Molecular mechanisms of membrane remodeling

膜重塑的分子机制

• 批准号: 10486914
• 负责人: Roberto Weigert
• 金额: $ 166.03万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486914
• 关键词: Ablation; Acinar Cell; Actins; Actomyosin; Address; Adhesions; Adult; Animals; Apical; Back; Biological Process; Blood Vessels; CD18 Antigens; Caliber; Cell Adhesion; Cell Culture Techniques; Cell membrane; Cells; Cellular Membrane; Cellular Metabolic Process; Characteristics; Chemotactic Factors; Communication; Complement; Complex; Computing Methodologies; Confocal Microscopy; Convection; Coupled; Cytoplasmic Granules; Cytoskeleton; Data; Data Set; Development; EMS1 gene; Ear; Eicosanoids; Endocytosis; Endothelium; Environment; Event; Exhibits; Exocrine Glands; Exocrine pancreas; Exocytosis; Extracellular Matrix; Extravasation; F-Actin; Family; Filament; Generations; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Image; Immune response; Impairment; Indirect Immunofluorescence; Individual; Inflammation; Injury; Investigation; Knock-in Mouse; LTB4R gene; Lateral; Lead; Leukotriene B4; Lipids; Location; Machine Learning; Mediating; Membrane; Metabolism; Microfilaments; Microscopy; Modality; Modeling; Molecular; Molecular Genetics; Mus; Myosin Light Chain Kinase; Myosin Type II; Neoplasm Metastasis; Organ Culture Techniques; Paracrine Communication; Pathologic; Pharmacology; Phosphorylation; Physiological; Play; Process; Production; Protein Isoforms; Proteins; Proteomics; Recycling; Regulation; Resolution; Rodent; Role; Salivary Glands; Secretory Vesicles; Series; Shapes; Signal Transduction; Site; Speed; Sterility; Surface; System; Techniques; Time; Tissues; Vascular Endothelium; Wasps; Work; Wound Infection; autocrine; base; cell motility; crosslink; density; improved; in vivo; interstitial; light microscopy; mechanical force; member; migration; neutrophil; non-muscle myosin; novel; polymerization; protein kinase C zeta; receptor; recruit; response; response to injury; scaffold; screening; secretory protein; spatiotemporal; temporal measurement; tool; trafficking; tumor progression; tumorigenesis; two-photon

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.

质膜分泌过程中膜重塑的分子基础（PM）。外分泌腺中调节的胞吐作用。在外分泌腺的腺泡细胞中（即唾液腺，外分泌胰腺），分泌蛋白包装在大型颗粒中，这些颗粒被运输到细胞外围，它们将其与顶质质膜（APM）融合在受体刺激后，并将其内容释放到acinar Canaliculi中。同时，分泌颗粒的膜逐渐整合到经过重大重塑的APM中。我们旨在阐明调节此过程的分子机械。为此，我们在活啮齿动物中开发了一个实验系统，旨在成像和跟踪单个分泌颗粒。我们确定颗粒与APM融合，在短延迟后，由F-肌动蛋白和两种非肌肉肌球蛋白II（NMIIA和NMIIB）组成的复合物在其膜上募集。我们表明，肌动球蛋白收缩活性调节颗粒状膜的整合到APM中并完成胞吐作用。我们在体内使用超分辨率显微镜，我们发现F-肌动蛋白和NMII均以不同的多面体笼子在分泌颗粒周围组装，由五边形和六角形单位形成。 NMII笼子交联肌动蛋白丝，并将NMII收缩活性产生的力传输到F-肌动蛋白笼，从而将其传输到颗粒膜。值得注意的是，通过旋转光盘显微镜提供的改进的时间分辨率使我们能够在体内集成过程中首次捕获笼子动力学的4D数据集。这表明F-肌动蛋白和NMII逐渐募集到保持恒定直径和固定形状的稳定笼中。此步骤之后是1）F-肌动蛋白从肌动蛋白笼子朝向颗粒膜的快速聚合，以及2）NMII分子的表面密度的增加。我们的数据支持基于多步骤过程的新型模型，在该过程中，首先，Actomyosin Cages抵消了APM中脂质的对流流动。其次，F-肌动蛋白聚合产生驱动整合的力，使用笼子作为杠杆作用，将膜推向APM。第三，NMII驱动的收缩产生了额外的力量以促进整合。此外，我们确定F-肌动蛋白和NMII笼都是独立组装的。具体而言，F-肌动蛋白分为两个步骤。首先，通过MDIA1/2的激活组装了初始的F-肌动蛋白笼，这是肌动蛋白成核的两个成员，它们产生线性丝。其次，通过激活ARP2/3复合物，N-WASP和Cortactin组装分支细丝。 MDIA1/2或ARP2/3复合物的药理或遗传消融破坏了分泌颗粒的整合到APM中。为了阐明NMII在分泌颗粒上的募集和调节的模态，我们研究了选定的分子，这些分子是在与纯化分泌颗粒相关的蛋白质蛋白初步蛋白质筛选中所鉴定的。使用超分辨率显微镜和间接免疫荧光，我们发现GTPase的SEPTIN家族的3个成员，以及Septins 2、6和7（Sept2，Sept2，Sept6和Sept7），存在于熔融颗粒的表面上，也存在于笼子中的笼子中，这些固定片类似于Cage类似于cage ocage of cage-lige nmii nmii nMii。表达荧光标记的Sept2和Sept7的敲入小鼠的生成验证了这一发现。对SEPT2的药理抑制作用导致活化的NMII和肌球蛋白轻链激酶（MLCK）的水平显着降低，而F-肌动蛋白组装的破坏会导致颗粒大小的扩大而不会损害NMII或septin的锻炼。最后，成年小鼠腺泡细胞中SEPT7的遗传消融损害了分泌颗粒的整合和表面上的肌动蛋白水平。基于我们的数据，我们建议新观察到的septins笼子：1）提供脚手架来募集和曲线表面上分泌颗粒的肌动蛋白丝，而2）可能通过MLCK介导的磷酸化来激活NMII。活动物中嗜中性粒细胞迁移期间膜重塑的机制。细胞迁移是一个基本的生物学过程，需要通过肌动蛋白细胞骨架的恒定重新安排膜重塑。由于中性粒细胞在免疫反应和肿瘤进展中的作用，对中性粒细胞的迁移进行了特别研究。在生理条件下，中性粒细胞在脉管系统中循环，而在病理状态（例如伤口，感染，炎症，肿瘤发生）期间，化学吸引者梯度的产生促进了其对血管内皮的粘附，并将其渗出到间质中，并最终朝向目标部位。 1）嗜中性粒细胞渗入过程中肌动菌素细胞骨架的作用和调节。 eicosanoid白细胞B4（LTB4）传递趋化信号，以通过其受体BLT1响应损伤而导致中性粒细胞迁移。然而，在体内尚未在体内解决LTB4-BLT1轴是否在血管内中性粒细胞反应期间调节肌动蛋白细胞骨架。因此，我们在小鼠脚下开发了一个炎症模型，以直接可视化LTB4-BLT1轴对血管内中性粒细胞动力学的影响。我们发现，中性粒细胞生产的LTB4通过BLT1充当自分泌/旁分泌信号，以推动其招募，逮捕和渗出。我们发现LTB4在中性粒细胞停滞期间引起细胞粘附和极化。具体而言，LTB4信号传导通过控制beta2- integrin（ITGB2）向PM面对中性粒细胞 - 内皮粒细胞界面的运输来控制NMIIA向细胞背部的动态重新分布，以促进其收缩活性。 2）通过产生在中性粒细胞穿过血管壁时形成中性粒细胞膜所需的机械力来驱动渗出。与这些发现一致，我们观察到阻断LTB4信号传导或NMIIA激活会抑制ITGB2回收到PM。总体而言，我们的研究揭示了LTB4在促进脉管中嗜中性粒细胞通信在对炎症的早期反应过程中通过控制肌动肌球蛋白细胞骨架的信号传导电路的至关重要功能。 2）肌动球蛋白细胞骨架在体内迁移期间的作用。我们已经使用了与新型计算方法耦合的ISMIC，以了解PM重塑，肌动蛋白细胞骨架和细胞代谢之间在小鼠耳朵损伤的小鼠耳朵模型中的基础机制。迁移的嗜中性粒细胞表现出非常动态的膜重塑，并连续形成微米尺度的膜突起，并与组织微环境相互作用（即细胞外基质）。与先前描述的不同，我们发现NMIIA不仅存在于细胞的uropod上，而且存在于前缘和大型侧向突起。在这些新位置，NMIIA不会主动缩回膜，而是为与ECM的相互作用而稳定它们。此外，我们发现，NMIIA领先地位的NMIIA募集是Rhoa/Rock独立的，并且由PKC-Zeta的激活控制，表明中性粒细胞中NMIIA的募集方式是新的。 PKC-ZETA的药理抑制作用减少了迁移中性粒细胞的前沿NMIIA的募集，破坏了膜的突起，并诱发了方向性和迁移速度的丧失。