Molecular mechanisms of membrane remodeling

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Molecular basis of membrane remodeling during secretion at the plasma membrane. In the acinar cells of exocrine glands, secretory proteins are packed in large granules that fuse with the apical plasma membrane (APM) upon receptor stimulation and release their content into the acinar canaliculi. The membranes of the secretory granules integrate into the APM undergoing substantial remodeling. We aim at elucidating the machinery regulating this process. To this end, we developed an experimental system in live mice aimed at imaging and tracking individual secretory granules. We established that granules fuse with the APM and a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB) is recruited on their membranes. The actomyosin contractile activity regulates the integration of the granular membranes into the APM and the completion of exocytosis. We showed that both F-actin and NMII assemble around the secretory granules in distinct polyhedral cages, formed by triskelion-like units. The NMII cage crosslinks F-actin and transmits the forces generated by the NMII contractile activity to the F-actin cage, and therefore to the granules' membranes. High resolution time-lapse imaging revealed that, initially, F-actin and NMII are 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. This supports a model based on a multi-step process in which i) the actomyosin cages counteract the convective flow of the lipids from the APM; ii) F-actin polymerization generates forces that drive the integration, using the cage as leverage to push the membranes toward the APM; and iii) NMII-driven contractions generate additional forces to facilitate the integration. We determined that the F-actin and NMII cages are assembled independently. First, the F-actin cage is assembled by activation of mDia1, a member of the Formin family of linear actin nucleators. Second, branched filaments are assembled by activation of the Arp2/3 complex. Pharmacological or genetic ablation of mDia1 disrupt the assembly of the cage and results in the expansion of the fused granules. On the other hand, pharmacological or genetic disruption of the Arp2/3 complex delays the integration of the secretory granules into the APM and inhibit the inward polymerization of F-actin, without altering the cage assembly. We also discovered that the Arp2/3-dependent branched filaments control the integration through Ezrin, a membrane tension regulator that links F-actin to the membranes. To further understand the modality of recruitment of NMII on the secretory granules, we investigated selected molecules, chosen among those identified in a proteomic screening of 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 organized into cage-like cages which co-align with the NMII and F-actin cages. Knock-in mice expressing fluorescently tagged versions of SEPT2 and SEPT7 validated these 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 septins recruitment. 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. Accordingly, 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 define the underlying mechanisms of the coordination among PM remodeling and actomyosin cytoskeleton during interstitial neutrophil migration in a mouse ear model of sterile injury. Migrating neutrophils exhibit very dynamic membrane remodeling with the continuous formation of micron-scale membrane protrusions at the PM, which interact with the tissue microenvironment. Differently from what previously described in 2D model systems, NMIIA localized in protrusions at the leading edge, where it organizes in lattices composed of the same triskelia units forming the cages in secretory granules. NMIIA is recruited at the leading edge though a mechanism that is RhoA/ROCK independent and it is controlled by the activation of PI3-kinase. We discovered that pharmacological inhibition of PI3-kinase disrupts the assembly of NMIIA lattices and specifically of the triskelia at the leading edge of the migrating neutrophils. This resulted in the destabilization of the membrane protrusions and loss of directionality. This modality of neutrophils migration occurs also during inflammation and tumor progression, but not during injuries involving vasculature breakage.