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Victor RacineProbing the Golgi apparatus using RNAi screening coupled to high content image analysis.

Abstract :

The Golgi apparatus in mammalian cells is a dynamic and complex organelle composed of several sub-compartments and regulated by multiple fluxes of membranes and material. The molecular pathways that orchestrate this complex organization are only partially understood and it is likely that many players still remain to identify. We have combined a high-throughput RNAi approach with high-throughput automated microscopy to characterize the genes that regulate Golgi organization. Hela cells were stained for nuclear DNA and three different Golgi markers specific for the cis, medial and trans cisternae. The four channels images were automatically segmented and 600 morphological features were extracted. We have next investigated several ways to transform features into understandable phenotypical descriptions. Using normalization and classification schemes, each cell is described as diffuse, fragmented and condensed. This technique makes possible the automatic annotation of gene knock-down for the different cisternae. We have validated our approach on the systematic screen covering the human kinome and phosphatome. Several kinases and phosphatase previously proposed to be associated with Golgi membranes did induce significant morphological perturbations when knocked down. Our preliminary results also point to numerous novel kinases/phosphatases that, upon knock-down, induce significant disorganization of the Golgi apparatus. Interestingly, several of them appear to be novel cisternae specific regulators. We have optimized and parallelized data analysis algorithms to be able to annotating the full human genome. We believe the proposed phenotype annotation strategy is extensible to all membrane organelles.

Selected publications

Activity-dependent tuning of inhibitory neurotransmission based on GABAAR diffusion dynamics. Bannai H, Lévi S, Schweizer C, Inoue T, Launey T, Racine V, Sibarita JB, Mikoshiba K, Triller A. Neuron. 2009 Jun 11;62(5):670-82.
Antipsychotics increase vesicular glutamate transporter 2 (VGLUT2) expression in thalamolimbic pathways.Moutsimilli L, Farley S, El Khoury MA, Chamot C, Sibarita JB, Racine V, El Mestikawy S, Mathieu F, Dumas S, Giros B, Tzavara ET.Neuropharmacology. 2008 Mar;54(3):497-508.
Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies.Groc L, Lafourcade M, Heine M, Renner M, Racine V, Sibarita JB, Lounis B, Choquet D, Cognet L.J