Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites

The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex

Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.journal articleresearch support, non-u.s. gov't2014 Oct 152014 08 20importe

The Value of High Performance Networking on International Distance Education - Team Building, Technology Selection, and Innovative Teaching Methodology

The University of Pennsylvania in the United States and its affiliated GigaPoP, MAGPI, together with the University of Grenoble in France are in the planning stages of a collaborative effort to demonstrate the educational potential of utilizing the new, high performance research networks, Renater 2 and Internet 2. Through the creative use of interactive video, video archived material, and web tools such as "Blackboard," this project will open new areas of opportunity for distance education by combining the elements of networking and video technology with multimedia teaching skills. A pilot demonstration is proposed initially to bring two classes of students together to negotiate a business transaction. This will require a proficiency in the foreign language and the ability to transact business across time zones and cultural obstacles. Both aspects of the lesson are very real requirements in the commercial world we live in today. The use of live, high quality video, and the reference to hypothetical archived video clips and web material will make the experience even more meaningful for the students. Faculty from both Universities will contribute to the structure and content of the material under the premise that any technological enhancements will be made available to them. Upon a successful completion of the demonstration, funding will be sought for a full term course that may include multiple sites and multiple age groups

Clonal population of flucytosine-resistant Candida tropicalis from blood cultures, Paris, France

Candida tropicalis is a diploid ascomycetes yeast responsible for 4%-24% of candidemia. Resistance to flucytosine is rarely described for this species but was observed for 45 (35%) of 130 C. tropicalis isolates recovered from blood cultures in the Paris area in a 4-year survey. The aims of this study were to test the hypothesis that the flucytosine-resistant isolates could represent a subgroup and to determine the relationship between epidemiologic and genomic data. Epidemiologic data and gene sequences were analyzed, and molecular typing was performed. Our results suggest that a clone of flucytosine-resistant isolates, associated with malignancies and a lower mortality than that for other C. tropicalis isolates, is widespread in the Paris area. We propose the analysis of 2 polymorphic microsatellite markers coupled with URA3 sequencing to track the clone

Myrip couples the capture of secretory granules by the actin-rich cell cortex and their attachment to the plasma membrane.

International audienceExocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa-defective mice