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Team Publications Space-time Imaging of Organelles and Endomembranes Dynamics Year of publication 2011 Svetlana Dokudovskaya, Francois Waharte, Avner Schlessinger, Ursula Pieper, Damien P Devos, Ileana M Cristea, Rosemary Williams, Jean Salamero, Brian T Chait, Andrej Sali, Mark C Field, Michael P Rout, Catherine Dargemont (2011 Apr 2) A conserved coatomer-related complex containing Sec13 and Seh1 dynamically associates with the vacuole in Saccharomyces cerevisiae. Molecular & cellular proteomics : MCP : M110.006478 : DOI : 10.1074/mcp.M110.006478 Summary The presence of multiple membrane-bound intracellular compartments is a major feature of eukaryotic cells. Many of the proteins required for formation and maintenance of these compartments share an evolutionary history. Here, we identify the SEA (Seh1-associated) protein complex in yeast that contains the nucleoporin Seh1 and Sec13, the latter subunit of both the nuclear pore complex and the COPII coating complex. The SEA complex also contains Npr2 and Npr3 proteins (upstream regulators of TORC1 kinase) and four previously uncharacterized proteins (Sea1-Sea4). Combined computational and biochemical approaches indicate that the SEA complex proteins possess structural characteristics similar to the membrane coating complexes COPI, COPII, the nuclear pore complex, and, in particular, the related Vps class C vesicle tethering complexes HOPS and CORVET. The SEA complex dynamically associates with the vacuole in vivo. Genetic assays indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, and response to nitrogen starvation. These data demonstrate that the SEA complex is an additional member of a family of membrane coating and vesicle tethering assemblies, extending the repertoire of protocoatomer-related complexes. Year of publication 2010 Jérôme Boulanger, Alexandre Gidon, Charles Kervran, Jean Salamero (2010 Oct 27) A patch-based method for repetitive and transient event detection in fluorescence imaging. PloS one : e13190 : DOI : 10.1371/journal.pone.0013190 Summary Automatic detection and characterization of molecular behavior in large data sets obtained by fast imaging in advanced light microscopy become key issues to decipher the dynamic architectures and their coordination in the living cell. Automatic quantification of the number of sudden and transient events observed in fluorescence microscopy is discussed in this paper. We propose a calibrated method based on the comparison of image patches expected to distinguish sudden appearing/vanishing fluorescent spots from other motion behaviors such as lateral movements. We analyze the performances of two statistical control procedures and compare the proposed approach to a frame difference approach using the same controls on a benchmark of synthetic image sequences. We have then selected a INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1 Team Publications Space-time Imaging of Organelles and Endomembranes Dynamics molecular model related to membrane trafficking and considered real image sequences obtained in cells stably expressing an endocytic-recycling trans-membrane protein, the Langerin-YFP, for validation. With this model, we targeted the efficient detection of fast and transient local fluorescence concentration arising in image sequences from a data base provided by two different microscopy modalities, wide field (WF) video microscopy using maximum intensity projection along the axial direction and total internal reflection fluorescence microscopy. Finally, the proposed detection method is briefly used to statistically explore the effect of several perturbations on the rate of transient events detected on the pilot biological model. Atsushi Matsuda, Lin Shao, Jerome Boulanger, Charles Kervrann, Peter M Carlton, Peter Kner, David Agard, John W Sedat (2010 Sep 22) Condensed mitotic chromosome structure at nanometer resolution using PALM and EGFP- histones. PloS one : e12768 : DOI : 10.1371/journal.pone.0012768 Summary Photoactivated localization microscopy (PALM) and related fluorescent biological imaging methods are capable of providing very high spatial resolutions (up to 20 nm). Two major demands limit its widespread use on biological samples: requirements for photoactivatable/photoconvertible fluorescent molecules, which are sometimes difficult to incorporate, and high background signals from autofluorescence or fluorophores in adjacent focal planes in three-dimensional imaging which reduces PALM resolution significantly. We present here a high-resolution PALM method utilizing conventional EGFP as the photoconvertible fluorophore, improved algorithms to deal with high levels of biological background noise, and apply this to imaging higher order chromatin structure. We found that the emission wavelength of EGFP is efficiently converted from green to red when exposed to blue light in the presence of reduced riboflavin. The photon yield of red-converted EGFP using riboflavin is comparable to other bright photoconvertible fluorescent proteins that allow <20 nm resolution. We further found that image pre-processing using a combination of denoising and deconvolution of the raw PALM images substantially improved the spatial resolution of the reconstruction from noisy images. Performing PALM on Drosophila mitotic chromosomes labeled with H2AvD-EGFP, a histone H2A variant, revealed filamentous components of ∼70 nm. This is the first observation of fine chromatin filaments specific for one histone variant at a resolution approximating that of conventional electron microscope images (10-30 nm). As demonstrated by modeling and experiments on a challenging specimen, the techniques described here facilitate super-resolution fluorescent imaging with common biological samples. Peter M Carlton, Jérôme Boulanger, Charles Kervrann, Jean-Baptiste Sibarita, Jean Salamero, Susannah Gordon-Messer, Debra Bressan, James E Haber, Sebastian Haase, Lin Shao, Lukman Winoto, Atsushi Matsuda, Peter Kner, Satoru Uzawa, Mats Gustafsson, Zvi Kam, David A Agard, John W Sedat (2010 Aug 14) INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2 Team Publications Space-time Imaging of Organelles and Endomembranes Dynamics Fast live simultaneous multiwavelength four-dimensional optical microscopy. Proceedings of the National Academy of Sciences of the United States of America : 16016-22 : DOI : 10.1073/pnas.1004037107 Summary Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading. INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3