Download Exporter la page en pdf

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