Sabine Bardin

Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex

Trafficking between membrane compartments is a characteristic of eukaryotic cells and relies on transport carriers that bud and fission from a donor membrane, before being transported and fusing with the correct acceptor compartment. Rab GTPases ensure specificity and directionality of trafficking steps by regulating the movement of transport carriers along cytoskeletal tracks, and the recruitment of tethering factors required for the docking and fusion processes. Here we show that Rab6, a Golgi-associated Rab, forms a complex with myosin II, contributes to its localization at the Golgi complex and, unexpectedly, controls the fission of Rab6 vesicles. Inhibition of either Rab6 or myosin II function impairs both the fission of Rab6 transport carriers from Golgi membranes and the trafficking of anterograde and retrograde cargo from the Golgi. These effects are consistent with myosin II being an effector of Rab6 in these processes. Our results provide evidence that the actomyosin system is required in vesicle biogenesis at the Golgi, and uncover a function for Rab GTPases in vesicle fission.

A role for the Rab6A′ GTPase in the inactivation of the Mad2‐spindle checkpoint

The two isoforms of the Rab6 GTPase, Rab6A and Rab6A′, regulate a retrograde transport route connecting early endosomes and the endoplasmic reticulum via the Golgi complex in interphasic cells. Here we report that when Rab6A′ function is altered cells are unable to progress normally through mitosis. Such cells are blocked in metaphase, despite displaying a normal Golgi fragmentation and with the Mad2‐spindle checkpoint activated. Furthermore, the Rab6 effector p150Glued, a subunit of the dynein/dynactin complex, remains associated with some kinetochores. A similar phenotype was observed when GAPCenA, a GTPase‐activating protein of Rab6, was depleted from cells. Our results suggest that Rab6A′ likely regulates the dynamics of the dynein/dynactin complex at the kinetochores and consequently the inactivation of the Mad2‐spindle checkpoint. Rab6A′, through its interaction with p150Glued and GAPCenA, may thus participate in a pathway involved in the metaphase/anaphase transition.

Gain-of-function mutant of angiotensin II receptor, type 1A, causes hypertension and cardiovascular fibrosis in mice

The role of the renin-angiotensin system has been investigated by overexpression or inactivation of its different genes in animals. However, there is no data concerning the effect of the constitutive activation of any component of the system. A knockin mouse model has been constructed with a gain-of-function mutant of the Ang II receptor, type 1A (AT(1A)), associating a constitutively activating mutation (N111S) with a C-terminal deletion, which impairs receptor internalization and desensitization. In vivo consequences of this mutant receptor expression in homozygous mice recapitulate its in vitro characteristics: the pressor response is more sensitive to Ang II and longer lasting. These mice present with a moderate (~20 mmHg) and stable increase in BP. They also develop early and progressive renal fibrosis and cardiac fibrosis and diastolic dysfunction. However, there was no overt cardiac hypertrophy. The hormonal parameters (low-renin and inappropriately normal aldosterone productions) mimic those of low-renin human hypertension. This new model reveals that a constitutive activation of AT(1A) leads to cardiac and renal fibrosis in spite of a modest effect on BP and will be useful for investigating the role of Ang II in target organs in a model similar to some forms of human hypertension.

A Rab11A/Myosin Vb/Rab11-FIP2 Complex Frames Two Late Recycling Steps of Langerin from the ERC to the Plasma Membrane

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.

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging

Significance Recent progress has pushed forward the resolving capacity of optical microscopy at the expense of a low acquisition rate and use of specific probes. Such limitations make these techniques incompatible with dynamics localization of multiple elements in single cell. We report here a method to recover 3D volumes from images obtained using several total internal reflection fluorescence (TIRF) incidence angles at dense regime of acquisition. This approach allows investigating several dynamical processes occurring in depth of the cell up to 800 nm from the plasma membrane such as actin remodeling. The study of time-correlated molecular behaviors at the very late steps of vesicle docking–fusion during exocytosis of two distinct recycling transport intermediates, in 3D and at high axial resolution, is also accessible. Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

Mechanical Role of Actin Dynamics in the Rheology of the Golgi Complex and in Golgi-Associated Trafficking Events

BACKGROUND In vitro studies have shown that physical parameters, such as membrane curvature, tension, and composition, influence the budding and fission of transport intermediates. Endocytosis in living cells also appears to be regulated by the mechanical load experienced by the plasma membrane. In contrast, how these parameters affect intracellular membrane trafficking in living cells is not known. To address this question, we investigate here the impact of a mechanical stress on the organization of the Golgi complex and on the formation of transport intermediates from the Golgi complex. RESULTS Using confocal microscopy, we visualize the deformation of Rab6-positive Golgi membranes applied by an internalized microsphere trapped in optical tweezers and simultaneously measure the corresponding forces. Our results show that the force necessary to deform Golgi membranes drops when actin dynamics is altered and correlates with myosin II activity. We also show that the applied stress has a long-range effect on Golgi membranes, perturbs the dynamics of Golgi-associated actin, and induces a sharp decrease in the formation of Rab6-positive vesicles from the Golgi complex as well as tubulation of Golgi membranes. CONCLUSIONS We suggest that acto-myosin contractility strongly contributes to the local rigidity of the Golgi complex and regulates the mechanics of the Golgi complex to control intracellular membrane trafficking.

The CryoCapsule: simplifying correlative light to electron microscopy.

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.

Computational Geometry-Based Scale-Space and Modal Image Decomposition

In this paper a framework for defining scale-spaces, based on the computational geometry concepts of *** -shapes, is proposed. In this approach, objects (curves or surfaces) of increasing convexity are computed by selective sub-sampling, from the original shape to its convex hull. The relationships with the Empirical Mode Decomposition (EMD), the curvature motion-based scale-space and some operators from mathematical morphology, are studied. Finally, we address the problem of additive image/signal decomposition in fluorescence video-microscopy. An image sequence is mainly considered as a collection of 1D temporal signals, each pixel being associated with its temporal intensity variation.

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function.

A detection-based framework for the analysis of recycling in TIRF microscopy

Endocytosis/recycling and exocytosis aremechanisms conserved through evolution allowing cells to communicate with their external medium. In order to study these dynamic processes, the present work proposes a patch-based method for detecting recycling or exocytotic events at the Plasma membrane in fast TIRF microscopy combined with the computation of normalized temporal representations of those events. Evaluation, performed on TIRF sequences showing Transferrin receptor (TfR) recycling, validates a high detection rate fully compatible with an automatic data extraction and analysis of the plasma membrane recycling process.