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 | $1,060,871 | Project Dates | 00/00/0000 - 00/00/0000 |
Fiscal Year | 2017 | 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 (SGs), mammary glands (MGs) and the liver represent three robust model systems to study various aspects of the remodeling of membranes during intracellular trafficking processes, such as constitutive and regulated protein- and lipid-secretion, and plasma membrane homeostasis. 1) Regulated exocytosis in salivary glands In SG 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 APM upon stimulation of GPCRs, 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. Last year, we focused on elucidating the mechanisms of recruitment and regulation of NMII. We showed that NMIIA and NMIIB are recruited onto the SGs after their fusion with the APM, and that their contractile activity drives the gradual integration of the granules into the APM. This contrasts with other cellular processes where actomyosin-based contractions employ only one isoform of NMII. By using conditional knock-out mice we determined that NMIIB is required to control the initial steps of the integration of the granular membrane, by stabilizing the F-actin scaffold and providing a continuous contractile activity that pushes the membranes towards the APM. On the other hand, NMIIA is required at later stages of the process to control the expansion of the fusion pore. Since both NMIIA and NMIIB are recruited after the formation of the F-actin scaffold, we assumed that this process would be mediated by their well-characterized actin-binding site. Unexpectedly, we found that both NMII isoforms are recruited in an actin-independent fashion and that the main role of F-actin is to facilitate the proper assembly of the NMII filaments. Indeed, F-actin facilitates the recruitment of the myosin light chain kinase (MLCK), which in turn activates both NMII isoforms via the phosphorylation of two residues (S19 and T18) which initiate the formation of contractile filaments. Finally, we discovered that three members of the Septin family of GTPases, Septin 2, 6, and 7, are recruited on the SGs after their fusion with the APM, and control the activation of MLCK. These results provide a springboard to begin investigating the biophysical basis underlying the process of membrane integration. 2) Lipid droplets secretion in mammary glands In MGs, the lipid droplet (LD) fraction of milk supplies preformed lipids for neonatal development, and the assembled LDs are secreted by a unique apocrine mechanism, that has never been investigated in vivo. To this end, we developed a method for the intravital imaging of mammary cells in transgenic mice that express fluorescently tagged marker proteins. For the first time, we described the kinetic analysis of LD growth and secretion at peak lactation in real time. LD transit from basal to apical regions was slow (0-2 um/min) and frequently intermittent. Droplets grew by the fusion of preexisting droplets, with no restriction on the size of fusogenic partners. Most droplet expansion took several hours and occurred in APM nucleation centers, either close to or in association with the |