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LSSS 2014-2015

2014LSSS2015

Life Sciences Seminar Series

 

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Pierre Gönczy

EPFL SV ISREC UPGON, Lausanne

Mechanisms of centriole formation

Selected Publications

SAS-1 is a C2 domain protein critical for centriole integrity in C. elegans.von Tobel L, Mikeladze-Dvali T, Delattre M, Balestra FR, Blanchoud S, Finger S, Knott G, Müller-Reichert T, Gönczy P
PLoS Genet. 2014 Nov; 10(11):e1004777

Abstract

Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome.

Multiciliogenesis: multicilin directs transcriptional activation of centriole formation.Balestra FR, Gönczy P
Curr. Biol. 2014 Aug 18; 24(16):R746-9

Abstract

During differentiation of multiciliated cells, numerous centrioles are generated in each cell to act as templates for the formation of a corresponding number of cilia. A new study reveals that multicilin, a protein required for multiciliogenesis, is a key component of a regulatory complex that activates the transcription of genes required for centriole formation.

NuMA interacts with phosphoinositides and links the mitotic spindle with the plasma membrane.Kotak S, Busso C, Gönczy P
EMBO J. 2014 Aug 18; 33(16):1815-30

Abstract

The positioning and the elongation of the mitotic spindle must be carefully regulated. In human cells, the evolutionary conserved proteins LGN/Gαi1-3 anchor the coiled-coil protein NuMA and dynein to the cell cortex during metaphase, thus ensuring proper spindle positioning. The mechanisms governing cortical localization of NuMA and dynein during anaphase remain more elusive. Here, we report that LGN/Gαi1-3 are dispensable for NuMA-dependent cortical dynein enrichment during anaphase. We further establish that NuMA is excluded from the equatorial region of the cell cortex in a manner that depends on the centralspindlin components CYK4 and MKLP1. Importantly, we reveal that NuMA can directly associate with PtdInsP (PIP) and PtdInsP2 (PIP2) phosphoinositides in vitro. Furthermore, chemical or enzymatic depletion of PIP/PIP2 prevents NuMA cortical localization during mitosis, and conversely, increasing PIP2 levels augments mitotic cortical NuMA. Overall, our study uncovers a novel function for plasma membrane phospholipids in governing cortical NuMA distribution and thus the proper execution of mitosis.

Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos.Spiró Z, Thyagarajan K, De Simone A, Träger S, Afshar K, Gönczy P
Development 2014 Jul; 141(13):2712-23

Abstract

Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension.

Mechanisms of HsSAS-6 assembly promoting centriole formation in human cells.Keller D, Orpinell M, Olivier N, Wachsmuth M, Mahen R, Wyss R, Hachet V, Ellenberg J, Manley S, Gönczy P
J. Cell Biol. 2014 Mar 3; 204(5):697-712

Abstract

SAS-6 proteins are thought to impart the ninefold symmetry of centrioles, but the mechanisms by which their assembly occurs within cells remain elusive. In this paper, we provide evidence that the N-terminal, coiled-coil, and C-terminal domains of HsSAS-6 are each required for procentriole formation in human cells. Moreover, the coiled coil is necessary and sufficient to mediate HsSAS-6 centrosomal targeting. High-resolution imaging reveals that GFP-tagged HsSAS-6 variants localize in a torus around the base of the parental centriole before S phase, perhaps indicative of an initial loading platform. Moreover, fluorescence recovery after photobleaching analysis demonstrates that HsSAS-6 is immobilized progressively at centrosomes during cell cycle progression. Using fluorescence correlation spectroscopy and three-dimensional stochastic optical reconstruction microscopy, we uncover that HsSAS-6 is present in the cytoplasm primarily as a homodimer and that its oligomerization into a ninefold symmetrical ring occurs at centrioles. Together, our findings lead us to propose a mechanism whereby HsSAS-6 homodimers are targeted to centrosomes where the local environment and high concentration of HsSAS-6 promote oligomerization, thus initiating procentriole formation.

Stereotyped distribution of midbody remnants in early C. elegans embryos requires cell death genes and is dispensable for development.Ou G, Gentili C, Gönczy P
Cell Res. 2014 Feb; 24(2):251-3

Abstract