Endocytosis & exocytosis

Les GTPases de la famille Ypt/Rab sont impliquées dans la régulation du trafic vésiculaire, et plus particulièrement dans les étapes de transport, d’arrimage et de fusion des vésicules. La régulation de leur activité GTPase est importante pour la régulation de leurs fonctions. L’activité GTPase des protéines Ypt/Rab est stimulée par des protéines activatrices de GTPase. Chez la levure Saccharomyces cerevisiae, il existe 10 protéines portant un domaine catalytique des protéines activatrices des GTPases Ypt/Rab. Gyp5p et Gyl1p sont deux protéines paralogues qui font partie de cette famille de protéines. In vitro, Gyp5p stimule l’activité GTPase de Ypt1p, qui est impliquée dans le transport entre le réticulum endoplasmique et l’appareil de Golgi, et de Sec4p, impliquée dans l’exocytose. Gyl1p, qui possède un domaine catalytique partiellement dégénéré, n’a pas d’activité stimulatrice de GTPase connue.

L’objectif de mon travail de thèse était de rechercher les fonctions biologiques de Gyp5p et de Gyl1p.

Nous avons montré que Gyp5p et Gyl1p sont situées aux sites de croissance polarisée, où elles sont co-localisées avec Sec4p. Gyp5p est présente dans un complexe contenant Sec4p dans une fraction enrichie en vésicules post-golgiennes, et Gyp5p et Gyl1p sont présentes dans un complexe contenant Sec4p dans une fraction enrichie en membrane plasmique. SEC2, qui code pour le facteur d’échange de Sec4p, et GYP5 présentent un effet génétique antagoniste par rapport à la croissance cellulaire. Les cellules dépourvues de Gyp5p et Gyl1p présentent des défauts de sécrétion, et accumulent des vésicules uniquement chez les jeunes bourgeons à 13°C. Ces résultats montrent que Gyp5p et Gyl1p sont impliquées dans l’exocytose polarisée.
Gyp5p et Gyl1p présentent une localisation dynamique au cours du cycle cellulaire. Gyp5p et Gyl1p sont co-localisées aux étapes de croissance polarisée, mais sont dissociées au cours de la croissance isotropique. Nous avons montré que Gyp5p est délocalisée en l’absence de Gyl1p, et Gyl1p est délocalisée en l’absence de Gyp5p, ce qui montre que la localisation de Gyp5p et Gyl1p est interdépendante. Nous avons montré que Gyp5p et Gyl1p sont capables d’interagir directement in vitro. Nous avons observé que les sous-unités du polarisome Bni1p, Bud6p et Spa2p sont nécessaires à la localisation de Gyp5p et Gyl1p aux sites de croissance polarisée. Le polarisome est un complexe
dont une des fonctions est de réguler la formation de câbles d’actine au sommet du bourgeon par la formine Bni1p. Des défauts de localisation de Gyp5p et Gyl1p ont également été observés en l’absence de l’autre formine Bnr1p. Ces résultats suggèrent que la localisation de Gyp5p et Gyl1p serait dépendante des câbles d’actine.
Nous avons également montré qu’une partie de Gyp5p et de Gyl1p est présente dans un complexe avec Rvs167p. Rvs167p est une protéine connue pour être impliquée dans l’endocytose. Une partie de Rvs167p est co-localisée avec Gyp5p et Gyl1p aux sites de croissance polarisée. Mais en l’absence de Gyp5p et de Gyl1p, Rvs167p n’est plus présente au sommet des jeunes bourgeons et au cou du bourgeon lors de la cytocinèse. Gyp5p et Gyl1p sont donc nécessaire à la localisation de Rvs167p aux sites de croissance polarisée.

Sensory hair cells rely on otoferlin as the calcium sensor for exocy-tosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins. exocytosis | membrane fusion | calcium | otoferlin | calcium channel

Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process, and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE mediated exocytotic events including cytokine release and acid sphingomyelinase secretion. H o w e ve r , i t i s u n c l e a r w h e t h e r d ys f e r l i n r e gu l a t e s S N A R E m e d i a t e d me m b r a n e f u s i o n. In this study we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin 4 and SNAP-23. In addition, analysis of FRET and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates syntaxin 4/SNAP-23 heterodimer formation and SNARE mediated lipid mixing in a calcium sensitive manner. These results support a function for dysferlin as a calcium sensing SNARE effector for membrane fusion events.

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae. We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro. In vivo, both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate tha...

The ferlin family proteins have emerged as multi-C2 domain regulators of calcium-triggered membrane fusion and fission events. While initially determined to share many of the features of members of the synaptotagmin family of calcium sensors, ferlins in more recent studies have been found to interact directly with non-neuronal voltage-gated calcium channels and nucleate the assembly of membrane-trafficking protein complexes, functions that distinguish them from the more well studied members of the synaptotagmin family. Here we highlight some of the recent findings that have advanced our understanding of ferlins and their functional differences with the synaptotagmin family.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25-95 μM) and low affinities (Kd = 400-700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'-myoinositol-4',5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

Gyp5p and Gyl1p are two members of the Ypt/Rab guanosine triphosphatases-activating proteins involved in the control of polarized exocytosis in Saccharomyces cerevisiae. We had previously shown that Gyp5p and Gyl1p colocalize at the sites of polarized growth and belong to the same complex in subcellular fractions enriched in plasma membrane or secretory vesicles. Here, we investigate the interaction between Gyp5p and Gyl1p as well as the mechanism of their localization to the sites of polarized growth. We show that purified recombinant Gyp5p and Gyl1p interact directly in vitro. In vivo, both Gyp5p and Gyl1p are mutually required to concentrate at the sites of polarized growth. Moreover, the localization of Gyp5p and Gyl1p to the sites of polarized growth requires the formins Bni1p and Bnr1p and depends on actin cables. We show that, in a sec6-4 mutant, blocking secretion leads to coaccumulation of Gyp5p and Gyl1p, together with Sec4p. Electron microscopy experiments demonstrate that Gyp5p is associated with secretory vesicles. Altogether, our results indicate that both Gyp5p and Gyl1p access the sites of polarized growth by transport on secretory vesicles. Two polarisome components, Spa2p and Bud6p, are involved in maintaining Gyp5p and Gyl1p colocalized at the sites of polarized growth.

Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo [1]. Membrane trafficking is crucial for animal cell cytokinesis [2-8], and several endocytic pathways regulated by distinct GTPases (Rab11, Rab21, Rab35, ARF6, RalA/B) [9-16] contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis [12]. Here, we report that the ARF6 GTPase [17, 18] negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis.

The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein-based assays indicate that Fer1l6 colocalizes with syntaxin 4 and vinculin, and that the putative C2 domains interact with lipid membranes. We conclude that Fer1l6 has diverged from other vertebrate ferlins to play an essential role in zebrafish skeletal and cardiac muscle development.

Vasopressin neurons generate distinctive phasic patterned spike activity in response to elevated extracellular osmotic
pressure. These spikes are generated in the cell body and are conducted down the axon to the axonal terminals where they
trigger Ca2+ entry and subsequent exocytosis of hormone-containing vesicles and secretion of vasopressin. This mechanism
is highly non-linear, subject to both frequency facilitation and fatigue, such that the rate of secretion depends on both the
rate and patterning of the spike activity. Here we used computational modelling to investigate this relationship and how it
shapes the overall response of the neuronal population. We generated a concise single compartment model of the
secretion mechanism, fitted to experimentally observed profiles of facilitation and fatigue, and based on representations of
the hypothesised underlying mechanisms. These mechanisms include spike broadening, Ca2+ channel inactivation, a Ca2+
sensitive K+ current, and releasable and reserve pools of vesicles. We coupled the secretion model to an existing integrateand-
fire based spiking model in order to study the secretion response to increasing synaptic input, and compared phasic
and non-phasic spiking models to assess the functional value of the phasic spiking pattern. The secretory response of
individual phasic cells is very non-linear, but the response of a heterogeneous population of phasic cells shows a much
more linear response to increasing input, matching the linear response we observe experimentally, though in this respect,
phasic cells have no apparent advantage over non-phasic cells. Another challenge for the cells is maintaining this linear
response during chronic stimulation, and we show that the activity-dependent fatigue mechanism has a potentially useful
function in helping to maintain secretion despite depletion of stores. Without this mechanism, secretion in response to a
steady stimulus declines as the stored content declines.

We report here elements for functional characterization of two members of the Saccharomyces cerevisiae Ypt/Rab GTPase activating proteins family (GAP): Gyp5p, a potent GAP in vitro for Ypt1p and Sec4p, and the protein Ymr192wp/APP2 that we propose to rename Gyl1p (GYp like protein). Immunofluorescence experiments showed that Gyp5p and Gyl1p partly colocalize at the bud emergence site, at the bud tip and at the bud neck during cytokinesis. Subcellular fractionation and co-immunoprecipitation experiments showed that Gyp5p and Gyl1p co-fractionate with post-Golgi vesicles and plasma membrane, and belong to the same protein complexes in both localizations. We found by co-immunoprecipitation experiments that a fraction of Gyp5p interacts with Sec4p, a small GTPase involved in exocytosis, and that a fraction of Gyl1p associates at the plasma membrane with the Gyp5p/Sec4p complexes. We showed also that GYP5 genetically interacts with SEC2, which encodes the Sec4p exchange factor. Examination of the gyp5Deltagyl1Delta mutants grown at 13 degrees C revealed a slight growth defect, a secretion defect and an accumulation of secretory vesicles in the small-budded cells. These data suggest that Gyp5p and Gyl1p are involved in control of polarized exocytosis.