ENDOMYOSHAPE · Identification of the molecular mechanisms whereby actin and myosin1b shape sorting endosomes

Identification of the molecular mechanisms whereby actin and myosin1b shape sorting endosomes

The trans-Golgi network (TGN) is one of the main protein sorting stations of the cell at the cross road of the exocytic and endocytic pathways. This organelle is composed of a complex tubular network that emanates from the trans-Golgi cisternae, and generates pleiomorphic carriers targeted to different destinations. Dramatic changes in its membrane shape underlie the exit of cargos. This exit involves concentration of cargo in membrane domains of the TGN, membrane deformation or budding, elongation of tubular-carrier precursors and scission leading to the formation of the post-Golgi carriers. There are various possible mechanisms that can regulate membrane deformation, which include the cytoskeleton and its motors, which can change the mechanical properties of membranes leading to membrane deformation. Myosin 1b (Myo1b) was shown to regulate the traffic of cargo along the endocytic pathway. Myo1b had been localized at the plasma membrane in regions enriched for actin filaments and at early endosomes, multivesicular endosomes and lysosomes. We now identified a pool of Myo1b in the perinuclear region of HeLa cells. This pool colocalizes partially at the TGN with CI-mannose-6-phosphate receptor (MPR) that carries cargos from the TGN to sorting endosomes and recycles back to TGN. Our results demonstrated that Myo1b controls the TGN exit of MPR and p75 but not of a GPI-anchored protein independently of its role along the endocytic pathway and that MPR tubular-carrier precursors, post-Golgi carriers and F-actin foci formation depend on Myo1b expression. These observations together argued that Myo1b functions with F-actin foci for the formation of tubular-carrier precursors leading to the TGN exit of transmembrane proteins but not of GPI-anchored proteins that by distributing with distribute with "lipid rafts" may depend on lipid segregation. Actin dynamics and various proteins related to the actin-based system have been involved in carrier biogenesis. We identified the actin structures that participate in this function. We showed that absence of F-actin foci upon depletion of the Arp2/3 subunit p34 inhibits the formation of tubular-carriers precursors and post-Golgi carriers. Furthermore we provide evidence that the inhibition of MPR and p75 exit from the TGN upon Myo1b depletion is accompanied by a decrease in F-actin foci. Together these observations supported a role of the F-actin foci and the Arp2/3 complex in the post-Golgi traffic for the formation of tubular-carrier precursors. How does Myo1b/F-actin promote the formation of tubular-carrier precursors? The pulling force generated by microtubule-associated motors drive the extension of tubular-carrier precursors. Since Myo1b/F-actin foci were required for tubular-precursor formation it is likely that they function prior to kinesins. An attractive possibility based on Myo1b mechanochemical properties is that Myo1b/F-actin foci control TGN membrane tension thereby promoting membrane deformation. Membrane deformation at the TGN driven by Myo1b with F-actin would facilitate the function of a kinesin to extend tubular-carrier precursors. Our finding that Myo1b motor activity is required for the formation of F-actin foci and for the steady state MPR distribution suggests that Myo1b actively tethers and orients polymerising F-actin to the TGN membrane and generates a force leading to TGN membrane deformations. In addition, Myo1b motor domain may stabilize newly polymerized F-actin leading to the formation of F-actin foci. Altogether, our work revealed a new function for myosin 1b that is to couple actin assembly to organelles and control membrane deformation leading to the formation of transport carriers. Understanding the molecular mechanism by which Myo1b can achieve this function is an exciting future challenge.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз