Interphase Centrosomes Research Articles

Cilia born out of shock and stress

Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins. Centrioles are barrel‐shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule‐organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle‐dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule‐dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM‐1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet‐Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: …

PLP inhibits the activity of interphase centrosomes to ensure their proper segregation in stem cells

Centrosomes determine the mitotic axis of asymmetrically dividing stem cells. Several studies have shown that the centrosomes of the Drosophila melanogaster central brain neural stem cells are themselves asymmetric, organizing varying levels of pericentriolar material and microtubules. This asymmetry produces one active and one inactive centrosome during interphase. We identify pericentrin-like protein (PLP) as a negative regulator of centrosome maturation and activity. We show that PLP is enriched on the inactive interphase centrosome, where it blocks recruitment of the master regulator of centrosome maturation, Polo kinase. Furthermore, we find that ectopic Centrobin expression influenced PLP levels on the basal centrosome, suggesting it may normally function to regulate PLP. Finally, we conclude that, although asymmetric centrosome maturation is not required for asymmetric cell division, it is required for proper centrosome segregation to the two daughter cells.

Centrosomal Che-1 Protein Is Involved in the Regulation of Mitosis and DNA Damage Response by Mediating Pericentrin (PCNT)-dependent Chk1 Protein Localization

To combat threats posed by DNA damage, cells have evolved mechanisms, collectively termed DNA damage response (DDR). These mechanisms detect DNA lesions, signal their presence, and promote their repair. Centrosomes integrate G2/M checkpoint control and repair signals in response to genotoxic stress, acting as an efficient control mechanism when G2/M checkpoint function fails and mitosis begins in the presence of damaged DNA. Che-1 is an RNA polymerase II-binding protein involved in the regulation of gene transcription, induction of cell proliferation, and DDR. Here we provide evidence that in addition to its nuclear localization, Che-1 localizes at interphase centrosomes, where it accumulates following DNA damage or spindle poisons. We show that Che-1 depletion generates supernumerary centrosomes, multinucleated cells, and multipolar spindle formation. Notably, Che-1 depletion abolishes the ability of Chk1 to bind pericentrin and to localize at centrosomes, which, in its turn, deregulates the activation of centrosomal cyclin B-Cdk1 and advances entry into mitosis. Our results reinforce the notion that Che-1 plays an important role in DDR and that its contribution seems to be relevant for the spindle assembly checkpoint.

Merlin/ERM proteins establish cortical asymmetry and centrosome position

The ability to generate asymmetry at the cell cortex underlies cell polarization and asymmetric cell division. Here we demonstrate a novel role for the tumor suppressor Merlin and closely related ERM proteins (Ezrin, Radixin, and Moesin) in generating cortical asymmetry in the absence of external cues. Our data reveal that Merlin functions to restrict the cortical distribution of the actin regulator Ezrin, which in turn positions the interphase centrosome in single epithelial cells and three-dimensional organotypic cultures. In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles, and aberrant epithelial architecture. Furthermore, in tumor cells with centrosome amplification, the failure to restrict cortical Ezrin abolishes centrosome clustering, yielding multipolar mitoses. These data uncover fundamental roles for Merlin/ERM proteins in spatiotemporally organizing the cell cortex and suggest that Merlin's role in restricting cortical Ezrin may contribute to tumorigenesis by disrupting cell polarity, spindle orientation, and, potentially, genome stability.

A cell cycle role for the epigenetic factor CTCF-L/BORIS.

CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.

Tankyrase 1 regulates centrosome function by controlling CPAP stability

CPAP--a gene mutated in primary microcephaly--is required for procentriole formation. Here we show that CPAP degradation and function is controlled by the poly(ADP-ribose) polymerase tankyrase 1. CPAP is PARsylated by tankyrase 1 in vitro and in vivo. Overexpression of tankyrase 1 leads to CPAP proteasomal degradation, preventing centriole duplication, whereas depletion of tankyrase 1 stabilizes CPAP in G1, generating elongated procentrioles and multipolarity. Tankyrase 1 localizes to centrosomes exclusively in G1, coinciding with CPAP degradation. Hence, tankyrase 1-mediated PARsylation regulates CPAP levels during the cell cycle to limit centriole elongation and ensure normal centrosome function.

Actin and Arp2/3 localize at the centrosome of interphase cells

Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel (“lower dimer”) actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.

The γTuRC Revisited: A Comparative Analysis of Interphase and Mitotic Human γTuRC Redefines the Set of Core Components and Identifies the Novel Subunit GCP8

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.

-catenin and Early Development in the Gastropod, Crepidula fornicata

This study describes the early expression and function of β-catenin in the gastropod, Crepidula fornicata. In other bilaterians β-catenin functions in cell adhesion, gastrulation, and cell signaling, which is related to the establishment of the dorso-ventral axis and mesendoderm. Here, we studied the distribution of β-catenin mRNA and protein in C. fornicata via whole mount in situ hybridization and by expressing GFP-tagged β-catenin in vivo. During early cleavage, β-catenin mRNA and protein appear to be broadly localized to all cells in the early embryo. The mRNA tends to be concentrated at inter-phase centrosomes in these cells. At later stages, the mRNA is predominantly in the vegetal macromeres, and subsequently in the rudiment of the hindgut, stomodeum, and velar lobes. Expression of full-length GFP-tagged protein suggests that there is no active mechanism to degrade β-catenin within cells of the early embryos prior to the 25-cell stage. However, by the second day of development, when the fourth quartet micromeres have formed, β-catenin becomes selectively stabilized in the progeny of the 4d mesentoblast (e.g., ML and MR and their daughters) and is missing from most other blastomeres, including vegetal macromeres. Over the next 2 days of development, during subsequent divisions of 4d, β-catenin protein becomes progressively degraded, along the proximo-distal axes, within the progeny of the paired mesendodermal bands. The cells located at the tips of the mesodermal bands (2 mL² and 2 mR²) are the last to contain this protein, which is no longer detected after 4 days of development. In animals like C. fornicata, which undergo a spiral cleavage program (e.g., molluscs, annelids, nemerteans, and polyclad flatworms), the mesentoblast or 4d cell represents the progenitor of endomesoderm (forming hindgut, internal and external kidneys, and various muscles). Therefore, the selective stabilization of β-catenin in the progeny of 4d in C. fornicata is consistent with arguments that a basic, ancestral role of β-catenin lies in the formation of endomesodermal fates. Experiments using a truncated β-catenin clone show that the regions located in the C-terminus, distal to the 11th armadillo repeat, are required for normal stabilization/degradation of β-catenin protein within the embryo. Microinjection of translation blocking β-catenin morpholinos into zygotes led to the down-regulation of β-catenin expression. This resulted in the subsequent failure of gastrulation, but did not interfere with the formation and early cleavage of 4d, although there were no discernable differentiated cell fates in these defective embryos. These results are compared with those obtained in other metazoans.

NudC-like protein 2 regulates the LIS1/dynein pathway by stabilizing LIS1 with Hsp90

The type I lissencephaly gene product LIS1, a key regulator of cytoplasmic dynein, is critical for cell proliferation, survival, and neuronal migration. However, little is known about the regulation of LIS1. Here, we identify a previously uncharacterized mammalian homolog of Aspergillus NudC, NudCL2 (NudC-like protein 2), as a regulator of LIS1. NudCL2 is localized to the centrosome in interphase, and spindle poles and kinetochores during mitosis, a pattern similar to the localization of LIS1 and cytoplasmic dynein. Depletion of NudCL2 destabilized LIS1 and led to phenotypes resembling those of either dynein or LIS1 deficiency. NudCL2 complexed with and enhanced the interaction between LIS1 and Hsp90. Either disruption of the LIS1-Hsp90 interaction with the C terminus of NudCL2 or inhibition of Hsp90 chaperone function by geldanamycin decreased LIS1 stability. Thus, our results suggest that NudCL2 regulates the LIS1/dynein pathway by stabilizing LIS1 with Hsp90 chaperone. This represents a hitherto undescribed mechanism of the LIS1/dynein regulation in mammalian cells.

HAUS, the 8-Subunit Human Augmin Complex, Regulates Centrosome and Spindle Integrity

The assembly of a robust microtubule-based mitotic spindle is a prerequisite for the accurate segregation of chromosomes to progeny. Spindle assembly relies on the concerted action of centrosomes, spindle microtubules, molecular motors, and nonmotor spindle proteins. Here we use an RNA-interference screen of the human centrosome proteome to identify novel regulators of spindle assembly. One such regulator is HAUS, an 8-subunit protein complex that shares homology to Drosophila Augmin. HAUS localizes to interphase centrosomes and to mitotic spindle microtubules, and its disruption induces microtubule-dependent fragmentation of centrosomes along with an increase in centrosome size. HAUS disruption results in the destabilization of kinetochore microtubules and the eventual formation of multipolar spindles. These severe mitotic defects are alleviated by codepletion of NuMA, indicating that both factors regulate opposing activities. HAUS disruption alters NuMA localization, suggesting that mislocalized NuMA activity contributes to the spindle and centrosome defects observed. The human Augmin complex (HAUS) is a critical and evolutionary conserved multisubunit protein complex that regulates centrosome and spindle integrity.

Centrosome Function: Sometimes Less Is More

Tight regulation of centrosome duplication is critical to ensure that centrosome number doubles once and only once per cell cycle. Superimposed onto this centrosome duplication cycle is a functional centrosome cycle in which they alternate between phases of quiescence and robust microtubule (MT) nucleation and MT-anchoring activities. In vertebrate cycling cells, interphase centrioles accumulate less pericentriolar material (PCM), reducing their MT nucleation capacity. In mitosis, centrosomes mature, accumulating more PCM to increase their nucleation and anchoring capacities to form robust MT asters. Interestingly, functional cycles of centrosomes can be altered to suit the cell's needs. Some interphase centrosomes function as a microtubule-organizing center by increasing their ability to anchor MTs to form centrosomal radial arrays. Other interphase centrosomes maintain their MT nucleation capacity but reduce/eliminate their MT-anchoring capacity. Recent work demonstrates that Drosophila cells take this to the extreme, whereby centrioles lose all detectable PCM during interphase, offering an explanation as to how centrosome-deficient flies develop to adulthood. Drosophila stem cells further modify the functional cycle by differentially regulating their two centrioles - a situation that seems important for stem cell asymmetric divisions, as misregulation of centrosome duplication in stem/progenitor cells can promote tumor formation. Here, we review recent findings that describe variations in the functional cycle of centrosomes.

Multiple Localization of Endogenous MARK4L Protein in Human Glioma

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma.Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines.Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines.Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.

Multiple localization of endogenous MARK4L protein in human glioma.

Background: We have previously shown that the sustained expression of MARK4L transcripts in glioma and neural progenitors (NHNPs) declines after exposure to antisense MARK4L oligonucleotides in glioblastoma cell lines. Array-CGH confirmed the genomic duplication of MARK4L identified by FISH in a glioblastoma cell line. This background together with literature data on the exogenous association of MARK4 with interphase centrosome prompted us to investigate the sub-cellular localization of the endogenous MARK4L protein aiming at achieving insights on its possible role in the pathomechanisms of glioma. Methods: Immunodetection was carried out to validate the specificity of MARK4L antibody in gliomas and NHNPs. Mass spectrometry was applied for MARK4L protein identification in a representative glioblastoma cell line. Combined biochemical fractionation and immunodetection analyses were performed to confirm the sub-cellular localization of MARK4L achieved by immunofluorescence in glioma cell lines. Results: By assigning MARK4L protein within the band immunoprecipitated by the specific antibody we validated our anti-MARK4L antibody. We demonstrated that the endogenous MARK4L: (i) colocalizes with centrosomes at all mitotic stages and resides in centrosome-enriched fractions; (ii) associates with the nucleolus and the midbody and respective fractions, and (iii) co-stains the aberrant centrosome configurations observed in glioma cell lines. Conclusions: The overall data merge on the multiplex entry of MARK4L into the cell cycle and link it to the aberrant centrosomes in glioma cell lines suggesting a possible role of this kinase in the abnormal mitotic processes of human glioma.

GM130-dependent Control of Cdc42 Activity at the Golgi Regulates Centrosome Organization

The physical proximity of the Golgi apparatus and the centrosome is a unique feature of mammalian cells whose functional significance is only poorly understood. Here, we demonstrate that the previously described regulation of centrosome organization and function by the Golgi protein, GM130, involves a Golgi-associated complex consisting of GM130, the Rho GTPase, Cdc42, and its guanine nucleotide exchange factor, Tuba. We identified Tuba as a novel GM130-interacting protein and showed that this association controls Tuba-mediated activation of Cdc42 at the Golgi apparatus. Blocking either Tuba or Cdc42 activity reproduced the GM130 depletion phenotype of aberrant, nonfunctional centrosomes. Expression of constitutively active Cdc42 bypassed the requirement for GM130 in centrosome regulation, indicating that Cdc42 functions downstream of GM130. Our studies demonstrate that Cdc42 has a novel role in controlling centrosome organization in unstimulated cells in addition to its known function as a regulator of centrosome reorientation in stimulated cells. This first description of a regulatory pathway between the Golgi apparatus and the interphase centrosome that complements the known role of Golgi proteins in controlling spindle formation during mitosis and may provide an explanation for the pericentriolar position of the mammalian Golgi apparatus during interphase.

Localization of Aurora A and Aurora B kinases during interphase: Role of the N-Terminal domain

Aurora kinases possess a conserved catalytic domain (CD) and a N-terminal domain (ND) that varies in size and sequence. We have previously reported that the N-terminal domain of AuroraA (AurA) participates in the localization of the kinase to the centrosome in interphase. AuroraB (AurB) is a chromosome passenger protein and its N-terminal domain is not necessary for its localization or function during mitosis. Using various combinations of GFP-AurA and AurB protein domains we show that AurB N-terminal domain is required for nuclear localization in Xenopus XL2 cells in interphase. In human cells, however, we found both AurA and AurB kinases in the nucleus, AurA being mainly cytoplasmic and AurB mainly nuclear. Both proteins are actively excluded from the nucleus by a CRM1 dependent pathway. Interestingly, at a functional level, in interphase, every combination of Aurora kinase domains (ND-CD) rescues histone H3 Serine10 phosphorylation defect induced by AurB knockdown. This clearly indicates the presence of a functional AurA in the nucleus. However, the chimera ND-AurA/CD-AurB was much more efficient than the ND-AurB/CD-AurA to rescue multinucleation also induced by AurB knockdown. This indicates that the catalytic domain of AurB is required to fulfill specific functions during mitosis that cannot be fulfilled by the catalytic domain of AurA, probably for localization reasons during mitosis.

Isolation of Centrosomes from Cultured Mammalian Cells

INTRODUCTIONThe centrosome is a cell's primary microtubule-organizing center. In most mammalian cells, the centrosome is composed of a pair of centrioles and surrounding pericentriolar material. The centrosome is duplicated exactly once per cell cycle such that at the onset of mitosis, a cell has two centrosomes, which serve as poles of the mitotic spindle. During cytokinesis, one centrosome is segregated to each daughter cell. This protocol describes the isolation of centrosomes from asynchronous cells, and thus the purified material will consist primarily of interphase centrosomes. Isolated centrosomes can be used in a variety of assays, including studies of microtubule function and the identification of centrosome-associated proteins and their interactions.

Hice1, a Novel Microtubule-Associated Protein Required for Maintenance of Spindle Integrity and Chromosomal Stability in Human Cells

Spindle integrity is critical for efficient mitotic progression and accurate chromosome segregation. Deregulation of spindles often leads to structural and functional aberrations, ultimately promoting segregation errors and aneuploidy, a hallmark of most human cancers. Here we report the characterization of a previously identified human sarcoma antigen (gene located at 19p13.11), Hice1, an evolutionarily nonconserved 46-kDa coiled-coil protein. Hice1 shows distinct cytoplasmic localization and associates with interphase centrosomes and mitotic spindles, preferentially at the spindle pole vicinity. Depletion of Hice1 by RNA interference resulted in abnormal and unstable spindle configurations, mitotic delay at prometaphase and metaphase, and elevated aneuploidy. Conversely, loss of Hice1 had minimal effects on interphase centrosome duplication. We also found that both full-length Hice1 and Hice1-N1, which is composed of 149 amino acids of the N-terminal region, but not the mutant lacking the N-terminal region, exhibited activities of microtubule bundling and stabilization at a near-physiological concentration. Consistently, overexpression of Hice1 rendered microtubule bundles in cells resistant to nocodazole- or cold-treatment-induced depolymerization. These results demonstrate that Hice1 is a novel microtubule-associated protein important for maintaining spindle integrity and chromosomal stability, in part by virtue of its ability to bind, bundle, and stabilize microtubules.

SSgo1, a Major Splice Variant of Sgo1, Functions in Centriole Cohesion Where It Is Regulated by Plk1

Shugoshin 1 (Sgo1) functions as a protector of centromeric cohesion of sister chromatids in higher eukaryotes. Here, we provide evidence for a previously unrecognized role for Sgo1 in centriole cohesion. Sgo1 depletion via RNA interference induces the formation of multiple centrosome-like structures in mitotic cells that result from the separation of paired centrioles. Sgo1+/- mitotic murine embryonic fibroblasts display split centrosomes. Localization study of two major endogenous splice variants of Sgo1 indicates that the smaller variant, sSgo1, is found at the centrosome in interphase and at spindle poles in mitosis. sSgo1 interacts with Plk1 and its spindle pole localization is Plk1 dependent. Centriole splitting induced by Sgo1 depletion or expression of a dominant negative mutant is suppressed by ectopic expression of sSgo1 or by Plk1 knockdown. Our studies strongly suggest that sSgo1 plays an essential role in protecting centriole cohesion, which is partly regulated by Plk1.

Regulation of Self-renewal and Differentiation in Adult Stem Cell Lineages: Lessons from the Drosophila Male Germ Line

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.