Molecular mechanisms of membrane remodeling

膜重塑的分子机制

• 批准号: 10926269
• 负责人: Roberto Weigert
• 金额: $ 227.95万
• 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/10926269
• 关键词: Ablation; Acinar Cell; Actins; Actomyosin; Address; Adhesions; Adult; Animals; Apical; Back; Biological Models; Biological Process; Blood Vessels; CD18 Antigens; Cell Adhesion; Cell Culture Techniques; Cell membrane; Cells; Cellular Membrane; Characteristics; Chemotactic Factors; Communication; Complement; Complex; Computing Methodologies; Confocal Microscopy; Coupled; Cytoplasmic Granules; Cytoskeleton; Data; Data Analyses; Development; Diameter; Ear; Eicosanoids; Endocytosis; Endothelium; Environment; Event; Exhibits; Exocrine Glands; Exocytosis; Extravasation; F-Actin; Family; Filament; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Image; Immune response; Impairment; Indirect Immunofluorescence; Individual; Infection; Inflammation; Injury; Invaded; Investigation; Knock-in Mouse; LTB4R gene; Lead; Leukotriene B4; Link; Lipids; Machine Learning; Mediating; Membrane; Metabolism; Microscopy; Modality; Modeling; Molecular; Mus; Myosin Light Chain Kinase; Myosin Type II; Neoplasm Metastasis; Organ Culture Techniques; PIK3CG gene; Paracrine Communication; Pathologic; Phosphorylation; Physiological; Play; Polymers; Process; Production; Protein Isoforms; Proteins; Proteomics; Recycling; Regulation; Resolution; Rodent; Role; Secretory Vesicles; Series; Shapes; Signal Transduction; Site; Sterility; Surface; System; Techniques; Tissues; Vascular Endothelium; Visualization; autocrine; cell motility; crosslink; density; ezrin; in vivo; interstitial; light microscopy; mechanical force; member; migration; neutrophil; non-muscle myosin; novel; pharmacologic; polymerization; receptor; recruit; response; response to injury; scaffold; screening; secretory protein; spatiotemporal; superresolution microscopy; tool; trafficking; transmission process; tumor progression; tumorigenesis; two-photon; wound

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.

质膜分泌过程中膜重塑的分子基础。在外分泌腺的腺泡细胞中，分泌蛋白被包装在大型颗粒中，与受体刺激后与顶端质膜（APM）融合在一起，并将其含量释放到腺泡管中。分泌颗粒的膜整合到经过重大重塑的APM中。我们旨在阐明调节此过程的机械。为此，我们在活小鼠中开发了一种实验系统，该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，并在其膜上募集由F-肌动蛋白和两种非肌肉肌球蛋白II（NMIIA和NMIIB）组成的同工型。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。我们表明，F-肌动蛋白和NMII都在分泌颗粒周围组装在不同的多面体笼子中，由Triskelion样单位形成。 NMII CAGE交叉链接F-肌动蛋白并将NMII收缩活性产生的力传输到F-肌动蛋白笼，因此向颗粒的膜。高分辨率的延时成像表明，最初，F-肌动蛋白和NMII被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。这支持了基于多步骤过程的模型，在该过程中，i）肌动蛋白笼子抵消了APM的脂质的对流流动； ii）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； iii）NMII驱动的收缩产生了额外的力量来促进整合。我们确定F-肌动蛋白和NMII笼子是独立组装的。首先，F-肌动蛋白笼是通过线性肌动蛋白核定剂的Formin家族的成员MDIA1的激活组装的。其次，通过激活ARP2/3复合物组装分支细丝。 MDIA1的药理或遗传消融破坏了笼子的组装，并导致熔融颗粒的扩张。另一方面，ARP2/3复合物的药理或遗传破坏延迟了分泌颗粒在APM中的整合，并抑制F-肌动蛋白的内向聚合，而无需改变笼子组件。我们还发现，ARP2/3依赖性分支细丝通过将F-肌动蛋白连接到膜的膜张力调节剂Ezrin（Ezrin）来控制整合。为了进一步了解NMII在分泌颗粒上募集的方式，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筛选中鉴定出的蛋白质中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，Septin GTPase家族的3个成员，以及Septins 2、6和7（Sept2，Sept2，Sept6和Sept7），存在于融合的颗粒表面上，并组织成与Nmii和F-Actirin cages的笼子样的笼子，并被组织到类似于NMII和F-Actirin cages的笼子样箱中。表达荧光标记的Sept2和Sept7版本的敲入小鼠验证了这些发现。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SESTINS招募。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及其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）的情况下，化学吸引者梯度的产生促进了其对血管内皮的粘附，将其渗出，渗出到间质中，最终导向目标部位。 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。因此，我们观察到阻塞LTB4信号传导或NMIIA激活抑制了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ISMIC与新型计算方法耦合，以定义PM重塑和肌动球蛋白细胞骨架之间在间质性嗜中性粒细胞迁移过程中的基础机制。迁移的中性粒细胞表现出非常动态的膜重塑，并在PM处的微米尺度膜突起的连续形成，并与组织微环境相互作用。与先前在2D模型系统中所描述的不同，NMIIA位于前缘的突起中，在该突起中，它以由相同的Triskelia单位组成的晶格组织，形成了分泌颗粒的笼子。 NMIIA是在Rhoa/Rock独立的机制中募集的，它受PI3-激酶的激活控制。我们发现，PI3-激酶的药理抑制会破坏NMIIA晶格的组装，特别是在迁移的中性粒细胞前缘的Triskelia的组装。这导致膜突起的稳定和方向性丧失。中性粒细胞的这种方式迁移也发生在炎症和肿瘤进展过程中，但在涉及脉管系统破裂的损伤期间不发生。

项目成果

The LTB4-BLT1 axis regulates actomyosin and β2-integrin dynamics during neutrophil extravasation.

• DOI: 10.1083/jcb.201910215
• 发表时间: 2020-10-05
• 期刊: The Journal of cell biology
• 作者: Subramanian BC;Melis N;Chen D;Wang W;Gallardo D;Weigert R;Parent CA
• 通讯作者: Parent CA

Dynamic polyhedral actomyosin lattices remodel micron-scale curved membranes during exocytosis in live mice.

• DOI: 10.1038/s41556-019-0365-7
• 发表时间: 2019-08
• 期刊: Nature cell biology
• 影响因子: 21.3
• 作者: Ebrahim S;Chen D;Weiss M;Malec L;Ng Y;Rebustini I;Krystofiak E;Hu L;Liu J;Masedunskas A;Hardeman E;Gunning P;Kachar B;Weigert R
• 通讯作者: Weigert R

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions.

• DOI: 10.3791/63581
• 发表时间: 2022-05-10
• 期刊: Journal of visualized experiments : JoVE
• 作者: Melis N;Subramanian B;Chen D;Weigert R
• 通讯作者: Weigert R

Nanoarchitecture and dynamics of the mouse enteric glycocalyx examined by freeze-etching electron tomography and intravital microscopy.

通过冷冻蚀刻电子断层扫描和活体显微镜检查小鼠肠糖萼的纳米结构和动力学。

• DOI: 10.1038/s42003-019-0735-5
• 发表时间: 2020
• 期刊: Communications biology
• 影响因子: 5.9
• 作者: Sun,WillyW;Krystofiak,EvanS;Leo-Macias,Alejandra;Cui,Runjia;Sesso,Antonio;Weigert,Roberto;Ebrahim,Seham;Kachar,Bechara
• 通讯作者: Kachar,Bechara

Cdc42 controls secretory granules morphology in rodent salivary glands in vivo

• DOI: 10.1080/19420889.2020.1724605
• 发表时间: 2020-01
• 期刊: Communicative & Integrative Biology
• 作者: Akiko Shitara;Christopher K. E. Bleck;R. Weigert