ZIA BC 011682 (ZIA) | |||
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Title | Molecular mechanisms of membrane remodeling | ||
Institution | NCI, Bethesda, MD | ||
Principal Investigator | Weigert, Roberto | NCI Program Director | N/A |
Cancer Activity | N/A | Division | CCR |
Funded Amount | $988,411 | Project Dates | null - null |
Fiscal Year | 2018 | Project Type | Intramural |
Research Topics w/ Percent Relevance | Cancer Types w/ Percent Relevance | ||
Cancer (100.0%) |
Buccal Cavity (20.0%) Head and Neck (100.0%) Salivary Glands (60.0%) |
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Research Type | |||
Normal Functioning Cancer Progression & Metastasis |
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Abstract | |||
Molecular basis of membrane remodeling during secretion at the plasma membrane. Secretory epithelia such as salivary glands represent a robust model systems to study various aspects of the remodeling of membranes during intracellular trafficking processes, such as regulated protein-secretion and plasma membrane homeostasis. 1) Regulated exocytosis in salivary glands. In salivary glands acinar cells, secretory proteins are packed in large granules at the trans-Golgi network (TGN) and transported to the cell periphery where they fuse with the apical plasma membrane (APM) upon stimulation of G-Protein coupled receptors, thus releasing their content into the acinar canaliculi. Concomitantly, the membranes of the secretory granules gradually integrate into the APM, thus undergoing substantial remodeling. Our aim is to elucidate the molecular machinery regulating the integration of the secretory granules with the APM. To this end, we developed an experimental system in live rodents aimed at imaging and tracking individual secretory granules. We established that upon stimulation of the beta-adrenergic receptor, the granules fuse with the APM, followed, after a short delay, by the recruitment of a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB). We showed that actomyosin contractile activity regulates the integration of the granular membranes into the APM and the completion of exocytosis. Modeling of this process based on the EM ultrastructural analysis of the secretory granules and the APM, revealed that the integration is energetically unfavorable, since it is constantly opposed by a convective flow of membranes directed from the APM to the granule membranes. This process is driven by the fact the membrane tension of the APM bilayer is higher than that of the secretory granules membranes. In order to understand how the actomyosin complex drives the integration, we focused on determining how F-actin and NMII are structurally arranged on the secretory granules. To this end, we used a series of selected light microscopy techniques with higher resolution than conventional confocal and two-photon microscopy, such as Spinning Disk and Stimulated Emission Depletion Microscopy (STED). Strikingly, we discovered that both F-actin and NMII assemble around the secretory granules in distinct polyhedral cages, formed by pentagonal and hexagonal units like those described for clathrin around the endocytic vesicles, although one order of magnitude larger. This represents a novel structural organization for the actomyosin cytoskeleton, never described before. Our data suggested that the NMII cage could function to crosslink actin filaments and/or transmit the forces generated by the contractile activity to the F-actin cage, and therefore to the granules membranes. Notably, the improved temporal resolution afforded by the spinning disc microscope, enabled us to capture, for the first time, 4D datasets of the dynamics of the cages during the integration process in vivo. This revealed that F-actin and NMII are gradually recruited into stable cages which maintain constant diameter and fixed shape (assembly phase). This step is followed by 1) the rapid polymerization of F-actin directed from the actomyosin cage towards the granule membranes (compression phase), and 2) the increase of the surface density of the NMII molecules (contractile phase). Finally, both cages disassemble with NMII being released in large filaments. Our data support a novel model based on a multi-step process in which first, the actomyosin cages counteract the convective flow of the lipids from the APM and prevent compound exocytosis; second, F-actin polymerization generates forces that drive the integration, using the cage as a leverage to push the membranes toward the APM; and third, NMIIA-driven contractions generate additional forces to facilitate the integration. In addition, we further confirmed that both the F-actin and NMII cages are as |