Molecular mechanisms of membrane remodeling

Division Of Basic Sciences - Nci

Investigators

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Abstract

Molecular basis of membrane remodeling during secretion at the plasma membrane (PM). Regulated exocytosis in exocrine glands. In the acinar cells of exocrine glands (i.e. salivary glands, exocrine pancreas), secretory proteins are packed in large granules that are transported to the cell periphery where they fuse with the apical plasma membrane (APM) upon receptor stimulation and release their content into the acinar canaliculi. Concomitantly, the membranes of the secretory granules gradually integrate into the APM undergoing substantial remodeling. We aim at elucidating the molecular machinery regulating this process. To this end, we developed an experimental system in live rodents aimed at imaging and tracking individual secretory granules. We established that granules fuse with the APM and after a short delay, a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB) is recruited on their membranes. We showed that actomyosin contractile activity regulates the integration of the granular membranes into the APM and the completion of exocytosis. Using super-resolution microscopy in vivo we discovered that both F-actin and NMII assemble around the secretory granules in distinct polyhedral cages, formed by pentagonal and hexagonal units. The NMII cage crosslinks actin filaments and transmits the forces generated by the NMII contractile activity to the F-actin cage, and therefore to the granules membranes. Notably, the improved temporal resolution afforded by spinning disc microscopy 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 that maintain constant diameter and fixed shape. This step is followed by 1) the rapid polymerization of F-actin directed from the actomyosin cage towards the granule membranes, and 2) the increase of the surface density of the NMII molecules. 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; second, F-actin polymerization generates forces that drive the integration, using the cage as leverage to push the membranes toward the APM; and third, NMII-driven contractions generate additional forces to facilitate the integration. Besides, we determined that both the F-actin and NMII cages are assembled independently. Specifically, F-actin is assembled in two steps. First, the initial F-actin cage is assembled by activation of mDia1/2, two members of the Formin family of actin nucleators which generate linear filaments. Second, branched filaments are assembled by activation of the Arp2/3 complex, N-Wasp, and cortactin. Pharmacological or genetic ablation of mDia1/2 or the Arp2/3 complex disrupt the integration of the secretory granules into the APM. To elucidate the modality of recruitment and regulation of NMII on the secretory granules, we investigated selected molecules, that were chosen among those identified in a preliminary proteomic screening of the proteins associated with purified secretory granules. Using super-resolution microscopy and indirect immunofluorescence, we discovered that 3 members of the Septin family of GTPase, and namely septins 2, 6, and 7 (SEPT2, SEPT6, and SEPT7), are present on the surface of fused granules and are also organized into cage-like cages which co-align NMII. Generation of knock-in mice expressing fluorescently tagged version of SEPT2 and SEPT7 validated this finding. Pharmacological inhibition of SEPT2 resulted in a significant decrease in the levels of activated NMII and myosin light chain kinase (MLCK) on fused granules, whereas, disruption of F-actin assembly lead to an expansion in granules size without impairing NMII or septin recruitment. Finally, genetic ablation of SEPT7 in the acinar cells of adult mice compromised the integration of the secretory granules and the levels of actomyosin on their surface. Based on our data, we propose that the newly observed septins cages: 1) provide a scaffold to recruit and curve acto-myosin filaments on the surface of the secretory granules, and 2) are needed for the activation of NMII, likely through MLCK-mediated phosphorylation. Mechanisms of membrane remodeling during neutrophil migration in live animals. Cell migration is a fundamental biological process that requires membrane remodeling through the constant re-arrangement of the actomyosin cytoskeleton. Neutrophil migration has been particularly studied due to its role in the immune response, and tumor progression. Under physiological conditions, neutrophils circulate in the vasculature, whereas during pathological states (e.g. wounds, infections, inflammation, tumorigenesis) the production of chemo-attractants gradients promote their adhesion to the vascular endothelium, extravasation into the interstitium, and finally directed migration towards the target site. 1)Role and regulation of the actomyosin cytoskeleton during neutrophil extravasation. The eicosanoid Leukotriene B4 (LTB4) relays chemotactic signals to direct neutrophil interstitial migration in response to injury through its receptor, BLT1. However, whether the LTB4-BLT1 axis modulates the actomyosin cytoskeleton during intravascular neutrophil response has not been addressed in vivo. Hence, we developed an inflammation model in the mouse footpad to directly visualize the impact of the LTB4-BLT1 axis on the intravascular neutrophil dynamics. We found that LTB4 produced by neutrophils acts as an autocrine/paracrine signal via BLT1 to drive their recruitment, arrest, and extravasation. We discovered that LTB4 elicits cell adhesion and polarization during neutrophil arrest. Specifically, LTB4 signaling coordinates the dynamic redistribution of NMIIA to the back of the cell, where its contractile activity is required to 1) promote neutrophil arrest and adhesion, by controlling the transport of beta2-integrin (Itgb2) to the PM facing the neutrophil-endothelial interface; and 2) drive extravasation by generating the mechanical forces required to deform the neutrophil membranes, as they pass through the vascular wall. Consistent with these findings, we observed that blocking LTB4 signaling or NMIIA activation inhibits Itgb2 recycling to the PM. Overall, our study unraveled a crucial function for LTB4 in promoting neutrophil communication in the vasculature during the early response to inflammation by the activation of signaling circuits that control the actomyosin cytoskeleton. 2)Role of the actomyosin cytoskeleton during interstitial migration in vivo. We have used ISMic coupled to novel computational methods to understand the underlying mechanisms of the coordination among PM remodeling, actomyosin cytoskeleton, and cell metabolism during interstitial neutrophil migration in a mouse ear model of sterile injury. Migrating neutrophils exhibit a very dynamic membrane remodeling with a continuous formation of micron-scale membrane protrusions, which interact with the tissue microenvironment (i.e. extracellular matrix). Differently from what previously described, we found that NMIIA is not only present at the uropod of the cell but also at the leading edge and in large lateral protrusions. In these new locations, NMIIA does not actively retract the membranes but works to stabilize them in response to interactions with the ECM. Furthermore, we found that NMIIA recruitment at the leading edge is RhoA/ROCK independent, and it is controlled by the activation of PKC-zeta, indicating a new modality of recruitment of NMIIA in neutrophils. Pharmacological inhibition of PKC-zeta, reduces the recruitment of NMIIA at the leading edge of the migrating neutrophils, destabilizing the membrane protrusion and inducing a loss of directionality and migration speed.