both sexes. Animals with a null mutation in zpg are viable but sterile and have tiny gonads. Adult zpg-null gonads contain small numbers of early germ cells, resembling stem cells or early spermatogonia or oogonia, but lack later stages of germ cell differentiation. In the male, Zpg protein localizes to the surface of spermatogonia, primarily on the sides adjacent to the somatic cyst cells. In the female, Zpg protein localizes to germ cell surfaces, both those adjacent to surrounding somatic cells and those adjacent to other germ cells. We propose that Zpg-containing gap junctional hemichannels in the germ cell plasma membrane may connect with hemichannels made of other innexin isoforms on adjacent somatic cells. Gap junctional intercellular communication via these channels may mediate passage of crucial small molecules or signals between germline and somatic support cells required for survival and differentiation of early germ cells in both sexes.
View details for Web of Science ID 000176063600019
View details for PubMedID 11973283
Genetic analysis of dPsa, the Drosophila orthologue of puromycin-sensitive aminopeptidase, suggests redundancy of aminopeptidases
DEVELOPMENT GENES AND EVOLUTION
2001; 211 (12): 581-588
Abstract. The Drosophila genome contains a single orthologue of mammalian puromycin-sensitive aminopeptidases, dPsa. Even though dPsa was expressed in many tissues during development, animals lacking dPsa activity were viable. Ubiquitous overexpression of dPsa during embryonic or larval development resulted in lethality and overexpression in isolated tissues during development resulted in localized lesions. These results suggest that even though dPsa function was not essential for viability, dPsa expression must be tightly regulated for normal development. By screening the Drosophila genome we found 43 predicted aminopeptidases and generated a phylogenetic tree of aminopeptidases related to dPsa by sequence. We discuss possible functions of dPsa and the idea that other Drosophila aminopeptidases might perform redundant functions with dPsa for regulating protein turnover.
View details for DOI 10.1007/s00427-001-0194-z
View details for Web of Science ID 000174249000002
View details for PubMedID 11819115
Developmental regulation of transcription by a tissue-specific TAF homolog
GENES & DEVELOPMENT
2001; 15 (8): 1021-1030
Alternate forms of the general transcription machinery have been described in several tissues or cell types. However, the role of tissue-specific TBP-associated factors (TAF(II)s) and other tissue-specific transcription components in regulating differential gene expression during development was not clear. Here we show that the cannonball gene of Drosophila encodes a cell type-specific homolog of a more ubiquitously expressed component of the general transcription factor TFIID. cannonball is required in vivo for high level transcription of a set of stage- and tissue-specific target genes during male gametogenesis. Regulation of transcription by cannonball is absolutely required for spermatogenesis, as null mutations block meiotic cell cycle progression and result in a complete failure of spermatid differentiation. Our results demonstrate that cell type-specific TAF(II)s play an important role in developmental regulation of gene expression.
View details for Web of Science ID 000168271800010
View details for PubMedID 11316795
Control of mitochondrial morphology by a human mitofusin
JOURNAL OF CELL SCIENCE
2001; 114 (5): 867-874
Although changes in mitochondrial size and arrangement accompany both cellular differentiation and human disease, the mechanisms that mediate mitochondrial fusion, fission and morphogenesis in mammalian cells are not understood. We have identified two human genes encoding potential mediators of mitochondrial fusion. The mitofusins (Mfn1 and Mfn2) are homologs of the Drosophila protein fuzzy onion (Fzo) that associate with mitochondria and alter mitochondrial morphology when expressed by transient transfection in tissue culture cells. An internal region including a predicted bipartite transmembrane domain (TM) is sufficient to target Mfn2 to mitochondria and requires hydrophobic residues within the TM. Co-expression of Mfn2 with a dominant interfering mutant dynamin-related protein (Drp1(K38A)) proposed to block mitochondrial fission resulted in long mitochondrial filaments and networks. Formation of mitochondrial filaments and networks required a wild-type Mfn2 GTPase domain, suggesting that the Mfn2 GTPase regulates or mediates mitochondrial fusion and that mitofusins and dynamin related GTPases play opposing roles in mitochondrial fusion and fission in mammals, as in yeast.
View details for Web of Science ID 000167569000005
View details for PubMedID 11181170
Transcription of meiotic cell cycle and terminal differentiation genes depends on a conserved chromatin associated protein, whose nuclear localisation is regulated
2000; 127 (24): 5463-5473
The Drosophila always early (aly) gene coordinately regulates meiotic cell cycle progression and terminal differentiation during male gametogenesis. aly is required for transcription of key G2-M cell cycle control genes and of spermatid differentiation genes, and for maintenance of normal chromatin structure in primary spermatocytes. We show that aly encodes a homologue of the Caenorhabditis elegans gene lin-9, a negative regulator of vulval development that acts in the same SynMuvB genetic pathway as the LIN-35 Rb-like protein. The aly gene family is conserved from plants to humans. Aly protein is both cytoplasmic and nuclear in early primary spermatocytes, then resolves to a chromatin-associated pattern. It remains cytoplasmic in a loss-of-function missense allele, suggesting that nuclear localisation is critical for Aly function, and that other factors may alter Aly activity by controlling its subcellular localisation. MAPK activation occurs normally in aly mutant testes. Therefore aly, and by inference lin-9, act in parallel to, or downstream of, activation of MAPK by the RTK-Ras signalling pathway. We favour a model where aly may regulate cell cycle progression and terminal differentiation during male gametogenesis by regulating chromatin conformation in primary spermatocytes.
View details for Web of Science ID 000166204800022
View details for PubMedID 11076766
A phospholipid kinase regulates actin organization and intercellular bridge formation during germline cytokinesis
2000; 127 (17): 3855-3864
The endgame of cytokinesis can follow one of two pathways depending on developmental context: resolution into separate cells or formation of a stable intercellular bridge. Here we show that the four wheel drive (fwd) gene of Drosophila melanogaster is required for intercellular bridge formation during cytokinesis in male meiosis. In fwd mutant males, contractile rings form and constrict in dividing spermatocytes, but cleavage furrows are unstable and daughter cells fuse together, producing multinucleate spermatids. fwd is shown to encode a phosphatidylinositol 4-kinase (PI 4-kinase), a member of a family of proteins that perform the first step in the synthesis of the key regulatory membrane phospholipid PIP2. Wild-type activity of the fwd PI 4-kinase is required for tyrosine phosphorylation in the cleavage furrow and for normal organization of actin filaments in the constricting contractile ring. Our results suggest a critical role for PI 4-kinases and phosphatidylinositol derivatives during the final stages of cytokinesis.
View details for Web of Science ID 000089430700020
View details for PubMedID 10934029
Regulation of meiosis and spermatid differentiation in Drosophila primary spermatocytes
XVth Testis Workshop on the Testis - From Stem Cell to Sperm Function
SPRINGER-VERLAG. 2000: 120–132
View details for Web of Science ID 000089810900011
Developmental genetics of the essential Drosophila nucleoporin nup154: Allelic differences due to an outward-directed promoter in the P-element 3 ' end
1999; 153 (2): 799-812
Drosophila nup154 encodes a predicted nucleoporin homologous to yeast Nup170p, Nup157p, and vertebrate Nup155, all of which are major components of the nuclear pore complex (NPC). Unlike its yeast homologs, nup154 is essential for viability. Animals with strong loss-of-function nup154 mutations caused by P-element insertion in the 5'-UTR of the gene died as larvae with small discs, brains, and testes. nup154 mRNA expression appeared developmentally regulated in tissues of wild-type embryos, larvae, and adults, suggesting that new nup154 synthesis is required when assembly of new NPCs is required, as in proliferating or growing tissues. Two additional nup154 alleles also associated with different P-element inserts in the 5'-UTR were viable but had strong loss-of-function sterile phenotypes, including failure to maintain spermatogenic stem cells and failure to progress into vitellogenic stages of oogenesis. Lethality vs. viability correlated with orientation of the P-element inserts in the different alleles. Transcript analysis by 5'-RACE suggested a mechanism for allelic differences: an outward-directed promoter internal to the P-element 3' end able to drive sufficient expression of the nup154 transcript for viability but not for fertility.
View details for Web of Science ID 000083051400024
View details for PubMedID 10511559
Molecular characterization of mutant alleles of the DNA repair basal transcription factor haywire/ERCC3 in Drosophila
1999; 152 (1): 291-297
The haywire gene of Drosophila encodes a putative helicase essential for transcription and nucleotide excision repair. A haywire allele encoding a dominant acting poison product, lethal alleles, and viable but UV-sensitive alleles isolated as revertants of the dominant acting poison allele were molecularly characterized. Sequence analysis of lethal haywire alleles revealed the importance of the nucleotide-binding domain, suggesting an essential role for ATPase activity. The viable haync2 allele, which encodes a poison product, has a single amino acid change in conserved helicase domain VI. This mutation results in accumulation of a 68-kD polypeptide that is much more abundant than the wild-type haywire protein.
View details for Web of Science ID 000080219100024
View details for PubMedID 10224261
Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p.
CELL PRESS. 1999: A2–A2
View details for Web of Science ID 000081085900008
Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p
JOURNAL OF CELL BIOLOGY
1998; 143 (2): 359-373
Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.
View details for Web of Science ID 000076618200008
View details for PubMedID 9786948
View details for PubMedCentralID PMC2132826
Genetic control of cell proliferation and differentiation in Drosophila spermatogenesis
SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY
1998; 9 (4): 433-444
Outlines of the genetic circuitry regulating male gametogenesis in Drosophila have begun to appear. Cessation of mitotic proliferation and onset of the meiotic program is regulated by the bam and bgcn genes acting within male germ cells and a TGF-beta class signaling cascade in surrounding somatic cells. Onset of spermatid differentiation is regulated by a stage- and tissue-specific transcriptional program controlled by the aly, can, mia and sa genes. A cross-regulatory mechanism might act, in part by controlling expression of the twine cell cycle phosphatase, to delay the G2/M transition of meiosis I until genes required for spermatid differentiation have been transcribed.
View details for Web of Science ID 000077088400007
View details for PubMedID 9813190
The zero population growth (zpg) gene is required for mitotic amplification stage of early germ cells in Drosophila.
ACADEMIC PRESS INC ELSEVIER SCIENCE. 1998: 197–97
View details for Web of Science ID 000074281900233
The DUG gene of Drosophila melanogaster encodes a structural and functional homolog of the S-cerevisiae SUG1 predicted ATPase associated with the 26S proteasome
1998; 206 (2): 165-174
The DUG gene of Drosophila encodes a putative ATPase that is a structural and functional homolog of the yeast SUG1 product. When introduced into S. cerevisiae, the Drosophila DUG gene rescued the lethality associated with a SUG1 mutant. Anti-DUG antibodies recognized a protein that migrated in high molecular weight complexes, along with components of the 26S proteasome, and also immunoprecipitated components of the 26S proteasome from embryonic extracts. Proteins recognized by the affinity-purified antibody raised against DUG were localized in either a punctate cytoplasmic distribution or in the nucleus, depending on the cell type, consistent with the subcellular localization of the 26S proteasome in various cell types.
View details for Web of Science ID 000072104800002
View details for PubMedID 9469929
Transcriptional and post-transcriptional control mechanisms coordinate the onset of spermatid differentiation with meiosis I in Drosophila
1998; 125 (1): 125-134
The aly, can, mia and sa genes of Drosophila are essential in males both for the G2-meiosis I transition and for onset of spermatid differentiation. Function of all four genes is required for transcription in primary spermatocytes of a suite of spermatid differentiation genes. aly is also required for transcription of the cell cycle control genes cyclin B and twine in primary spermatocytes. In contrast can, mia and sa are required for accumulation of twine protein but not twine transcript. We propose that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. We also propose that control of translation or protein stability regulates entry into the first meiotic division. We suggest that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of twine protein, thus coordinating meiotic division with onset of spermatid differentiation.
View details for Web of Science ID 000071652400013
View details for PubMedID 9389670
A chromatin-associated kinesin-related protein required for normal mitotic chromosome segregation in Drosophila
JOURNAL OF CELL BIOLOGY
1997; 139 (6): 1361-1371
The tiovivo (tio) gene of Drosophila encodes a kinesin-related protein, KLP38B, that colocalizes with condensed chromatin during cell division. Wild-type function of the tio gene product KLP38B is required for normal chromosome segregation during mitosis. Mitotic cells in tio larval brains displayed circular mitotic figures, increased ploidy, and abnormal anaphase figures. KLP38B mRNA is maternally provided and expressed in cells about to undergo division. We propose that KLP38B, perhaps redundantly with other chromosome-associated microtubule motor proteins, contributes to interactions between chromosome arms and microtubules important for establishing bipolar attachment of chromosomes and assembly of stable bipolar spindles.
View details for Web of Science ID 000071147400001
View details for PubMedID 9396743
Differential expression of two gamma-tubulin isoforms during gametogenesis and development in Drosophila
1997; 184 (2): 207-221
Previous work identified a gamma-tubulin gene, gamma Tub23C, in Drosophila (Zheng et al., 1991). We now report identification of a second gamma-tubulin gene, gamma Tub37CD. Immunoblot analysis and immunolocalization show that gamma Tub37CD and gamma Tub23C are differentially expressed during gametogenesis and development. During oogenesis, gamma Tub23C was detected at centrosomes and in the cytoplasm of mitotic germ cells, but was not detected in germ cells following completion of mitosis. Conversely, gamma Tub37CD was not detected in proliferating germ cells, but appeared to accumulate in germ cells during egg chamber development. Neither gamma-tubulin isoform was detected at the anterior or posterior poles of developing oocytes. During spermatogenesis, only gamma Tub23C was detected at centrosomes, where it showed cell cycle- and differentiation-dependent organization. During the transition into the first meiotic division, gamma Tub23C became organized as a corpuscular focus at centrioles until completion of meiosis II. During postmeiotic spermatid differentiation, gamma Tub23C was detected first as a rod and then as a collar-like structure near the juncture of the nucleus and the elongating flagellum, but was not detected in bundles of mature sperm. The germline-specific CDC25 encoded by twine is required for organization of gamma Tub23C into corpuscular focus in spermatocytes, but not for separation of centriole pairs in M-phase or postmeiotic organization of gamma Tub23C at centrioles. Following reconstitution of a canonical centrosome at fertilization, only gamma Tub37CD was detected at centrosomes in syncytial embryos, but both gamma Tub37CD and gamma Tub23C were detected at centrosomes in cellularized embryos. Colocalization of these two isoforms suggests that gamma Tub23C and gamma Tub37CD both contain structural features of gamma-tubulins essential for localization to centrosomes.
View details for Web of Science ID A1997WX47200003
View details for PubMedID 9133431
Monastral bipolar spindles: Implications for dynamic centrosome organization
JOURNAL OF CELL SCIENCE
1997; 110: 451-464
Implicit to all models for mitotic spindle assembly is the view that centrosomes are essentially permanent structures. Yet, immunofluorescence revealed that spindles in larval brains of urchin mutants in Drosophila were frequently monastral but bipolar; the astral pole contained a centrosome while the opposing anastral pole showed neither gamma tubulin nor a radial array of astral microtubules. Thus, mutations in the urchin gene seem to uncouple centrosome organization and spindle bipolarity in mitotic cells. Hypomorphic mutants showed a high frequency of monastral bipolar spindles but low frequencies of polyploidy, suggesting that monastral bipolar spindles might be functional. To test this hypothesis, we performed pedigree analysis of centrosome distribution and spindle structure in the four mitotic divisions of gonial cells. Prophase gonial cells showed two centrosomes, suggesting cells entered mitosis with the normal number of centrosomes and that centrosomes separated during prophase. Despite a high frequency of monastral bipolar spindles, the end products of the four mitotic divisions were equivalent in size and chromatin content. These results indicate that monastral bipolar spindles are functional and that the daughter cell derived from the anastral pole can assemble a functional bipolar spindle in the subsequent cell cycle. Cell proliferation despite high frequencies of monastral bipolar spindles can be explained if centrosome structure in mitotic cells is dynamic, allowing transient and benign disorganization of pericentriolar components. Since urchin proved to be allelic to KLP61F which encodes a kinesin related motor protein (Heck et al. (1993) J. Cell Biol. 123, 665-671), our results suggest that motors influence the dynamic organization of centrosomes.
View details for Web of Science ID A1997WL46000006
View details for PubMedID 9067597
Assembly of ring canals in the male germ line from structural components of the contractile ring
JOURNAL OF CELL SCIENCE
1996; 109: 2779-2788
Stable intercellular bridges called ring canals form following incomplete cytokinesis, and interconnect mitotically or meiotically related germ cells. We show that ring canals in Drosophila melanogaster males are surprisingly different from those previously described in females. Mature ring canal walls in males lack actin and appear to derive directly from structural proteins associated with the contractile ring. Ring canal assembly in males, as in females, initiates during cytokinesis with the appearance of a ring of phosphotyrosine epitopes at the site of the contractile ring. Following constriction, actin and myosin II disappear. However, at least four proteins present at the contractile ring remain: the three septins (Pnut, Sep1 and Sep2) and anillin. In sharp contrast, in ovarian ring canals, septins have not been detected, anillin is lost from mature ring canals and filamentous actin is a major component. In both males and females, a highly branched vesicular structure, termed the fusome, interconnects developing germ cells via the ring canals and is thought to coordinate mitotic germ cell divisions. We show that, in males, unlike females, the fusome persists and enlarges following cessation of the mitotic divisions, developing additional branches during meiosis. During differentiation, the fusome and its associated ring canals localize to the distal tip of the elongating spermatids.
View details for Web of Science ID A1996WB43700001
View details for PubMedID 9013326
Coordinate developmental control of the meiotic cell cycle and spermatid differentiation in Drosophila males
1996; 122 (4): 1331-1341
Wild-type function of four Drosophila genes, spermatocyte arrest, cannonball, always early and meiosis I arrest, is required both for cell-cycle progression through the G2/M transition of meiosis I in males and for onset of spermatid differentiation. In males mutant for any one of these meiotic arrest genes, mature primary spermatocytes with partially condensed chromosomes accumulate and postmeiotic cells are lacking. The arrest in cell-cycle progression occurs prior to degradation of cyclin A protein. The block in spermatogenesis in these mutants is not simply a secondary consequence of meiotic cell-cycle arrest, as spermatid differentiation proceeds in males mutant for the cell cycle activating phosphatase twine. Instead, the arrest of both meiosis and spermiogenesis suggests a control point that may serve to coordinate the male meiotic cell cycle with the spermatid differentiation program. The phenotype of the Drosophila meiotic arrest mutants is strikingly similar to the histopathological features of meiosis I maturation arrest infertility in human males, suggesting that the control point may be conserved from flies to man.
View details for Web of Science ID A1996UF83100029
View details for PubMedID 8620860
- RIDING THE POLAR WINDS - CHROMOSOMES MOTOR DOWN EAST CELL 1995; 81 (1): 5-8
A CYTOPLASMIC DYNEIN MOTOR IN DROSOPHILA - IDENTIFICATION AND LOCALIZATION DURING EMBRYOGENESIS
JOURNAL OF CELL SCIENCE
1994; 107: 1557-1569
We have characterized a cytoplasmic dynein motor isoform that is present in extracts of Drosophila embryos. A prominent high molecular weight (HMW) polypeptide (> 400 kDa) is enriched in microtubules prepared from nucleotide-depleted embryonic extracts. Based on its ATP-sensitive microtubule binding activity, 20 S sedimentation coefficient, sensitivity to UV-vanadate and nucleotide specificity, the HMW polypeptide resembles cytoplasmic dyneins prepared from other organisms. The Drosophila cytoplasmic dynein acts as a minus-end motor that promotes microtubule translocation in vitro. A polyclonal antibody raised against the dynein heavy chain polypeptide was used to localize the dynein antigen in whole-mount preparations of embryos by immunofluorescence microscopy. These studies show that the dynein motor is associated with microtubules throughout embryogenesis, including mitotic spindle microtubules and microtubules of the embryonic nervous system.
View details for Web of Science ID A1994NW49500021
View details for PubMedID 7962198
A DROSOPHILA MODEL FOR XERODERMA-PIGMENTOSUM AND COCKAYNES-SYNDROME - HAYWIRE ENCODES THE FLY HOMOLOG OF ERCC3, A HUMAN EXCISION REPAIR GENE
1992; 71 (6): 925-937
The haywire gene of Drosophila encodes a protein with 66% identity to the product of the human ERCC3 gene, associated with xeroderma pigmentosum B (XP-B) and Cockayne's syndrome (CS). XP is a human autosomal recessive disease characterized by extreme sensitivity to ultraviolet irradiation and marked susceptibility to skin cancer. In addition, XP and CS patients often exhibit a variety of defects, ranging from central nervous system disorders to hypogonadism. Phenotypes of haywire mutants mimic some of the effects of XP. Many haywire alleles are recessive lethal, viable alleles cause ultraviolet sensitivity, and files expressing marginal levels of haywire display motor defects and reduced life span. Progeny of females carrying a maternal effect allele show central nervous system defects.
View details for Web of Science ID A1992KB99000006
View details for PubMedID 1458540
- FORCE AND COUNTERFORCE IN THE MITOTIC SPINDLE CELL 1992; 71 (4): 547-550
DIFFERENTIAL EXPRESSION AND BEHAVIOR OF 2 GAMMA TUBULIN ISOFORMS IN DROSOPHILA
AMER SOC CELL BIOLOGY. 1992: A346–A346
View details for Web of Science ID A1992JR25502012
2 TYPES OF GENETIC INTERACTION IMPLICATE THE WHIRLIGIG GENE OF DROSOPHILA-MELANOGASTER IN MICROTUBULE ORGANIZATION IN THE FLAGELLAR AXONEME
1990; 126 (4): 961-973
The mutant nc4 allele of whirligig (3-54.4) of Drosophila melanogaster fails to complement mutations in an alpha-tubulin locus, alpha 1t, mutations in a beta-tubulin locus, B2t, or a mutation in the haywire locus. However, wrl fails to map to any of the known alpha- or beta-tubulin genes. The extragenic failure to complement could indicate that the wrl product participates in structural interactions with microtubule proteins. The whirligig locus appears to be haploinsufficient for male fertility. Both a deficiency of wrl and possible loss of function alleles obtained by reverting the failure to complement between wrlnc4 and B2tn are dominant male sterile in a genetic background wild type for tubulin. The dominant male sterility of the revertant alleles is suppressed if the flies are also heterozygous for B2tn, for a deficiency of alpha 1t, or for the haync2 allele. These results suggest that it is not the absolute level of wrl gene product but its level relative to tubulin or microtubule function that is important for normal spermatogenesis. The phenotype of homozygous wrl mutants suggests that the whirligig product plays a role in postmeiotic spermatid differentiation, possibly in organizing the microtubules of the sperm flagellar axoneme. Flies homozygous for either wrlnc4 or revertant alleles are viable and female fertile but male sterile. Premeiotic and meiotic stages of spermatogenesis appear normal. However, in post-meiotic stages, flagellar axonemes show loss of the accessory microtubule on the B-subfiber of outer doublet microtubules, outer triplet instead of outer doublet microtubules, and missing central pair microtubules.
View details for Web of Science ID A1990EL33400016
View details for PubMedID 2127579
INTERACTING GENES THAT AFFECT MICROTUBULE FUNCTION IN DROSOPHILA-MELANOGASTER - 2 CLASSES OF MUTATION REVERT THE FAILURE TO COMPLEMENT BETWEEN HAYNC2 AND MUTATIONS IN TUBULIN GENES
1990; 125 (1): 77-90
The recessive male sterile mutation haync2 of Drosophila melanogaster fails to complement certain beta 2-tubulin and alpha-tubulin mutations, suggesting that the haywire product plays a role in microtubule function, perhaps as a structural component of microtubules. The genetic interaction appears to require the presence of the aberrant product encoded by haync2, which may act as a structural poison. Based on this observation, we have isolated ten new mutations that revert the failure to complement between haync2 and B2tn. The revertants tested behaved as intragenic mutations of hay in recombination tests, and fell into two phenotypic classes, suggesting two functional domains of the hay gene product. Some revertants were hemizygous viable and less severe than haync2 in their recessive phenotype. These mutations might revert the poison by restoring the aberrant product encoded by the haync2 allele to more wild-type function. Most of the revertants were recessive lethal mutations, indicating that the hay gene product is essential for viability. These more extreme mutations could revert the poison by destroying the ability of the aberrant haywirenc2 product to interact structurally with microtubules. Flies heterozygous for the original haync2 allele and an extreme revertant show defects in both the structure and the function of the male meiotic spindle.
View details for Web of Science ID A1990DA94700008
View details for PubMedID 2111265
INTERACTING PROTEINS IDENTIFIED BY GENETIC INTERACTIONS - A MISSENSE MUTATION IN ALPHA-TUBULIN FAILS TO COMPLEMENT ALLELES OF THE TESTIS-SPECIFIC BETA-TUBULIN GENE OF DROSOPHILA-MELANOGASTER
MOLECULAR AND CELLULAR BIOLOGY
1989; 9 (3): 875-884
In this paper we demonstrate that failure to complement between mutations at separate loci can be used to identify genes that encode interacting structural proteins. A mutation (nc33) identified because it failed to complement mutant alleles of the gene encoding the testis-specific beta 2-tubulin of Drosophila melanogaster (B2t) did not map to the B2t locus. We show that this second-site noncomplementing mutation is a missense mutation in alpha-tubulin that results in substitution of methionine in place of valine at amino acid 177. Because alpha- and beta-tubulin form a heterodimer, our results suggest that the genetic interaction, failure to complement, is based on the structural interaction between the protein products of the two genes. Although the nc33 mutation failed to complement a null allele of B2t (B2tn), a deletion of the alpha-tubulin gene to which nc33 mapped complemented B2tn. Thus, the failure to complement appears to require the presence of the altered alpha-tubulin encoded by the nc33 allele, which may act as a structural poison when incorporated into either the tubulin heterodimer or microtubules.
View details for Web of Science ID A1989T444300001
View details for PubMedID 2498648
- INTERACTING GENES IDENTIFY INTERACTING PROTEINS INVOLVED IN MICROTUBULE FUNCTION IN DROSOPHILA CELL MOTILITY AND THE CYTOSKELETON 1989; 14 (1): 128-135
MUTATIONS THAT ENCODE PARTIALLY FUNCTIONAL BETA-2 TUBULIN SUBUNITS HAVE DIFFERENT EFFECTS ON STRUCTURALLY DIFFERENT MICROTUBULE ARRAYS
JOURNAL OF CELL BIOLOGY
1988; 107 (1): 141-152
The testis-specific beta 2 tubulin of Drosophila is required for assembly and function of at least three architecturally different microtubule arrays (Kemphues et al., 1982). Two recessive male-sterile mutations in the B2t locus that encode partially functional, stable, variant forms of beta 2 tubulin cause defects in only certain microtubule-based processes during spermatogenesis. These mutations could thus identify aspects of beta tubulin primary structure critical for function only in specific microtubule arrays. In males carrying the B2t6 mutation, meiotic chromosome segregation and nuclear shaping are normal and flagellar axonemes are formed, but there is a subtle defect in axoneme structure; the outer doublet microtubules fill in with a central core normally seen only in the central pair and accessory microtubules. In homozygous B2t7 males, chromosome movement is usually normal during meiosis but cytokinesis often fails, cytoplasmic microtubules are assembled and nuclear shaping appears to be normal, but the flagellar axoneme lacks structural integrity. In contrast, the B2t8 allele affects a general property of tubulin, the ability to form normal side-to-side association of protofilaments (Fuller et al., 1987), and causes defects in meiosis, axoneme assembly and nuclear shaping. Certain combinations of these beta 2 tubulin mutations show interallelic complementation; in B2t6/B2t8 males functional sperm are produced and both variant subunits are incorporated into mature sperm, in the absence of wild-type beta 2 tubulin. Comparison of the phenotypes of the three partially functional beta 2 tubulin alleles reveals some aspects of tubulin primary structure more important for function in specific subsets of microtubule arrays, and other aspects required for the construction of microtubules in general.
View details for Web of Science ID A1988P399500014
View details for PubMedID 3134362
TEMPORAL AND SPATIAL PATTERN OF DIFFERENCES IN MICROTUBULE BEHAVIOR DURING DROSOPHILA EMBRYOGENESIS REVEALED BY DISTRIBUTION OF A TUBULIN ISOFORM
1988; 102 (2): 311-324
Immunofluorescence staining of Drosophila embryos with a monoclonal antibody specific for acetylated alpha-tubulin has revealed that acetylated and nonacetylated alpha-tubulin isoforms have different patterns of distribution during early development. Acetylated alpha-tubulin was not detected in either interphase or mitotic spindle microtubules during the rapid early cleavage or syncytial blastoderm divisions. Acetylated alpha-tubulin was first observed as interphase lengthened at the end of syncytial blastoderm, and at cycle 14 was localized to a ring of structures clustered around the interphase nuclei. These structures probably represent a set of stable microtubules involved in nuclear elongation. Absence of detectable acetylated alpha-tubulin prior to cellular blastoderm seems to be due to rapid turnover of microtubule arrays rather than to lack of the enzyme required for modification, since acetylated alpha-tubulin appeared in early embryos when micro-tubules were stabilized by taxol treatment or anoxia. Because acetylated alpha-tubulin seems to be characteristic of stable microtubule arrays, the appearance of the antigen at cycle 14 represents a fundamental change in microtubule behaviour in the somatic cells of the embryo. Acetylated alpha-tubulin was not detected in pole cells during the blastoderm or early gastrula stages, indicating that acetylation of alpha-tubulin is not merely a consequence of cellularization. After the onset of gastrulation, interphase microtubule arrays in most cell types contain acetylated alpha-tubulin. However, cells in mitosis lack antibody staining. The resulting unstained patches reveal the stereotyped spatial pattern of cell division during gastrulation. Although the cells that give rise to the amnioserosa have acetylated alpha-tubulin in their interphase arrays at early gastrulation, by germ band elongation these large, plastic cells completely lack staining with anti-acetylated alpha-tubulin. In contrast, differentiated cell types such as neurones, which have arrays of stable axonal microtubules, stain brightly with the specific antibody. Although acetylated and nonacetylated alpha-tubulin are present in roughly equal amounts by the late stages of embryogenesis, acetylated alpha-tubulin is partitioned into the pellet during centrifugation of extracts of embryos homogenized at 4 degrees C.
View details for Web of Science ID A1988M459400007
View details for PubMedID 3138100
INTERACTING GENES THAT AFFECT MICROTUBULE FUNCTION - THE NC2 ALLELE OF THE HAYWIRE LOCUS FAILS TO COMPLEMENT MUTATIONS IN THE TESTIS-SPECIFIC BETA-TUBULIN GENE OF DROSOPHILA
GENES & DEVELOPMENT
1988; 2 (1): 82-92
A mutation that fails to complement certain alleles of the testis-specific beta 2-tubulin gene (B2t) of Drosophila melanogaster maps to a separate locus, haywire, located at 3-34.4 map units in polytene region 67E3-F3. Second-site non-complementing mutations such as haync2 and B2t alleles could identify genes that encode products that participate in the same functions or that interact in the same structure. Consistent with a structural interaction between the hay gene product and beta 2-tubulin, the genetic interaction between haync2 and B2t requires the presence of the mutant hay gene product; a deficiency for the hay region complements the same alleles of B2t that haync2 fails to complement. haync2 is a recessive male sterile mutation in a genetic background that is wild type at the B2t locus. Homozygous males have defects in meiosis, flagellar elongation and nuclear shaping, the three major microtubule-based processes in which the testis-specific beta 2-tubulin participates. The haync2 allele also has effects outside of spermatogenesis. It is a temperature-sensitive semilethal mutation, and homozygous haync2 females have reduced fertility. These phenotypes are consistent with a role for the haywire gene product in general microtubule function. Analysis of second-site non-complementing mutations such as haync2 offers a genetic tool for analysis of interacting proteins in complex assemblies.
View details for Web of Science ID A1988M096400008
View details for PubMedID 3128461
GENETIC-ANALYSIS OF MICROTUBULE STRUCTURE - A BETA-TUBULIN MUTATION CAUSES THE FORMATION OF ABERRANT MICROTUBULES INVIVO AND INVITRO
JOURNAL OF CELL BIOLOGY
1987; 104 (3): 385-394
A recessive male sterile mutation (B2t8) that encodes a stable variant of the testis-specific beta 2-tubulin of Drosophila causes the assembly of aberrant microtubules both in vivo and in vitro. The B2t8 mutation appears to cause defects in the formation of interprotofilament bonds. In testes from homozygous mutant males, the most commonly observed aberrant structures were sheets of protofilaments curved to form an S in cross section rather than a normal, closed microtubule. These characteristic S-shaped structures appear in the meiotic spindle, in place of axonemes in differentiating spermatids, and in cytoplasmic microtubules, including those that lie next to the nucleus during nuclear elongation. Homozygous mutant males exhibit defects in chromosome movement and cytokinesis during meiosis, flagellar elongation, and nuclear shaping, indicating that the ability to form normal closed microtubules is required for each of these events. The presence of the aberrant microtubules in three architecturally different microtubule arrays demonstrates conclusively the multifunctional nature of the beta 2-tubulin gene product. Although the mutant beta 2-tubulin subunit causes assembly of aberrant microtubules in vitro and in homozygous males, in the presence of wild-type beta 2-tubulin in heterozygous males, the variant subunit coassembles with the wild-type subunit into functional sperm.
View details for Web of Science ID A1987G207900002
View details for PubMedID 3818786
- ASSEMBLY INVITRO OF BACTERIOPHAGE-P22 PROCAPSIDS FROM PURIFIED COAT AND SCAFFOLDING SUBUNITS JOURNAL OF MOLECULAR BIOLOGY 1982; 156 (3): 633-665
STUDIES OF VIRUS STRUCTURE BY LASER RAMAN-SPECTROSCOPY .10. STRUCTURAL STUDIES OF P22 PHAGE, PRECURSOR PARTICLES, AND PROTEINS BY LASER RAMAN-SPECTROSCOPY
1982; 21 (16): 3866-3878
For the study of the protein--protein and protein--nucleic acid interactions in the assembly of virus particles, laser Raman spectra have been obtained in H2O and D2O solutions and as a function of temperature for the following Salmonella phage P22 components: mature phage particles, isolated mature phage DNA, mature protein shells empty of DNA, precursor protein shells (procapsids), and purified coat, scaffolding and tail-spike proteins. The spectra confirm that the condensed DNA within the phage capsid assumes the B-form secondary structure similar to aqueous DNA and reveal no evidence of specific molecular interactions between subgroups of DNA and protein subunits of the phage capsid. No differences were detected in the highly irregular secondary structure of the major capsid protein in mature capsids, empty capsids (lacking DNA), procapsids, and empty procapsids (lacking scaffolding protein). Features of both primary and secondary structures of the viral scaffolding and tail-spike proteins are also revealed by the spectra. Differences in thermal stability of tyrosyl side-chain interactions were observed between scaffolding protein extracted from the procapsid and within the procapsid. These differences correspond to different hydrogen bonding configurations of p-hydroxyphenyl groups and provide indirect evidence for the participation of the scaffolding proteins in specific macromolecular interactions within the procapsid.
View details for Web of Science ID A1982PA28300023
REGULATION OF TUBULIN GENE-EXPRESSION DURING EMBRYOGENESIS IN DROSOPHILA-MELANOGASTER
1982; 28 (1): 33-40
Four different tubulins have been identified that are expressed during embryogenesis in Drosophila melanogaster. Two alpha-tubulin subunits (alpha 1 and alpha 2) and one beta-tubulin subunit (beta 1) are expressed throughout embryonic development. A second beta-tubulin subunit (beta 3) is expressed only for a short period in mid-embryonic development. Synthesis of beta 3-tubulin in vitro in a rabbit reticulocyte translation system is directed by RNA extracted from embryos only at the stage when the protein is expressed. Thus we conclude that the mRNA encoding beta 3-tubulin is transcribed only during the brief period of beta 3-tubulin synthesis. The expression of beta 3-tubulin is accompanied by a coordinate transient increase in the level of synthesis of the embryonic alpha-tubulins, thereby maintaining an approximately equimolar synthesis of alpha- and beta-tubulin subunits throughout embryogenesis.
View details for Web of Science ID A1982MY42100007
View details for PubMedID 6802501
- PURIFICATION OF THE COAT AND SCAFFOLDING PROTEINS FROM PROCAPSIDS OF BACTERIOPHAGE P22 VIROLOGY 1981; 112 (2): 529-547
Investigations of bacteriophage P22 by laser Raman spectroscopy.
Progress in clinical and biological research
1981; 64: 271-283
View details for PubMedID 7330048
Scaffolding proteins and the genetic control of virus shell assembly.
Quarterly review of biology
1980; 55 (4): 369-393
Historically a gap has existed between the study of the one-dimensional organization of hereditary information in genes, and of the three-dimensional organization of macromolecules in biological structures. In this article we describe progress in closing this gap through the genetic and biochemical analysis of the assembly of the icosahedral shells of spherical viruses, a class of subcellular structures whose subunit organization is relatively well understood. The genes specifying the proteins required for capsid assembly have been identified for many bacterial viruses. By using mutants defective in these genes, it has been possible to identify intermediates in shell morphogenesis and DNA condensation, and to unravel the different levels of the genetic control of macromolecular assembly processes. In general, a precursor shell or procapsid is first constructed, and the DNA is subsequently coiled within it. The construction of a closed shell poses as difficult a problem for a virus as for an architect. In the well-studied bacteriophage P22 of Salmonella typhimurium, the construction of the procapsid requires the interaction of about 200 molecules of the gene-8 scaffolding protein with 420 molecules of the gene-5 coat protein, forming a double-shelled structure with the scaffolding protein on the inside. Once completed, procapsids undergo substantial alteration in the course of encapsulating the viral DNA. In P22, the initiation of DNA packaging triggers the exit of all of the scaffolding molecules from within the capsid, probably through the coat-protein lattice. These released molecules are re-utilized, interacting with newly synthesized coat subunits to form further procapsids. Thus, the scaffolding protein functions catalytically in capsid assembly. All of the well-studied DNA phages require a scaffolding protein species for procapsid assembly, though their properties vary. Purified coat and scaffolding subunits by themselves show little tendency to polymerize, and are stable as monomers in solution. Upon mixing together under the appropriate conditions, however, the proteins copolymerize into double shells. Their interaction with each other appears to be critical for efficient assembly; this interaction probably occurs on the edges of growing shells, and not among subunits in solution. We have termed this kind of process, which we previously described in T4 tail morphogenesis, self-regulated assembly. The subunits are synthesized in a nonreactive form and are activated, not in solution, but upon incorporation into the growing substrate structure. A number of further transformations of the capsid subunits occur only within the organized structure and not as free subunits. Thus, aspects of the genetic information controlling the assembly process are not fully expressed at the level of the properties of protein subunits, but become manifest only through interactions with other proteins, or at a higher level, after completion of the correct organized structure.
View details for PubMedID 7267974
REGULATION OF COAT PROTEIN POLYMERIZATION BY THE SCAFFOLDING PROTEIN OF BACTERIOPHAGE-P22
1980; 32 (1): 381-401
In the morphogenesis of double stranded DNA phages, a precursor protein shell empty of DNA is first assembled and then filled with DNA. The assembly of the correctly dimensioned precursor shell (procapsid) of Salmonella bacteriophage P22 requires the interaction of some 420 coat protein subunits with approximately 200 scaffolding protein subunits to form a double shelled particle with the scaffolding protein on the inside. In the course of DNA packaging, all of the scaffolding protein subunits exit from the procapsid and participate in further rounds of procapsid assembly (King and Casjens. 1974. Nature (Lond.). 251:112-119). To study the mechanism of shell assembly we have purified the coat and scaffolding protein subunits by selective dissociation of isolated procapsids. Both proteins can be obtained as soluble subunits in Tris buffer at near neutral pH. The coat protein sedimented in sucrose gradients as a roughly spherical monomer, while the scaffolding protein sedimented as if it were an elongated monomer. When the two proteins were mixed together in 1.5 M guanidine hydrochloride and dialyzed back to buffer at room temperature, procapsids formed which were very similar in morphology, sedimentation behavior, and protein composition to procapsids formed in vivo. Incubation of either protein alone under the same conditions did not yield any large structures. We interpret these results to mean that the assembly of the shell involves a switching of both proteins from their nonaggregating to their aggregating forms through their mutual interaction. The results are discussed in terms of the general problem of self-regulated assembly and the control of protein polymerization in morphogenesis.
View details for Web of Science ID A1980KM03600053
View details for PubMedID 7018607
STUDIES OF VIRUS STRUCTURE BY LASER-RAMAN SPECTROSCOPY .5. INVESTIGATION OF SECONDARY STRUCTURES AND MACROMOLECULAR INTERACTIONS IN BACTERIOPHAGE-P22 BY LASER RAMAN-SPECTROSCOPY
1980; 32 (1): 234-237
Laser Raman spectra of the DNA bacteriophage P22 and of its precursor particles and related structures have been obtained using 514.5-nm excitation. The spectra show that P22 DNA exists in the B form both inside of the phage head and after extraction from the phage. The major coat protein (gp5) contains a secondary structure composed of 18% alpha-helix, 20% beta-sheet and 62% irregular conformations. The scaffolding protein (gp8) in the phage prohead is substantially richer than gp5 in alpha-helical content. Among the amino acid residues which give prominent Raman lines, the spectra show that tryptophans are exposed to solvent and most tyrosines are hydrogen bonded to positive donor groups. The above features of phage DNA and protein structures are nearly invariant to changes in temperature up to 80 degrees C, indicating a remarkable thermal stability of the phage head and its encapsulated DNA.
View details for Web of Science ID A1980KM03600035
GENETIC-CONTROL OF ORGANELLE ASSEMBLY AT THE MOLECULAR-LEVEL .4. SCAFFOLDING PROTEINS AND THE GENETIC-CONTROL OF VIRUS SHELL ASSEMBLY
QUARTERLY REVIEW OF BIOLOGY
1980; 55 (4): 369-393
View details for Web of Science ID A1980LD48000004