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。唾液腺和胰腺等分泌上皮细胞代表了一个强大的模型系统，可用于研究细胞内运输过程中膜重塑的各个方面，例如调节的蛋白质分泌和质膜稳态。 1）调节唾液腺的胞吐作用。在唾液腺腺泡细胞中，分泌蛋白在跨高尔基体网络 (TGN) 处堆积成大颗粒，并转运至细胞外周，在刺激 G 蛋白偶联受体后与顶端质膜 (APM) 融合，从而释放它们的内容物进入腺泡小管。与此同时，分泌颗粒的膜逐渐融入 APM，从而经历实质性的重塑。我们的目标是阐明调节分泌颗粒与 APM 整合的分子机制。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，在刺激 β-肾上腺素能受体时，颗粒与 APM 融合，短暂延迟后，募集由 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。 ）。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。基于分泌颗粒和 APM 的 EM 超微结构分析对这一过程进行建模，表明这种整合在能量上是不利的，因为它不断受到从 APM 引导到颗粒膜的膜对流的阻碍。该过程是由 APM 双层的膜张力高于分泌颗粒膜的膜张力这一事实驱动的。为了了解肌动球蛋白复合物如何驱动整合，我们重点确定 F-肌动蛋白和 NMII 在分泌颗粒上的结构排列方式。为此，我们使用了一系列比传统共焦和双光子显微镜具有更高分辨率的选定光学显微镜技术，例如转盘和受激发射损耗显微镜（STED）。引人注目的是，我们发现 F-肌动蛋白和 NMII 都在不同的多面体笼中围绕分泌颗粒组装，这些多面体笼由五边形和六边形单元形成，类似于内吞囊泡周围网格蛋白的那些单元，尽管要大一个数量级。这代表了肌动球蛋白细胞骨架的一种新颖的结构组织，以前从未被描述过。我们的数据表明，NMII 笼可以起到交联肌动蛋白丝和/或将收缩活动产生的力传递到 F-肌动蛋白笼，从而传递到颗粒膜的作用。值得注意的是，转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集成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（组装阶段）。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（压缩阶段），以及 2) NMII 分子表面密度的增加（收缩阶段）。最后，两个笼子都会分解，NMII 会以大丝的形式释放出来。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 APM 中脂质的对流流动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，利用笼作为杠杆将膜推向 APM；第三，NMIIA 驱动的收缩产生了额外的力量来促进整合。此外，我们进一步证实F-肌动蛋白和NMII笼是独立组装的。这些结果为开始研究膜整合过程的生物物理基础提供了一个跳板。 2) 唾液腺顶端质膜稳态 在管状器官中，管腔由 APM 形成。它们的建立和维持对其生理功能至关重要。大多数研究调节这一过程的机制都是在细胞培养物或较小的生物体中进行的，而在体内哺乳动物模型系统中进行的研究却很少。我们使用活体小鼠的唾液腺来检查小 GTP 酶 Cdc42 在调节细胞间小管稳态中所起的作用，细胞间小管是腺泡细胞的一个特殊的顶端区域，蛋白质和液体分泌发生在此处。我们发现，在成年小鼠中，Cdc42 的耗尽会诱导：1）APM 显着扩张，2）侧膜长度增加，3）对基底外侧和 APM 处的内吞运输产生强烈刺激，而这并没有影响。影响质膜特性和连接完整性。另一方面，胚胎晚期的 Cdc42 缺失导致 1) 细胞间小管出生后发育的完全抑制，2) 刺激内吞运输。总体而言，这些结果表明 Cdc42 在调节体内上皮腔的发育和维持中发挥着基础作用，并强调了 Cdc42 在膜重塑中的额外作用，即作为内吞运输途径的负调节因子。 3）细胞代谢与膜重塑之间的协调。膜重塑是一个能量上不利的过程。在蛋白质分泌过程中，单个胞吐事件需要大量能量来：1）将分泌颗粒和 PM 聚集在一起并融合，正如对各种系统的估计，并将颗粒膜整合到 APM 中并维持其稳态。我们试图确定细胞代谢与蛋白质分泌之间的关系。首先，我们证实调节的胞吐作用依赖于线粒体代谢。事实上，颗粒融合和整合需要线粒体 ATP 合酶的活性，而它们独立于复合物 I 的活性。这一发现表明，β-肾上腺素能信号以一种新颖的方式改变了线粒体代谢。其次，我们发现体内腺泡细胞中有两种空间上不同的线粒体群体：一种位于基底外侧PM，另一种分散在整个细胞质中。第三，我们发现胞吐作用的开始引发了细胞内 ATP 池的耗尽，正如细胞质能量传感器 AMP 激活蛋白激酶 (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库增加了运动性和大小。最后一个特征与线粒体产生 ATP 能力的增加有关。值得注意的是，我们发现，单个线粒体大小的增加是由 PKA 依赖性线粒体裂变蛋白 DRP1 活性抑制介导的。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，它们将为协调细胞代谢和重塑开辟新途径。