CONICET | Buscador de Institutos y Recursos Humanos

Autophagy is a lysosomal-based degradation process, involving double membrane vesicles (autophagosomes) recovered of LC3 protein. We previously described that ATP-loaded autophagosomes are redistributed to focal adhesions, where they exocyte in a VAMP-7 dependent way. Rab proteins are important in cellular trafficking and sorting. They cycle between an active (GTP) and inactive (GDP) form, due to GEF and GAP proteins respectively. VARP is a multifunctional protein: It is a GEF for Rab21 (an endosomal and golgian Rab) and an effector for Rab32 (an endosomal, reticular, mitochondrial and golgian Rab). VARP can also interact with VAMP-7, blocking its fusion motif.The aim of this work is to evaluate the role of Rab21, Rab32 and VARP in the redistribution of exocytic autophagosomes upon autophagic stimulus.We transfected HeLa cells with LC3, VARP, Rab32 or Rab 21 wild types plasmids. We also used mutants of this Rabs (T39N and T33N respectively).Then, we detected endogenous proteins by indirect immunofluorescence against LC3, VARP or GM130 in cells incubated in starvation or complete medium containing 100 µM of resveratrol or 50 µM of rapamycin. We distinguished two populations of Rab32 and Rab21: one perinuclear that colocalizes with GM130 in control conditions, and another that goes to the cell periphery, losing the GM130 positive mark. This suggests that autophagy may induce this Rabs relocation. Interestingly, when we overexpressed VARP, Rab21 wt and Rab32 T39N, the colocalization between VARP and Rab21 wt increased notably. We found that, after autophagic stimulation, LC3, Rab32, Rab21 or VARP positive vesicles were redistributed to the cell periphery, increasing the colocalization between Rabs and LC3 or VARP. This results may indicate a complex molecular mechanism involved in the autophagic exocytosis, being VARP the putative main protein involve in the sorting of vesicles that go to the cell periphery after induction of autophagy.