Emmanuel Boutant

In Vivo Dynamics and Differential Microtubule-Binding Activities of MAP65 Proteins

Plant cells produce different microtubule arrays that are essential for cell division and morphogenesis without equivalent in other eukaryotes. Microtubule-associated proteins influence the behavior of microtubules that is presumed to culminate into transitions from one array to another. We analyzed the microtubule-binding properties of three Arabidopsis (Arabidopsis thaliana) members, AtMAP65-1, AtMAP65-4, and AtMAP65-5, in live cells using laser scanning confocal microscopy. Depending on the overall organization of the cortical array, AtMAP65-1-GFP (green fluorescent protein) and AtMAP65-5-GFP associated with a subset of microtubules. In cells containing both coaligned and oblique microtubules, AtMAP65-1-GFP and AtMAP65-5-GFP tended to be associated with the coaligned microtubules. Cortical microtubules labeled with AtMAP65-1-GFP and AtMAP65-5-GFP appeared as thick bundles and showed more resistance to microtubule-destabilizing drugs. The polymerization rates of AtMAP65-1-GFP and AtMAP65-5-GFP microtubules were similar to those of tubulin-GFP marked microtubules but were different from AtEB1a-GFP, a microtubule plus-end-binding EB1-like protein that stimulated polymerization. By contrast, depolymerization rates of AtMAP65-1-GFP- and AtMAP65-5-GFP-labeled microtubules were reduced. AtMAP65-1-GFP associated with polymerizing microtubules within a bundle, and with fixed microtubule termini, suggesting that AtMAP65-1s function is to bundle and stabilize adjacent microtubules of the cortex. Polymerization within a bundle took place in either direction so that bundling occurred between parallel or antiparallel aligned microtubules. AtMAP65-4-GFP did not label cortical microtubules or the preprophase band, despite continuous expression driven by the 35S promoter, and its subcellular localization was restricted to microtubules that rearranged to form a spindle and the polar sides of the spindle proper. The expression of AtMAP65-4 peaked at mitosis, in agreement with a function related to spindle formation, whereas AtMAP65-1 and AtMAP65-5 were expressed throughout the cell cycle.

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

Tobacco Mosaic Virus Movement Protein Interacts with Green Fluorescent Protein-Tagged Microtubule End-Binding Protein 1

The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.

Fluorescent protein recruitment assay for demonstration and analysis of in vivo protein interactions in plant cells and its application to Tobacco mosaic virus movement protein.

We describe a simple fluorescent protein-based method to investigate interactions with a viral movement protein in living cells that relies on the in vivo re-localization of proteins in the presence of their interaction partners. We apply this method in combination with fluorescence lifetime imaging microscopy (FLIM) to demonstrate that a domain of the Tobacco mosaic virus (TMV) movement protein (MP) previously predicted to mediate protein:protein interactions is dispensable for these contacts. We suggest that this method can be generalized for analysis of other protein interactions in planta.

Investigating the Cellular Distribution and Interactions of HIV-1 Nucleocapsid Protein by Quantitative Fluorescence Microscopy

The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime imaging microscopy, fluorescence recovery after photobleaching, fluorescence correlation and cross-correlation spectroscopy, and raster imaging correlation spectroscopy. HeLa cells transfected with eGFP-labeled NCp7 were used as a model system. We found that NCp7-eGFP localizes mainly in the cytoplasm and the nucleoli, where it binds to cellular RNAs, and notably to ribosomal RNAs which are the most abundant. The binding of NCp7 to ribosomes was further substantiated by the intracellular co-diffusion of NCp7 with the ribosomal protein 26, a component of the large ribosomal subunit. Finally, gradient centrifugation experiments demonstrate a direct association of NCp7 with purified 80S ribosomes. Thus, our data suggest that NCp7 molecules released in newly infected cells may primarily bind to ribosomes, where they may exert a new potential role in HIV-1 infection.

Interaction of the Tobacco mosaic virus movement protein with microtubules during the cell cycle in tobacco BY-2 cells

Cell-to-cell movement of Tobacco mosaic virus (TMV) involves the interaction of virus-encoded 30-kDa movement protein (MP) with microtubules. In cells behind the infection front that accumulate high levels of MP, this activity is reflected by the formation of stabilized MP/microtubule complexes. The ability of MP to bind along and stabilize microtubules is conserved upon expression in mammalian cells. In mammalian cells, the protein also leads to inhibition of mitosis and cell division through a microtubule-independent process correlated with the loss of centrosomal γ-tubulin and of centrosomal microtubule-nucleation activity. Since MP has the capacity to interact with plant factors involved in microtubule nucleation and dynamics, we used inducible expression in BY-2 cells to test whether MP expression inhibits mitosis and cell division also in plants. We demonstrate that MP:GFP associates with all plant microtubule arrays and, unlike in mammalian cells, does not interfere with mitosis. Thus, MP function and the interaction of MP with factors of the cytoskeleton do not entail an inhibition of mitosis in plants. We also report that the protein targets primary plasmodesmata in BY-2 cells immediately upon or during cytokinesis and that the accumulation of MP in plasmodesmata occurs in the presence of inhibitors of the cytoskeleton and the secretory pathway.

The NC domain of HIV-1 Gag contributes to the interaction of Gag with TSG101

BACKGROUND HIV-1 Gag polyprotein orchestrates the assembly of viral particles. Its C-terminus consists of the nucleocapsid (NC) domain that interacts with RNA, and the p6 domain containing the PTAP motif that binds the cellular ESCRT factor TSG101 and ALIX. Deletion of the NC domain of Gag (GagNC) results in defective Gag assembly, a decrease in virus production and, thus probably affects recruitment of the ESCRT machinery. To investigate the role of GagNC in this recruitment, we analysed its impact on TSG101 and ALIX localisations and interactions in cells expressing Gag. METHODS Cells expressing mCherry-Gag or derivatives, alone or together with eGFP-TSG101 or eGFP-ALIX, were analysed by confocal microscopy and FLIM-FRET. Chemical shift mapping between TSG101-UEV motif and Gag C-terminus was performed by NMR. RESULTS We show that deletion of NC or of its two zinc fingers decreases the amount of Gag-TSG101 interacting complexes in cells. These findings are supported by NMR data showing chemical shift perturbations in the NC domain in- and outside - of the zinc finger elements upon TSG101 binding. The NMR data further identify a large stretch of amino acids within the p6 domain directly interacting with TSG101. CONCLUSION The NC zinc fingers and p6 domain of Gag participate in the formation of the Gag-TSG101 complex and in its cellular localisation. GENERAL SIGNIFICANCE This study illustrates that the NC and p6 domains cooperate in the interaction with TSG101 during HIV-1 budding. In addition, details on the Gag-TSG101 complex were obtained by combining two high resolution biophysical techniques.

MemBright: a Family of Fluorescent Membrane Probes for Advanced Cellular Imaging and Neuroscience

The proper staining of the plasma membrane (PM) is critical in bioimaging as it delimits the cell. Herein, we developed MemBright: a family of six cyanine-based fluorescent turn-on PM probes that emit from orange to near-infrared when reaching the PM, and enable homogeneous and selective PM staining with excellent contrast in mono and two-photon microscopy. These probes are compatible with long-term live cell imaging and immunostaining. Moreover, MemBright label neurons in a brighter manner than surrounding cells allowing identification of neurons in acute brain tissue section and neuromuscular-junctions without any use of transfection or transgenic animals. At last, MemBright were used in super-resolution imaging to unravel the dendritic spines’ neck. 3D multicolor dSTORM in combination with immunostaining revealed en-passant synapse displaying endogenous glutamate receptors clustered at the axonal-dendritic contact site. MemBright probes thus constitute a universal toolkit for cell biology and neuroscience biomembrane imaging with a variety of microscopy techniques.

Optimized protocol for combined PALM-dSTORM imaging

Multi-colour super-resolution localization microscopy is an efficient technique to study a variety of intracellular processes, including protein-protein interactions. This technique requires specific labels that display transition between fluorescent and non-fluorescent states under given conditions. For the most commonly used label types, photoactivatable fluorescent proteins and organic fluorophores, these conditions are different, making experiments that combine both labels difficult. Here, we demonstrate that changing the standard imaging buffer of thiols/oxygen scavenging system, used for organic fluorophores, to the commercial mounting medium Vectashield increased the number of photons emitted by the fluorescent protein mEos2 and enhanced the photoconversion rate between its green and red forms. In addition, the photophysical properties of organic fluorophores remained unaltered with respect to the standard imaging buffer. The use of Vectashield together with our optimized protocol for correction of sample drift and chromatic aberrations enabled us to perform two-colour 3D super-resolution imaging of the nucleolus and resolve its three compartments.