Centrosome duplication; Structural determination; Novel in vivo labeling approaches; Functional genomics; Super-resolution microscopy
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.
Lukinavičius Gražvydas, Lavogina Darja, Gönczy Pierre, Johnsson Kai (2013), Commercial Cdk1 antibodies recognize the centrosomal protein Cep152., in BioTechniques
, 55(3), 111-4.
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.
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.
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).