Imagerie spatio-temporelle de la dynamique des organelles et des endomembranes

Publications de l’équipe

Année de publication : 2013

Xavier Heiligenstein, Jérôme Heiligenstein, Cédric Delevoye, Ilse Hurbain, Sabine Bardin, Perrine Paul-Gilloteaux, Lucie Sengmanivong, Gilles Régnier, Jean Salamero, Claude Antony, Graca Raposo (2013 Aug 28)

The CryoCapsule: simplifying correlative light to electron microscopy.

Traffic (Copenhagen, Denmark) : 700-16 : DOI : 10.1111/tra.12164 En savoir plus
Résumé

Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light microscopy and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin.

Replier
Deepali N Shinde, Dominik P Elmer, Peter Calabrese, Jérôme Boulanger, Norman Arnheim, Irene Tiemann-Boege (2013 Jun 7)

New evidence for positive selection helps explain the paternal age effect observed in achondroplasia.

Human molecular genetics : 4117-26 : DOI : 10.1093/hmg/ddt260 En savoir plus
Résumé

There are certain de novo germline mutations associated with genetic disorders whose mutation rates per generation are orders of magnitude higher than the genome average. Moreover, these mutations occur exclusively in the male germ line and older men have a higher probability of having an affected child than younger ones, known as the paternal age effect (PAE). The classic example of a genetic disorder exhibiting a PAE is achondroplasia, caused predominantly by a single-nucleotide substitution (c.1138G>A) in FGFR3. To elucidate what mechanisms might be driving the high frequency of this mutation in the male germline, we examined the spatial distribution of the c.1138G>A substitution in a testis from an 80-year-old unaffected man. Using a technology based on bead-emulsion amplification, we were able to measure mutation frequencies in 192 individual pieces of the dissected testis with a false-positive rate lower than 2.7 × 10(-6). We observed that most mutations are clustered in a few pieces with 95% of all mutations occurring in 27% of the total testis. Using computational simulations, we rejected the model proposing an elevated mutation rate per cell division at this nucleotide site. Instead, we determined that the observed mutation distribution fits a germline selection model, where mutant spermatogonial stem cells have a proliferative advantage over unmutated cells. Combined with data on several other PAE mutations, our results support the idea that the PAE, associated with a number of Mendelian disorders, may be explained primarily by a selective mechanism.

Replier
Cédric Lenormand, Coralie Spiegelhalter, Bertrand Cinquin, Sabine Bardin, Huguette Bausinger, Catherine Angénieux, Anita Eckly, Fabienne Proamer, David Wall, Ben Lich, Sylvie Tourne, Daniel Hanau, Yannick Schwab, Jean Salamero, Henri de la Salle (2013 Apr 12)

Birbeck granule-like « organized smooth endoplasmic reticulum » resulting from the expression of a cytoplasmic YFP-tagged langerin.

PloS one : e60813 : DOI : 10.1371/journal.pone.0060813 En savoir plus
Résumé

Langerin is required for the biogenesis of Birbeck granules (BGs), the characteristic organelles of Langerhans cells. We previously used a Langerin-YFP fusion protein having a C-terminal luminal YFP tag to dynamically decipher the molecular and cellular processes which accompany the traffic of Langerin. In order to elucidate the interactions of Langerin with its trafficking effectors and their structural impact on the biogenesis of BGs, we generated a YFP-Langerin chimera with an N-terminal, cytosolic YFP tag. This latter fusion protein induced the formation of YFP-positive large puncta. Live cell imaging coupled to a fluorescence recovery after photobleaching approach showed that this coalescence of proteins in newly formed compartments was static. In contrast, the YFP-positive structures present in the pericentriolar region of cells expressing Langerin-YFP chimera, displayed fluorescent recovery characteristics compatible with active membrane exchanges. Using correlative light-electron microscopy we showed that the coalescent structures represented highly organized stacks of membranes with a pentalaminar architecture typical of BGs. Continuities between these organelles and the rough endoplasmic reticulum allowed us to identify the stacks of membranes as a form of « Organized Smooth Endoplasmic Reticulum » (OSER), with distinct molecular and physiological properties. The involvement of homotypic interactions between cytoplasmic YFP molecules was demonstrated using an A206K variant of YFP, which restored most of the Langerin traffic and BG characteristics observed in Langerhans cells. Mutation of the carbohydrate recognition domain also blocked the formation of OSER. Hence, a « double-lock » mechanism governs the behavior of YFP-Langerin, where asymmetric homodimerization of the YFP tag and homotypic interactions between the lectin domains of Langerin molecules participate in its retention and the subsequent formation of BG-like OSER. These observations confirm that BG-like structures appear wherever Langerin accumulates and confirm that membrane trafficking effectors dictate their physiology and, illustrate the importance of molecular interactions in the architecture of intracellular membranes.

Replier
Agnès Miermont, François Waharte, Shiqiong Hu, Megan Nicole McClean, Samuel Bottani, Sébastien Léon, Pascal Hersen (2013 Mar 16)

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding.

Proceedings of the National Academy of Sciences of the United States of America : 5725-30 : DOI : 10.1073/pnas.1215367110 En savoir plus
Résumé

Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

Replier

Année de publication : 2012

Catherine Angénieux, François Waharte, Alexandre Gidon, François Signorino-Gelo, Virginie Wurtz, Rim Hojeij, Fabienne Proamer, Christian Gachet, Alain Van Dorsselaer, Daniel Hanau, Jean Salamero, Henri de la Salle (2012 Aug 11)

Lysosomal-associated transmembrane protein 5 (LAPTM5) is a molecular partner of CD1e.

PloS one : e42634 : DOI : 10.1371/journal.pone.0042634 En savoir plus
Résumé

The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

Replier
Jérôme Boulanger, Leila Muresan, Irene Tiemann-Boege (2012 May 5)

Massively parallel haplotyping on microscopic beads for the high-throughput phase analysis of single molecules.

PloS one : e36064 : DOI : 10.1371/journal.pone.0036064 En savoir plus
Résumé

In spite of the many advances in haplotyping methods, it is still very difficult to characterize rare haplotypes in tissues and different environmental samples or to accurately assess the haplotype diversity in large mixtures. This would require a haplotyping method capable of analyzing the phase of single molecules with an unprecedented throughput. Here we describe such a haplotyping method capable of analyzing in parallel hundreds of thousands single molecules in one experiment. In this method, multiple PCR reactions amplify different polymorphic regions of a single DNA molecule on a magnetic bead compartmentalized in an emulsion drop. The allelic states of the amplified polymorphisms are identified with fluorescently labeled probes that are then decoded from images taken of the arrayed beads by a microscope. This method can evaluate the phase of up to 3 polymorphisms separated by up to 5 kilobases in hundreds of thousands single molecules. We tested the sensitivity of the method by measuring the number of mutant haplotypes synthesized by four different commercially available enzymes: Phusion, Platinum Taq, Titanium Taq, and Phire. The digital nature of the method makes it highly sensitive to detecting haplotype ratios of less than 1:10,000. We also accurately quantified chimera formation during the exponential phase of PCR by different DNA polymerases.

Replier
I Izeddin, J Boulanger, V Racine, C G Specht, A Kechkar, D Nair, A Triller, D Choquet, M Dahan, J B Sibarita (2012 Feb 15)

Wavelet analysis for single molecule localization microscopy.

Optics express : 2081-95 : DOI : 10.1364/OE.20.002081 En savoir plus
Résumé

Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

Replier

Année de publication : 2011

Alexandre Gidon, Sabine Bardin, Bertrand Cinquin, Jerome Boulanger, François Waharte, Laurent Heliot, Henri de la Salle, Daniel Hanau, Charles Kervrann, Bruno Goud, Jean Salamero (2011 Sep 23)

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

Traffic (Copenhagen, Denmark) : 815-33 : DOI : 10.1111/j.1600-0854.2012.01354.x En savoir plus
Résumé

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane.

Replier
Nancy Abou-Zeid, Rudy Pandjaitan, Lucie Sengmanivong, Violaine David, Gwenaelle Le Pavec, Jean Salamero, Ahmed Zahraoui (2011 Jul 29)

MICAL-like1 mediates epidermal growth factor receptor endocytosis.

Molecular biology of the cell : 3431-41 : DOI : 10.1091/mbc.E11-01-0030 En savoir plus
Résumé

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.

Replier
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 En savoir plus
Résumé

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.

Replier

Année de 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 En savoir plus
Résumé

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 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.

Replier
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 En savoir plus
Résumé

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.

Replier
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)

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 En savoir plus
Résumé

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.

Replier

Année de publication : 2009

Jérôme Boulanger, Charles Kervrann, Patrick Bouthemy, Peter Elbau, Jean-Baptiste Sibarita, Jean Salamero (2009 Nov 11)

Patch-based nonlocal functional for denoising fluorescence microscopy image sequences.

IEEE transactions on medical imaging : 442-54 : DOI : 10.1109/TMI.2009.2033991 En savoir plus
Résumé

We present a nonparametric regression method for denoising 3-D image sequences acquired via fluorescence microscopy. The proposed method exploits the redundancy of the 3-D+time information to improve the signal-to-noise ratio of images corrupted by Poisson-Gaussian noise. A variance stabilization transform is first applied to the image-data to remove the dependence between the mean and variance of intensity values. This preprocessing requires the knowledge of parameters related to the acquisition system, also estimated in our approach. In a second step, we propose an original statistical patch-based framework for noise reduction and preservation of space-time discontinuities. In our study, discontinuities are related to small moving spots with high velocity observed in fluorescence video-microscopy. The idea is to minimize an objective nonlocal energy functional involving spatio-temporal image patches. The minimizer has a simple form and is defined as the weighted average of input data taken in spatially-varying neighborhoods. The size of each neighborhood is optimized to improve the performance of the pointwise estimator. The performance of the algorithm (which requires no motion estimation) is then evaluated on both synthetic and real image sequences using qualitative and quantitative criteria.

Replier
Cédric Delevoye, Ilse Hurbain, Danièle Tenza, Jean-Baptiste Sibarita, Stéphanie Uzan-Gafsou, Hiroshi Ohno, Willie J C Geerts, Arie J Verkleij, Jean Salamero, Michael S Marks, Graça Raposo (2009 Oct 21)

AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.

The Journal of cell biology : 247-64 : DOI : 10.1083/jcb.200907122 En savoir plus
Résumé

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1- and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type-specific positioning of endosomes that facilitate endosome-LRO contacts and are required for organelle maturation.

Replier