Biophysical Society Thematic Meeting| Padova 2019

Quantitative Aspects of Membrane Fusion and Fission

Poster Abstracts

19-POS Board 19 REGULATION OF VESICLE ACIDIFICATION BY RABCONNECTIN-3A Sindhuja Gowrisankaran 1 ; Andrea Raimondi 2 ; Nicolas de Roux 3 ; Ira Milosevic 1 ; 1 European Neuroscience Institute, University Medical Center Göttingen (UMG), Göttingen, Niedersachsen, Germany 2 Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC), Hospital San Raffaele, Milano, Italy 3 Inserm U1141, Hospital Robert Debré, University Paris Diderot, Paris, France The regulation and timing of vesicle acidification is essential for numerous cellular processes, from macro-molecule degradation in lysosomes to refilling of synaptic vesicles (SVs) at the neuronal synapse. Acidification of vesicles is achieved by vacuolar ATPases (vATPases), a family of proton pumps that controls the pH gradient across organelle membranes. Despite their critical importance at the synapse and in many intracellular trafficking routes, the regulation of vATPase activity is poorly understood. In a search for vATPase regulators, we cloned human Dmxl2 gene encoding Rabconnectin-3a (Rbcn-3a). Rbcn-3a encodes a large 340kDa protein, whose function at the mammalian synapse remains largely unknown. An alteration in the gene dosage of Dmxl2 in humans resulted in a complex pathology called as poly-endocrine- polyneuropathy syndrome (PEPNS). We found Rbcn-3a to be present on every organelle that acidifies; including SVs. Loss of Rbcn-3a in mice resulted in early embryonic lethality. When Rbcn-3a is eliminated from neuronal cells in culture, neurons developed normally, yet their activity was perturbed. In addition, pHluorin-based imaging revealed an alteration in SV recycling in neurons. Single vesicle analysis with a voltage sensor (Fluorovolt dye) showed that SVs from neurons without Rbcn-3a failed to acidify fully. Additionally, an ultrastructural examination of neurons without Rbcn-3a showed a reduction in the number of SVs, as well as accumulation of lysosomes-like structures and lysosomal markers. Our results suggests an unanticipated connection between the machinery for acidification, vesicle recycling and cellular homeostasis.

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