Biomedical subjects

Pierre Gönczy

Publications and source records attributed to Pierre Gönczy.

At least 19 recordsLinked to original sources

PubMed2006

Sequential protein recruitment in C. elegans centriole formation.

Formation of the microtubule-based centriole is a poorly understood process that is crucial for duplication of the centrosome, the principal microtubule-organizing center of animal cells . Five proteins have been identified as being essential for centriole formation in Caenorhabditis elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6, and SPD-2 . The relationship between these proteins is incompletely understood, limiting understanding of how they contribute to centriole formation. In this study, we established the order in which these five proteins are recruited to centrioles, and we conducted molecular epistasis experiments expanding on earlier work. We find that SPD-2 is loaded first and is needed for the centriolar localization of the four other proteins. ZYG-1 recruitment is required thereafter for the remaining three proteins to localize to centrioles. SAS-5 and SAS-6 are recruited next and are needed for the presence of SAS-4, which is incorporated last. Our results indicate in addition that the presence of SAS-5 and SAS-6 allows diminution of centriolar ZYG-1. Moreover, astral microtubules appear dispensable for the centriolar recruitment of all five proteins. Several of these proteins have homologs in other metazoans, and we expect the assembly pathway that stems from our work to be conserved.

Animals↗

PubMed2005

Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division.

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.

Animals↗

PubMed2005

Centrosome duplication and nematodes: recent insights from an old relationship.

Centrosome duplication is required for proper cell division, and centriole formation is a key step in this process. This review discusses recent studies in C. elegans that have identified five core proteins required for centriole formation, thus shedding light into the mechanisms underlying centrosome duplication in nematodes and beyond.

Animals↗

PubMed2005

SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells.

The mechanisms that ensure centrosome duplication are poorly understood. In Caenorhabditis elegans, ZYG-1, SAS-4, SAS-5 and SPD-2 are required for centriole formation. However, it is unclear whether these proteins have functional homologues in other organisms. Here, we identify SAS-6 as a component that is required for daughter centriole formation in C. elegans. SAS-6 is a coiled-coil protein that is recruited to centrioles at the onset of the centrosome duplication cycle. Our analysis indicates that SAS-6 and SAS-5 associate and that this interaction, as well as ZYG-1 function, is required for SAS-6 centriolar recruitment. SAS-6 is the founding member of an evolutionarily conserved protein family that contains the novel PISA motif. We investigated the function of the human homologue of SAS-6. GFP-HsSAS-6 localizes to centrosomes and its overexpression results in excess foci-bearing centriolar markers. Furthermore, siRNA-mediated inactivation of HsSAS-6 in U2OS cells abrogates centrosome overduplication following aphidicolin treatment and interferes with the normal centrosome duplication cycle. Therefore, HsSAS-6 is also required for centrosome duplication, indicating that the function of SAS-6-related proteins has been widely conserved during evolution.

Amino Acid Sequence↗

PubMed2004

RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos.

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.

Animals↗

PubMed2004

lis-1 is required for dynein-dependent cell division processes in C. elegans embryos.

We investigated the role of the evolutionarily conserved protein Lis1 in cell division processes of Caenorhabditis elegans embryos. We identified apparent null alleles of lis-1, which result in defects identical to those observed after inactivation of the dynein heavy chain dhc-1, including defects in centrosome separation and spindle assembly. We raised antibodies against LIS-1 and generated transgenic animals expressing functional GFP-LIS-1. Using indirect immunofluorescence and spinning-disk confocal microscopy, we found that LIS-1 is present throughout the cytoplasm and is enriched in discrete subcellular locations, including the cell cortex, the vicinity of microtubule asters, the nuclear periphery and kinetochores. We established that lis-1 contributes to, but is not essential for, DHC-1 enrichment at specific subcellular locations. Conversely, we found that dhc-1, as well as the dynactin components dnc-1 (p150Glued) and dnc-2 (p50/dynamitin), are essential for LIS-1 targeting to the nuclear periphery, but not to the cell cortex nor to kinetochores. These results suggest that dynein and Lis1, albeit functioning in identical processes, are targeted partially independently of one another.

Alleles↗

PubMed2004

Myosin assembly: the power of multiubiquitylation.

Ubiquitylation provides a means of targeting substrate proteins for degradation by the proteasome. Novel findings in C. elegans (Hoppe et al., 2004, this issue of Cell) establish that two ubiquitin-ligases team up to multiubiquitylate the myosin chaperone UNC-45, suggesting a novel link between regulated protein degradation and myosin assembly.

Myosins↗

PubMed2004

Zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans.

The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, we establish that zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in Caenorhabditis elegans. Our results indicate that ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. Our data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. We demonstrate that PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and that zyg-11 and cul-2 can regulate polarity establishment independently of a role in cell cycle progression. Furthermore, we find that microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. Our findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

Animals↗

PubMed2004

Centrosomes: hooked on the nucleus.

A recent study has shown that the Hook protein ZYG-12 of Caenorhabditis elegans is essential for attachment of the centrosome to the nucleus. The results suggest a novel mechanism for the tight coupling between these two organelles, and shed new light on its biological significance.

Animals↗

PubMed2004

The arithmetic of centrosome biogenesis.

How do cells regulate centrosome number? A canonical duplication cycle generates two centrosomes from one in most proliferating cells. Centrioles are key to this process, and molecules such as centrins, SAS-4 and ZYG-1 govern daughter centriole formation. Cdk2 activity probably couples centrosome duplication with the S phase, and a licensing mechanism appears to limit centrosome duplication to once per cell cycle. However, such mechanisms must be altered in some cells--for example, spermatocytes--in which centrosome duplication and DNA replication are uncoupled. There are also alternative pathways of centrosome biogenesis. For example, one centrosome is reconstituted from two gametes at fertilization; in this case, the most common strategy involves differential contributions of centrioles and pericentriolar material (PCM) from each gamete. Furthermore, centrioles can sometimes form de novo from no apparent template. This occurs, for instance, in the early mouse embryo and in parthenogenetic species and might rely on a pre-existing seed that resides within PCM but is not visible by ultrastructural analysis.

Animals↗

PubMed2004

Centriolar SAS-5 is required for centrosome duplication in C. elegans.

Centrosomes, the major microtubule-organizing centres (MTOCs) of animal cells, are comprised of a pair of centrioles surrounded by pericentriolar material (PCM). Early in the cell cycle, there is a single centrosome, which duplicates during S-phase to direct bipolar spindle assembly during mitosis. Although crucial for proper cell division, the mechanisms that govern centrosome duplication are not fully understood. Here, we identify the Caenorhabditis elegans gene sas-5 as essential for daughter-centriole formation. SAS-5 is a coiled-coil protein that localizes primarily to centrioles. Fluorescence recovery after photobleaching (FRAP) experiments with green fluorescent protein (GFP) fused to SAS-5 (GFP-SAS-5) demonstrated that the protein shuttles between centrioles and the cytoplasm throughout the cell cycle. Analysis of mutant alleles revealed that the presence of SAS-5 at centrioles is crucial for daughter-centriole formation and that ZYG-1, a kinase that is also essential for this process, controls the distribution of SAS-5 to centrioles. Furthermore, partial RNA-interference (RNAi)-mediated inactivation experiments suggest that both sas-5 and zyg-1 are dose-dependent regulators of centrosome duplication.

Active Transport, Cell Nucleus↗

PubMed2003

TAC-1 and ZYG-9 form a complex that promotes microtubule assembly in C. elegans embryos.

BACKGROUND: Modulation of microtubule dynamics is crucial for proper cell division. While a large body of work has made important contributions to our understanding of the mechanisms governing microtubule dynamics in vitro, much remains to be learned about how these mechanisms operate in vivo. RESULTS: We identified TAC-1 as the sole TACC (Transforming Acidic Coiled Coil) protein in C. elegans. TAC-1 consists essentially of a TACC domain, in contrast to the much larger members of this protein family in other species. We find that tac-1 is essential for pronuclear migration and spindle elongation in one-cell-stage C. elegans embryos. Using an in vivo FRAP-based assay, we establish that inactivation of tac-1 results in defective microtubule assembly. TAC-1 is present in the cytoplasm and is enriched at centrosomes in a cell cycle-dependent manner. Centrosomal localization is independent of microtubules but requires the activity of gamma-tubulin and the Aurora-A kinase AIR-1. By conducting FRAP analysis in embryos expressing GFP-TAC-1, we find that centrosomal TAC-1 exchanges rapidly with the cytoplasmic pool. Importantly, we establish that TAC-1 physically interacts with ZYG-9, a microtubule-associated protein (MAP) of the XMAP215 family, both in vitro and in vivo. Furthermore, we also uncover that TAC-1 and ZYG-9 stabilize each other in C. elegans embryos. CONCLUSIONS: Our findings identify TAC-1 as a core structural and functional member of the evolutionarily conserved TACC family of proteins and suggest that mutual stabilization between TACC and XMAP215 proteins is a key feature ensuring microtubule assembly in vivo.

Animals↗

PubMed2003

Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos.

Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. We found that the GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, were essential for generation of proper pulling forces. GPR-1/2 interacted with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2.

Animals↗

PubMed2003

Differential activation of the DNA replication checkpoint contributes to asynchrony of cell division in C. elegans embryos.

BACKGROUND: Acquisition of lineage-specific cell cycle duration is a central feature of metazoan development. The mechanisms by which this is achieved during early embryogenesis are poorly understood. In the nematode Caenorhabditis elegans, differential cell cycle duration is apparent starting at the two-cell stage, when the larger anterior blastomere AB divides before the smaller posterior blastomere P(1). How anterior-posterior (A-P) polarity cues control this asynchrony remains to be elucidated. RESULTS: We establish that early C. elegans embryos possess a hitherto unrecognized DNA replication checkpoint that relies on the PI-3-like kinase atl-1 and the kinase chk-1. We demonstrate that preferential activation of this checkpoint in the P(1) blastomere contributes to asynchrony of cell division in two-cell-stage wild-type embryos. Furthermore, we show that preferential checkpoint activation is largely abrogated in embryos that undergo equal first cleavage following inactivation of Galpha signaling. CONCLUSION: Our findings establish that differential checkpoint activation contributes to acquisition of distinct cell cycle duration in two-cell-stage C. elegans embryos and suggest a novel mechanism coupling asymmetric division to acquisition of distinct cell cycle duration during development.

Animals↗

PubMed2003

SAS-4 is essential for centrosome duplication in C elegans and is recruited to daughter centrioles once per cell cycle.

The mechanisms governing centrosome duplication remain poorly understood. We identified a gene called sas-4 that is essential for this process in C. elegans. SAS-4 encodes a predicted coiled-coil protein that localizes to a tiny dot in the center of centrosomes throughout the cell cycle. FRAP experiments with GFP-SAS-4 transgenic embryos reveal that SAS-4 is recruited to the centrosome once per cell cycle, at the time of organelle duplication. Additional evidence indicates that SAS-4 is recruited to the daughter centriole or a closely associated structure. These findings identify SAS-4 recruitment as a key step in the centrosome duplication cycle.

Animals↗

PubMed2002

The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is gamma-tubulin dependent.

gamma-Tubulin-containing complexes are thought to nucleate and anchor centrosomal microtubules (MTs). Surprisingly, a recent study (Strome, S., J. Powers, M. Dunn, K. Reese, C.J. Malone, J. White, G. Seydoux, and W. Saxton. Mol. Biol. Cell. 12:1751-1764) showed that centrosomal asters form in Caenorhabditis elegans embryos depleted of gamma-tubulin by RNA-mediated interference (RNAi). Here, we investigate the nucleation and organization of centrosomal MT asters in C. elegans embryos severely compromised for gamma-tubulin function. We characterize embryos depleted of approximately 98% centrosomal gamma-tubulin by RNAi, embryos expressing a mutant form of gamma-tubulin, and embryos depleted of a gamma-tubulin-associated protein, CeGrip-1. In all cases, centrosomal asters fail to form during interphase but assemble as embryos enter mitosis. The formation of these mitotic asters does not require ZYG-9, a centrosomal MT-associated protein, or cytoplasmic dynein, a minus end-directed motor that contributes to self-organization of mitotic asters in other organisms. By kinetically monitoring MT regrowth from cold-treated mitotic centrosomes in vivo, we show that centrosomal nucleating activity is severely compromised by gamma-tubulin depletion. Thus, although unknown mechanisms can support partial assembly of mitotic centrosomal asters, gamma-tubulin is the kinetically dominant centrosomal MT nucleator.

Animals↗

PubMed2002

Nuclear envelope: torn apart at mitosis.

The findings of two recent studies suggest a novel mechanism for nuclear envelope breakdown in which cytoplasmic dynein anchored on the outside of the nucleus generates tension, thus triggering tearing of the nuclear envelope.

Animals↗

PubMed2002

Cytoskeletal regulation by the Nedd8 ubiquitin-like protein modification pathway.

The Nedd8 ubiquitin-like protein modification pathway regulates cell-cycle progression. Our analysis of Nedd8 requirements during Caenorhabditis elegans embryogenesis indicates that the cytoskeleton is another target. Nedd8 conjugation negatively regulated contractility of the microfilament-rich cell cortex during pronuclear migration and again during cytokinesis. The Nedd8 pathway also was required after meiosis to negatively regulate katanin, a microtubule-severing complex, permitting the assembly of a large mitotic spindle. We propose that Nedd8-modified cullin, as part of an E3 ubiquitin ligase complex, targets katanin for degradation during the transition from meiosis to mitosis.

Actin Cytoskeleton↗