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（PM）分泌过程中膜重塑的分子基础。调节外分泌腺的胞吐作用。在外分泌腺（即唾液腺、外分泌胰腺）的腺泡细胞中，分泌蛋白被包装成大颗粒，被运输到细胞外围，在受体刺激下与顶端质膜（APM）融合，并将其内容物释放到细胞中。腺泡小管。与此同时，分泌颗粒的膜逐渐融入 APM 中，经历实质性重塑。我们的目标是阐明调节这一过程的分子机制。为此，我们在活啮齿动物中开发了一个实验系统，旨在成像和跟踪个体分泌颗粒。我们确定颗粒与 APM 融合，并在短暂延迟后，由 F-肌动蛋白和两种非肌肉肌球蛋白 II 亚型（NMIIA 和 NMIIB）组成的复合物在其膜上募集。我们发现肌动球蛋白收缩活性调节颗粒膜与 APM 的整合以及胞吐作用的完成。使用体内超分辨率显微镜，我们发现 F-肌动蛋白和 NMII 都在由五边形和六边形单元形成的不同多面体笼中围绕分泌颗粒组装。 NMII 笼交联肌动蛋白丝，并将 NMII 收缩活动产生的力传递到 F-肌动蛋白笼，从而传递到颗粒膜。值得注意的是，旋转圆盘显微镜提供的改进的时间分辨率使我们能够首次捕获体内整合过程中笼子动力学的 4D 数据集。这表明 F-肌动蛋白和 NMII 逐渐被招募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-肌动蛋白从肌动球蛋白笼定向到颗粒膜的快速聚合，以及 2) NMII 分子表面密度的增加。我们的数据支持基于多步骤过程的新模型，其中首先，肌动球蛋白笼抵消来自 APM 的脂质对流；其次，F-肌动蛋白聚合产生驱动整合的力，利用笼作为杠杆将膜推向 APM；第三，NMII 驱动的收缩会产生额外的力量来促进整合。此外，我们确定 F-肌动蛋白和 NMII 笼都是独立组装的。具体来说，F-肌动蛋白的组装分两步进行。首先，通过激活 mDia1/2 组装初始 F-肌动蛋白笼，mDia1/2 是肌动蛋白成核剂 Formin 家族的两个成员，可产生线性丝。其次，通过激活 Arp2/3 复合体、N-Wasp 和 cortactin 来组装分支丝。 mDia1/2 或 Arp2/3 复合体的药理学或遗传消融会破坏分泌颗粒与 APM 的整合。为了阐明 NMII 在分泌颗粒上的募集和调节方式，我们研究了选定的分子，这些分子是在对与纯化的分泌颗粒相关的蛋白质进行初步蛋白质组筛选时鉴定出的分子中选择的。使用超分辨率显微镜和间接免疫荧光，我们发现 GTPase Septin 家族的 3 个成员，即 septin 2、6 和 7（SEPT2、SEPT6 和 SEPT7）存在于融合颗粒的表面，并且也存在组织成与 NMII 共同对齐的笼状笼子。表达荧光标记版本的 SEPT2 和 SEPT7 的敲入小鼠的产生验证了这一发现。 SEPT2 的药理学抑制导致融合颗粒上活化的 NMII 和肌球蛋白轻链激酶 (MLCK) 水平显着降低，而 F-肌动蛋白组装的破坏导致颗粒尺寸扩大，但不损害 NMII 或隔膜招募。最后，成年小鼠腺泡细胞中 SEPT7 的基因消除会损害分泌颗粒的整合及其表面肌动球蛋白的水平。根据我们的数据，我们提出新观察到的脓毒症笼：1）提供了一个支架，以募集和弯曲分泌颗粒表面的肌动球蛋白丝，2）可能是通过 MLCK 激活 NMII 所需要的。介导的磷酸化。活体动物中性粒细胞迁移过程中膜重塑的机制。细胞迁移是一个基本的生物过程，需要通过肌动球蛋白细胞骨架的不断重新排列来进行膜重塑。中性粒细胞迁移因其在免疫反应和肿瘤进展中的作用而受到特别研究。在生理条件下，中性粒细胞在脉管系统中循环，而在病理状态下（例如伤口、感染、炎症、肿瘤发生），化学引诱剂梯度的产生促进它们与血管内皮的粘附，外渗到间质中，并最终定向向血管内迁移。目标站点。 1）肌动球蛋白细胞骨架在中性粒细胞外渗过程中的作用和调节。类花生酸白三烯 B4 (LTB4) 通过其受体 BLT1 传递趋化信号，引导中性粒细胞间质迁移，以响应损伤。然而，LTB4-BLT1 轴是否在血管内中性粒细胞反应期间调节肌动球蛋白细胞骨架尚未在体内得到解决。因此，我们在小鼠足垫中开发了炎症模型，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产生的 LTB4 通过 BLT1 作为自分泌/旁分泌信号来驱动其募集、停滞和外渗。我们发现 LTB4 在中性粒细胞停滞期间引发细胞粘附和极化。具体来说，LTB4 信号传导协调 NMIIA 到细胞背面的动态重新分配，其中需要其收缩活性来 1) 通过控制 β2-整合素 (Itgb2) 向面向中性粒细胞的 PM 的转运，促进中性粒细胞停滞和粘附。内皮界面； 2）当中性粒细胞穿过血管壁时，通过产生使中性粒细胞膜变形所需的机械力来驱动外渗。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 再循环至 PM。总体而言，我们的研究揭示了 LTB4 在炎症早期反应过程中通过激活控制肌动球蛋白细胞骨架的信号通路来促进脉管系统中中性粒细胞通讯的关键功能。 2)肌动球蛋白细胞骨架在体内间质迁移过程中的作用。我们使用 ISMic 与新颖的计算方法相结合，以了解无菌损伤小鼠耳模型中间质中性粒细胞迁移过程中 PM 重塑、肌动球蛋白细胞骨架和细胞代谢之间协调的潜在机制。迁移的中性粒细胞表现出非常动态的膜重塑，连续形成微米级膜突起，与组织微环境（即细胞外基质）相互作用。与之前描述的不同，我们发现 NMIIA 不仅存在于细胞的尾足，而且还存在于前缘和大的侧向突起处。在这些新位置，NMIIA 不会主动收缩膜，而是稳定膜以响应与 ECM 的相互作用。此外，我们发现前沿的 NMIIA 募集与 RhoA/ROCK 无关，并且受 PKC-zeta 激活控制，表明中性粒细胞中 NMIIA 募集的新方式。 PKC-zeta 的药理抑制可减少迁移中性粒细胞前缘 NMIIA 的募集，破坏膜突出的稳定性并导致方向性和迁移速度丧失。