Project

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An integrated multidisciplinary approach towards a molecular understanding of centrosome duplication

Applicant Gönczy Pierre
Number 125463
Funding scheme Sinergia
Research institution Aztekin Lab SV ISREC EPFL
Institution of higher education EPF Lausanne - EPFL
Main discipline Cellular Biology, Cytology
Start/End 01.01.2010 - 31.12.2013
Approved amount 1'440'719.00
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All Disciplines (6)

Discipline
Cellular Biology, Cytology
Other disciplines of Physics
Biophysics
Organic Chemistry
Molecular Biology
Genetics

Keywords (5)

Centrosome duplication; Structural determination; Novel in vivo labeling approaches; Functional genomics; Super-resolution microscopy

Lay Summary (English)

Lead
Lay summary
Overall goalWe aim at gaining novel insights into the mechanisms of centrosome duplication, a process that is crucial for genome integrity and whose deregulation may contribute to tumor progression. BackgroundThe centrosome is an organelle that nucleates most microtubules in animal cells. Proliferating cells are born with a single centrosome that normally duplicates once and only once per cell cycle. The two resulting centrosomes assemble a bipolar spindle during mitosis, which serves to faithfully segregate chromosomes to daughter cells. If the centrosome does not duplicate, a monopolar spindle may assemble; conversely, if the centrosome duplicates more than once, a multipolar spindle may assemble. Both lead to incorrect chromosome segregation and thus threaten genome integrity. Despite their importance, however, the mechanisms governing centrosome duplication remain poorly understood and constitute a fundamental open question in biology.Specific aimsOwing to the unique expertise of the five participating laboratories, we will pursue four lines of investigation using innovative approaches in cell biology, structural biology, organic chemistry and advanced optics. First, we will conduct biophysical and structural analyses of the HsSAS-6 family of proteins, which are critical for centrosome duplication across evolution, and generate novel inhibitors of the kinase Plk4, which regulates HsSAS-6 function. Second, using a high-throughput fluorescence-based assay in human cells, we will conduct a genome-wide screen for genes essential for centrosome duplication and a chemical genetic screen for small molecules preventing this process. Third, we will utilize a novel selective chemical labeling method to determine the nature of HsSAS-6 and Plk4 interacting proteins; such interactions will be further characterized, notably by developing small molecule inhibitors. Finally, we will develop and implement super-high resolution microscopy methods to analyze with utmost precision the distribution of HsSAS-6, Plk4 and other centrosomal components.Anticipated impactWe anticipate that these four lines of research will increase in an unprecedented manner our understanding of the mechanisms governing centrosome duplication, thus contributing to solve a fundamental open question in biology. In addition, our research program is expected to generate knowledge and molecules that will serve as novel diagnostic and therapeutic tools in the fight against cancer.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry.
Hilbert Manuel, Erat Michèle C, Hachet Virginie, Guichard Paul, Blank Iris D, Flückiger Isabelle, Slater Leanne, Lowe Edward D, Hatzopoulos Georgios N, Steinmetz Michel O, Gönczy Pierre, Vakonakis Ioannis (2013), Caenorhabditis elegans centriolar protein SAS-6 forms a spiral that is consistent with imparting a ninefold symmetry., in Proceedings of the National Academy of Sciences of the United States of America, 110(28), 11373-8.
Commercial Cdk1 antibodies recognize the centrosomal protein Cep152.
Lukinavičius Gražvydas, Lavogina Darja, Gönczy Pierre, Johnsson Kai (2013), Commercial Cdk1 antibodies recognize the centrosomal protein Cep152., in BioTechniques, 55(3), 111-4.
Native architecture of the centriole proximal region reveals features underlying its 9-fold radial symmetry.
Guichard Paul, Hachet Virginie, Majubu Norbert, Neves Aitana, Demurtas Davide, Olieric Natacha, Fluckiger Isabelle, Yamada Akinori, Kihara Kumiko, Nishida Yuichiro, Moriya Shigeharu, Steinmetz Michel O, Hongoh Yuichi, Gönczy Pierre (2013), Native architecture of the centriole proximal region reveals features underlying its 9-fold radial symmetry., in Current biology : CB, 23(17), 1620-8.
Near-field ptychography: phase retrieval for inline holography using a structured illumination.
Stockmar Marco, Cloetens Peter, Zanette Irene, Enders Bjoern, Dierolf Martin, Pfeiffer Franz, Thibault Pierre (2013), Near-field ptychography: phase retrieval for inline holography using a structured illumination., in Scientific reports, 3, 1927-1927.

Collaboration

Group / person Country
Types of collaboration
University of Oxford Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Associated projects

Number Title Start Funding scheme
133845 Structured Illumination Light Microscope 01.06.2011 R'EQUIP
150840 Fluorescence Lifetime Imaging (FLIM) 01.12.2013 R'EQUIP
138659 Protein interactions regulating the microtubule cytoskeleton 01.01.2012 Project funding

Abstract

The centrosome is the major microtubule organizing center of animal cells. Just as for the replication of the genetic material, duplication of the centrosome must occur once per cell cycle to ensure genome integrity. The mechanisms governing centrosome duplication remain poorly understood and constitute a fundamental open question in biology.We propose to launch a multidisciplinary approach that draws on the unique expertise of the five team members to gain fundamental insights into the mechanisms of centrosome duplication in human cells. Four specific aims will be pursued using an array of innovative approaches in cell biology, structural biology, organic chemistry and advanced optics:1) Role of ZYG-1/Plk4 in modulating SAS-6/HsSAS-6 function [MS/PG/KJ]Phosphorylation by ZYG-1 of SAS-6 leads to a conformational change and triggers centriole formation in C. elegans. We will conduct biophysical and structural analyses of SAS-6 and of its human relative HsSAS-6 to characterize this conformational change and how it relates to function. We will also generate protein-based inhibitors of the ZYG-1-related human kinase Plk4 and use them to probe its requirement across the cell cycle.2) Functional genomic and chemical genetic screens for centrosome duplication [PG/KJ] We will use a high-throughput fluorescence-based assay in human cells to conduct a genome-wide siRNAi-based screen for genes essential for centrosome duplication, as well as a chemical genetic screen for small molecules preventing this process. Lead compounds will be further improved through the synthesis of appropriate derivatives.3) Systematic characterization of the centrosome interactome in vivo [KJ/PG/MS] We will exploit newly developed methods for the selective chemical labeling of proteins to determine the in vivo interactome of crucial human centrosomal proteins, including Plk4 and HsSAS-6. Key protein-protein interactions will be characterized further through structural methods and the development of small molecule inhibitors.4) Super-resolution microscopy of centrosomal proteins [TL/KJ/FP/PG]We will conduct super-high resolution microscopy to analyze with utmost precision the distribution of Plk4, HsSAS-6 and select centrosomal components identified in the course of this research program. We will use STED microscopy and develop two complementary innovative imaging methods, stochastic optical reconstruction microscopy (STORM) of protein-protein interactions and high-resolution scanning x-ray diffraction microscopy (SXDM).
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