Bruno Goud

Comparative Analysis Between Flaviviruses Reveals Specific Neural Stem Cell Tropism for Zika Virus in the Mouse Developing Neocortex

The recent Zika outbreak in South America and French Polynesia was associated with an epidemic of microcephaly, a disease characterized by a reduced size of the cerebral cortex. Other members of the Flavivirus genus, including West Nile virus (WNV), can cause encephalitis but were not demonstrated to cause microcephaly. It remains unclear whether Zika virus (ZIKV) and other flaviviruses may infect different cell populations in the developing neocortex and lead to distinct developmental defects. Here, we describe an assay to infect mouse E15 embryonic brain slices with ZIKV, WNV and dengue virus serotype 4 (DENV-4). We show that this tissue is able to support viral replication of ZIKV and WNV, but not DENV-4. Cell fate analysis reveals a remarkable tropism of ZIKV infection for neural stem cells. Closely related WNV displays a very different tropism of infection, with a bias towards neurons. We further show that ZIKV infection, but not WNV infection, impairs cell cycle progression of neural stem cells. Both viruses inhibited apoptosis at early stages of infection. This work establishes a powerful comparative approach to identify ZIKV-specific alterations in the developing neocortex and reveals specific preferential infection of neural stem cells by ZIKV.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

Constitutive resistance to viral infection in human CD141+ dendritic cells

Specialized human dendritic cells that resist viral infection activate T cells by relying on bystander cells for antigen production. See the related Focus by Wong et al. Divided, they conquer Dendritic cells (DCs) play a crucial role in priming T cell–driven antiviral responses. Silvin et al. have examined the paradox of how virus-infected DCs retain the ability to drive adaptive immune responses. In response to endocytic viruses, they found CD1c+ DCs to be susceptible to infection and death, whereas CD141+ DCs were not. They report that viral resistance of CD141+ DCs was conferred by the expression of an endocytic guanosine triphosphatase, RAB15, and that transfer of antigen from infected CD1c+ DCs by CD141+ DCs allowed these virus-resistant DCs to prime T cell responses. By documenting a division of labor between DC subsets that separates antigen acquisition from antigen presentation, Silvin et al. offer a solution to this long-standing puzzle. Dendritic cells (DCs) are critical for the launching of protective T cell immunity in response to viral infection. Viruses can directly infect DCs, thereby compromising their viability and suppressing their ability to activate immune responses. How DC function is maintained in light of this paradox is not understood. By analyzing the susceptibility of primary human DC subsets to viral infections, we report that CD141+ DCs have an innate resistance to infection by a broad range of enveloped viruses, including HIV and influenza virus. In contrast, CD1c+ DCs are susceptible to infection, which enables viral antigen production but impairs their immune functions and survival. The ability of CD141+ DCs to resist infection is conferred by RAB15, a vesicle-trafficking protein constitutively expressed in this DC subset. We show that CD141+ DCs rely on viral antigens produced in bystander cells to launch cross-presentation–driven T cell responses. By dissociating viral infection from antigen presentation, this mechanism protects the functional capacity of DCs to launch adaptive immunity against viral infection.

Phenotypic characterisation of RAB6A knockout mouse embryonic fibroblasts

Rab6 is one of the most conserved Rab GTPaes throughout evolution and the most abundant Rab protein associated with the Golgi complex. The two ubiquitous Rab isoforms, Rab6A and Rab6A′, that are generated by alternative splicing of the RAB6A gene, regulate several transport steps at the Golgi level, including retrograde transport between endosomes and Golgi, anterograde transport between Golgi and the plasma membrane, and intra‐Golgi and Golgi to endoplasmic reticulum transport.

Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. DOI: http://dx.doi.org/10.7554/eLife.17523.001

The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction.

Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions.

The actin and microtubule cytoskeletons play important roles in Golgi structure and function, but how they are connected remain poorly known. In this study, we investigated whether RAB6 GTPase, a Golgi-associated RAB involved in the regulation of several transport steps at the Golgi level, and two of its effectors, Myosin IIA and KIF20A participate in the coupling between actin and microtubule cytoskeleton. We have previously shown that RAB6–Myosin IIA interaction is critical for the fission of RAB6-positive transport carriers from Golgi/TGN membranes. Here we show that KIF20A is also involved in the fission process and serves to anchor RAB6 on Golgi/TGN membranes near microtubule nucleating sites. We provide evidence that the fission events occur at a limited number of hotspots sites. Our results suggest that coupling between actin and microtubule cytoskeletons driven by Myosin II and KIF20A ensures the spatial coordination between RAB6-positive vesicles fission from Golgi/TGN membranes and their exit along microtubules.Actin and microtubules play important roles in Golgi structure and function but how they are connected is poorly understood. Here the authors show that KIF20A is involved in the fission process and, in association with Myosin II, serves to anchor RAB6 on Golgi/TGN membranes near microtubules nucleating sites.

A pull-down procedure for the identification of unknown GEFs for small GTPases

ABSTRACT Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

Rab proteins as major determinants of the Golgi complex structure

ABSTRACT GTP-ases of the Rab family (about 70 in human) are key regulators of intracellular transport and membrane trafficking in eukaryotic cells. Remarkably, almost one third associate with membranes of the Golgi complex and TGN (trans-Golgi network). Through interactions with a variety of effectors that include molecular motors, tethering complexes, scaffolding proteins and lipid kinases, they play an important role in maintaining Golgi architecture.

ProLIF: quantitative integrin protein-protein interactions and synergistic membrane effects on proteoliposomes

ABSTRACT Integrin transmembrane receptors control a wide range of biological interactions by triggering the assembly of large multiprotein complexes at their cytoplasmic interface. Diverse methods have been used to investigate interactions between integrins and intracellular proteins, and predominantly include peptide-based pulldowns and biochemical immuno-isolations from detergent-solubilised cell lysates. However, quantitative methods to probe integrin–protein interactions in a more biologically relevant context where the integrin is embedded within a lipid bilayer have been lacking. Here, we describe ‘protein–liposome interactions by flow cytometry’ (denoted ProLIF), a technique to reconstitute recombinant integrin transmembrane domains (TMDs) and cytoplasmic tail (CT) fragments in liposomes as individual subunits or as αβ heterodimers and, via flow cytometry, allow rapid and quantitative measurement of protein interactions with these membrane-embedded integrins. Importantly, the assay can analyse binding of fluorescent proteins directly from cell lysates without further purification steps. Moreover, the effect of membrane composition, such as PI(4,5)P2 incorporation, on protein recruitment to the integrin CTs can be analysed. ProLIF requires no specific instrumentation and can be applied to measure a broad range of membrane-dependent protein–protein interactions with the potential for high-throughput/multiplex analyses. This article has associated First Person interviews with the first authors of the paper (see doi: 10.1242/jcs.223644 and doi: 10.1242/jcs.223719). Summary: This paper outlines a simple protocol to reconstitute integrin chimeras within liposomes and use flow cytometry to quantify the impact of lipid composition on integrin–talin interactions.