Molecular mechanisms of membrane remodeling

膜重塑的分子机制

• 批准号: 10014763
• 负责人: Roberto Weigert
• 金额: $ 159.81万
• 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/10014763
• 关键词: 5' -AMP-activated protein kinase; Acinar Cell; Actins; Actomyosin; Adrenergic Agents; Adult; Apical; Biological Models; Biophysics; Caliber; Cell Culture Techniques; Cell membrane; Cells; Cellular Membrane; Cellular Metabolic Process; Characteristics; Clathrin; Complement; Complex; Confocal Microscopy; Convection; Cyclic AMP-Dependent Protein Kinases; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; Data; Data Set; Development; Embryo; Endocytic Vesicle; Endocytosis; Environment; Epithelial; Epithelium; Event; Exocytosis; F-Actin; Filament; Fluids and Secretions; G-Protein-Coupled Receptors; Goals; Homeostasis; Image; Individual; Investigation; Lateral; Length; Link; Lipids; Maintenance; Mediating; Membrane; Metabolism; Microfilaments; Microscope; Microscopy; Mitochondria; Mitochondrial Proton-Translocating ATPases; Modeling; Molecular; Molecular Genetics; Monomeric GTP-Binding Proteins; Mus; Myosin Type II; Neoplasm Metastasis; Organ; Organ Culture Techniques; Organism; Pancreas; Pathologic; Pathway interactions; Pharmacology; Phase; Physiological; Play; Population; Process; Protein Isoforms; Protein Kinase; Protein Secretion; Proteins; Regulation; Resolution; Rodent; Role; Salivary Glands; Secretory Vesicles; Series; Shapes; Signal Transduction; Structure; Surface; System; Techniques; Time; Tissues; Tubular formation; base; beta-adrenergic receptor; cell motility; crosslink; density; improved; in vivo; light microscopy; mitochondrial metabolism; non-muscle myosin; novel; organizational structure; polymerization; prevent; recruit; secretory protein; sensor; spatiotemporal; temporal measurement; tool; trafficking; trans-Golgi Network; tumor progression; two photon microscopy; two-photon

Molecular basis of membrane remodeling during secretion at the plasma membrane. Secretory epithelia such as salivary glands and pancreas represent a robust model system to study various aspects of the remodeling of membranes during intracellular trafficking processes, such as regulated protein-secretion and plasma membrane homeostasis. 1) Regulated exocytosis in salivary glands. In salivary glands acinar cells, secretory proteins are packed in large granules at the trans-Golgi network (TGN) and transported to the cell periphery where they fuse with the apical plasma membrane (APM) upon stimulation of G-Protein coupled receptors, thus releasing their content into the acinar canaliculi. Concomitantly, the membranes of the secretory granules gradually integrate into the APM, thus undergoing substantial remodeling. Our aim is to elucidate the molecular machinery regulating the integration of the secretory granules with the APM. To this end, we developed an experimental system in live rodents aimed at imaging and tracking individual secretory granules. We established that upon stimulation of the beta-adrenergic receptor, the granules fuse with the APM, followed, after a short delay, by the recruitment of a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB). We showed that actomyosin contractile activity regulates the integration of the granular membranes into the APM and the completion of exocytosis. Modeling of this process based on the EM ultrastructural analysis of the secretory granules and the APM, revealed that the integration is energetically unfavorable, since it is constantly opposed by a convective flow of membranes directed from the APM to the granule membranes. This process is driven by the fact the membrane tension of the APM bilayer is higher than that of the secretory granules membranes. In order to understand how the actomyosin complex drives the integration, we focused on determining how F-actin and NMII are structurally arranged on the secretory granules. To this end, we used a series of selected light microscopy techniques with higher resolution than conventional confocal and two-photon microscopy, such as Spinning Disk and Stimulated Emission Depletion Microscopy (STED). Strikingly, we discovered that both F-actin and NMII assemble around the secretory granules in distinct polyhedral cages, formed by pentagonal and hexagonal units like those described for clathrin around the endocytic vesicles, although one order of magnitude larger. This represents a novel structural organization for the actomyosin cytoskeleton, never described before. Our data suggested that the NMII cage could function to crosslink actin filaments and/or transmit the forces generated by the contractile activity to the F-actin cage, and therefore to the granules membranes. Notably, the improved temporal resolution afforded by the spinning disc microscope, 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 which maintain constant diameter and fixed shape (assembly phase). This step is followed by 1) the rapid polymerization of F-actin directed from the actomyosin cage towards the granule membranes (compression phase), and 2) the increase of the surface density of the NMII molecules (contractile phase). Finally, both cages disassemble with NMII being released in large filaments. 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 and prevent compound exocytosis; second, F-actin polymerization generates forces that drive the integration, using the cage as a leverage to push the membranes toward the APM; and third, NMIIA-driven contractions generate additional forces to facilitate the integration. In addition, we further confirmed that both the F-actin and NMII cages are assembled independently. These results provide a springboard to begin investigating the biophysical basis underlying the process of membrane integration. 2) Apical plasma membrane homeostasis in salivary glands In tubular organs, lumens are formed by the APM. Their establishment and maintenance is fundamental for their physiological function. Most of the studies investigating the mechanisms regulating this process have been carried out in cell cultures or in smaller organisms, whereas little has been done in in vivo mammalian model systems. We used the salivary glands of live mice to examine the role of played by the small GTPase Cdc42 in the regulation of the homeostasis of the intercellular canaliculi, a specialized apical domain of the acinar cells, where protein and fluid secretion occur. We found that in adult mice, depletion of Cdc42 induced: 1) significant expansion of the APM, 2) increase in the length of the lateral membranes, and 3) robust stimulation of endocytic trafficking both at the basolateral and the APM, which did not affect plasma membrane identity and junctional integrity. On the other hand, Cdc42-depletion at late embryonic stages resulted in 1) complete inhibition of the post-natal development of the intercellular canaliculi, and 2) stimulation of endocytic trafficking. Overall, these results show that Cdc42 plays a fundamental role in regulating both development and maintenance of the epithelial lumen in vivo, and highlight an additional role of Cdc42 in membrane remodeling, as negative regulator of endocytic trafficking pathways. 3) Coordination between cell metabolism and membrane remodeling. Membrane remodeling is an energetically unfavorable process. During protein secretion, significant energy is required for a single exocytic event to: 1) bring together and fuse a secretory granule and the PM, as estimated for various systems and integrate the granule membranes into the APM and maintain its homeostasis. We sought to determine how cellular metabolism is linked to protein secretion. First, we confirmed that regulated exocytosis is dependent on mitochondrial metabolism. Indeed, granule fusion and integration required the activity of the mitochondrial ATP-synthase, whereas they were independent from the activity of complex I. This finding suggested that beta-adrenergic signaling altered mitochondrial metabolism in a novel fashion. Second, we discovered that in vivo there are two spatially distinct populations of mitochondria in the acinar cells: one localized at the basolateral PM, and the other dispersed throughout the cytoplasm. Third, we discovered that the onset of exocytosis triggered the depletion of the intracellular ATP pool, as revealed by the fast activation of the cytosolic energy sensor, AMP-activated protein kinase (AMPK). AMPK has been shown to upregulate mitochondrial metabolism and homeostasis, and consistently, we discovered that the cytoplasmic pool of mitochondria increased motility and size. This last feature has been associated with an increase in the mitochondrial capacity to produce ATP. Notably, we found that, the increase in size of the individual mitochondria was mediated by the PKA-dependent inhibition of the activity of the mitochondrial fission protein DRP1. These studies underscore the importance of studying the spatiotemporal regulation of mitochondrial structure and function in intact tissues in vivo and they will open new avenues in the coordination of cell metabolism and remodeling.

质膜分泌过程中膜重塑的分子基础。分泌性上皮（例如唾液腺和胰腺）代表了一个可靠的模型系统，可在细胞内运输过程中研究膜重塑的各个方面，例如调节的蛋白质 - 分泌和质膜膜稳态。 1）调节唾液腺中的胞吐作用。在唾液腺腺泡细胞中，分泌蛋白在反式高尔基网络（TGN）的大颗粒中堆积，并在刺激G蛋白耦合受体的刺激后将其与顶端质膜（APM）融合到细胞周围，从而将其内容释放到acinar CanaliCuli中。同时，分泌颗粒的膜逐渐整合到APM中，从而进行了大量的重塑。我们的目的是阐明调节分泌颗粒与APM的整合的分子机械。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，在刺激β-肾上腺素受体的刺激后，将颗粒与APM融合，然后在短延迟后，通过募集了由F-肌动蛋白组成的复合物和两种由非肌肉肌球蛋白II（NMIIA和NMIIB）组成的复合物。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。基于对分泌颗粒和APM的EM超微结构分析进行的该过程的建模表明，整合在能量上是不利的，因为它不断地反对从APM到颗粒膜的对流膜流动。该过程是由APM双层的膜张力高于分泌颗粒膜的事实。为了了解肌动球蛋白复合物如何驱动整合，我们专注于确定F-肌动蛋白和NMII在结构上是如何在分泌颗粒上排列的。为此，我们使用了一系列选择的光学显微镜技术，其分辨率高于常规共聚焦和两光子显微镜，例如旋转盘和刺激的发射消耗显微镜（STED）。令人惊讶的是，我们发现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上，这些笼子由五角形和六边形单元（如五角形和六角形单元）形成，例如围绕内吞囊泡周围的网状蛋白所描述的单位，尽管一个较大的顺序较大。这代表了肌动菌素细胞骨架的新结构组织，从未描述过。我们的数据表明，NMII笼可以起作用，可以交叉链接肌动蛋白丝和/或将收缩活性产生的力传递给F-肌动蛋白笼，从而将其转移到颗粒膜中。值得注意的是，旋转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逐渐募集到稳定的笼子中，这些笼子保持恒定直径和固定形状（装配阶段）。此步骤之后是1）F-肌动蛋白从肌动球蛋白笼子朝向颗粒膜的快速聚合（压缩阶段），以及2）NMII分子（收缩阶段）的表面密度的增加。最终，两个笼子都拆卸，NMII以大细丝释放。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，肌动蛋白笼子抵消了脂质从APM中的对流流，并防止复合胞吐作用。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，将膜推向APM。第三，NMIIA驱动的收缩产生了额外的力量以促进整合。此外，我们进一步证实了F-肌动蛋白和NMII笼均独立组装。这些结果提供了一个跳板，开始研究膜整合过程的生物物理基础。 2）垂体器官中的唾液腺中的顶质膜稳态，腔由APM形成。他们的建立和维护对于他们的生理功能至关重要。研究调节此过程的机制的大多数研究都是在细胞培养物或较小的生物中进行的，而在体内哺乳动物模型系统中几乎没有进行。我们使用活小鼠的唾液腺检查小型GTPase CDC42在调节细胞间管的体内稳态中扮演的作用，这是腺泡细胞的专门顶端结构域，蛋白质和流体分泌发生。我们发现，在成年小鼠中，Cdc42的耗竭诱导：1）APM的显着膨胀，2）增加侧膜的长度，3）3）强大的刺激基底外侧和APM的内吞运输，这不影响质膜膜的识别和连接完整性。另一方面，晚期胚胎阶段的cdc42止动物1）完全抑制了细胞间管的产后发育，以及2）刺激内吞运输的刺激。总体而言，这些结果表明，CDC42在体内调节上皮管腔的开发和维持中起着基本作用，并突出了Cdc42在膜重塑中的额外作用，是内吞运输途径的负调节剂。 3）细胞代谢和膜重塑之间的协调。膜重塑是一个充满能量的过程。在蛋白质分泌过程中，单个外囊肿事件需要大量能量，以：1）将分泌物颗粒和PM融合在一起，如估计各种系统，并将颗粒膜整合到APM中并保持其稳态。我们试图确定细胞代谢如何与蛋白质分泌有关。首先，我们证实调节的胞吐作用取决于线粒体代谢。实际上，颗粒融合和整合需要线粒体ATP合并酶的活性，而它们独立于复杂的活性I。这一发现表明β-肾上腺素能信号传导以新颖的方式改变了线粒体代谢。其次，我们发现在体内存在两个在空间上不同的线粒体中的线粒体：一种位于基底外侧PM，另一个位于整个细胞质中。第三，我们发现胞吐作用的发作引发了细胞内ATP池的耗竭，如胞质能传感器，AMP激活的蛋白激酶（AMPK）的快速激活所揭示的那样。 AMPK已被证明可以上调线粒体代谢和稳态，并且一致地，我们发现线粒体的细胞质池增加了运动性和大小。最后一个特征与线粒体产生ATP的能力的增加有关。值得注意的是，我们发现，单个线粒体的大小增加是由PKA依赖性抑制线粒体裂变蛋白DRP1介导的。这些研究强调了研究体内完整组织中线粒体结构和功能的时空调节的重要性，它们将在细胞代谢和重塑的协调方面开放新的途径。