Molecular Mechanisms regulating membrane trafficking in salivary glands

调节唾液腺膜运输的分子机制

• 批准号: 8743751
• 负责人: Roberto Weigert
• 金额: $ 136.69万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 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/8743751
• 关键词: Acinar Cell; Acinus organ component; Actins; Actomyosin; Adrenergic Agents; Affect; Animals; Apical; Caliber; Cell membrane; Cells; Cellular Structures; Complex; Confocal Microscopy; Cytochalasin D; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; Diffuse; Docking; Down-Regulation; Drosophila genus; Event; Exocrine Glands; Exocrine pancreas; Exocytosis; F-Actin; Fluids and Secretions; Food; Functional disorder; G-Protein-Coupled Receptors; Gene Silencing; Gene Transfer; Genetic; Green Fluorescent Proteins; Homeostasis; Hour; Hydrostatic Pressure; Image; Imagery; Impairment; Individual; Knock-in Mouse; Knockout Mice; Label; Larva; Length; Life; Major salivary gland structure; Mediating; Membrane; Membrane Protein Traffic; Modality; Molecular; Monomeric GTP-Binding Proteins; Morphology; Motor; Motor Activity; Movement; Mucins; Mus; Muscarinic Acetylcholine Receptor; Myosin ATPase; Myosin Light Chain Kinase; Myosin Type II; Organ; Pathway interactions; Peptides; Phosphorylation; Physiology; Play; Process; Protein Isoforms; Protein Secretion; Proteins; Proteomics; Rattus; Recruitment Activity; Resolution; Rodent; Role; Salivary Glands; Salivary Proteins; Scaffolding Protein; Scanning; Scanning Transmission Electron Microscopy Procedures; Secretory Vesicles; Series; Small Interfering RNA; Sorting - Cell Movement; Submandibular gland; Surface; System; Techniques; Tomatoes; Transgenic Mice; Transgenic Organisms; Water; Work; adrenergic; base; blebbistatin; in vivo; in vivo Model; inhibitor/antagonist; intravital microscopy; latrunculin A; light microscopy; mouse model; novel; polarized cell; prevent; tool; two-photon

1) Molecular machinery regulating protein secretion in the acinar cells of salivary glands. In the SGs, the major secretory units are the acini that are formed by pyramidal polarized cells, which form small canaliculi at the apical plasma membrane (APM) where salivary proteins and water are secreted. Proteins destined to secretion are packed in secretory granules (SCGs) that are released into the cytoplasm, and transported to the cell periphery. Here, upon stimulation of the appropriate G protein-coupled receptor (GPCR), the granules fuse with the APM, releasing their content into the lumen of the canaliculi. Our aim is to elucidate the molecular machinery regulating their fusion with the APM and how the homeostasis of the APM is maintained. We developed an experimental system in the SGs of live rodents aimed at imaging and tracking individual secretory granules, and visualizing the dynamics of the APM. We used high-resolution intravital microscopy performed on a series of transgenic mouse models, which ubiquitously express: i) the soluble green fluorescent protein (GFP-mouse), or ii) the tandem-Tomato fluorescent protein fused with a membrane-targeted peptide (m-Tomato mouse), iii) or both molecules (GFP/m-Tomato mouse). The GFP-mouse provided clear visualization of both the secretory granules and the APM, and enabled an accurate quantitative analysis of the exocytic events. On the other hand, the m-Tomato mouse enabled imaging the APM and the secretory granules after fusion has occurred. Although the morphology of the acinar cells has been analyzed by transmission and scanning electron microscopy, several key aspects of the structure of these cells and their three dimensional arrangement were not fully elucidated. By performing z-scans and generating volume renderings of individual acini, we determined that each acinar cell contains an average of 250-300 secretory granules with diameters between 1.0-1.5 m. Moreover, we found that each acinar cell contributes to the formation of at least two canaliculi that are 10-15 m in length, and have diameters of 0.3-0.4 m. We have shown that the stimulation of the β-adrenergic, but not the muscarinic receptors, enhances the mobility of the secretory granules and promotes their docking and subsequent fusion with the APM. Moreover, we determined that after the opening of the fusion pore the secretory granules completely collapse within 40-60 seconds and their membranes are integrated into the canaliculi. Overall, these results reveal two major differences between in vivo observations and ex-vivo models in which, i) muscarinic receptors have been shown to elicit exocytosis, and ii) compound exocytosis (i.e. the sequential fusion of strings of secretory granules) has been described as the primary modality of fusion. By using a mouse expressing both the m-Tomato and the small peptide lifeact fused with GFP (a novel tool to label dynamically F-actin) we determined that upon the opening of the fusion pore, the APM diffuses into the limiting membrane of the granules triggering the recruitment of F-actin onto their surface. The assembly of F-actin initiates and facilitates the gradual collapse of the secretory granules, as shown by impairing the dynamics of the actin cytoskeleton through pharmacological agents, such as cytochalasin D (cyto D) or latrunculin A (lat A). Indeed, under these conditions the fused granules were arrested at the APM after the opening of the fusion pore, and expanded in size 2-3 fold with respect to their original diameters. This expansion is the result of two processes: i) the increase of the hydrostatic pressure in the acinar canaliculi due to the stimulation of fluid secretion, and ii) the sequential fusion of adjacent secretory granules. These observations strongly suggest that actin plays three major roles during regulated exocytosis: i) facilitates the completion of the gradual collapse of the secretory granules at the APM, ii) protects the granules from the hydrostatic pressure, and iii) prevents the unregulated fusion of the adjacent granules. We postulated that in order to produce the contractile activity necessary to drive the gradual collapse of the secretory granules, a myosin motor would be required as well. Indeed, we found that two of the three isoforms of the myosin II actin motor (IIa and IIb) were recruited onto the secretory granules upon stimulation of regulated exocytosis. Interestingly, blocking the motor activity of myosin II with blebbistatin resulted in a delay in the gradual collapse of the secretory granules. We sought to determine how both isoforms of myosin II are recruited onto the membrane of the secretory granules. We first asked whether myosin II was recruited through F-actin. To this end, we crossed knock-in mice expressing either GFP-myosin IIa or GFP-myosin IIb with mice expressing RFP-lifeact. We found that both myosin IIa and IIb are recruited after the assembly of F-actin onto the granules. However, we observed that i) both myosins continue to be actively recruited onto the membranes even after F-actin disassembles, and ii) the recruitment of both myosins is only slightly affected by actin depolymerizing agents. These results suggested that other molecules may be required for myosin II recruitment and retention and in order to identify them we performed the proteomic analysis of secretory granules purified from rat submandibular glands. Among the proteins associated with the granules we identified Septin2, a small GTPase previously suggested to work as a scaffold protein facilitating the phosphorylation of myosin II via the recruitment of the myosin light chain kinase (MLCK). Septin2 was indeed expressed in the SGs, and recruited onto the secretory granules after their fusion with the plasma membrane. Moreover, inhibition of the septin2 activity with the specific inhibitor forchlorfenuron (FCF) resulted in the impairment of the assembly of the actin cytoskeleton during regulated exocytosis. Interestingly, we found that in the SGs of Drosophila larvae, a system that shares similarities with the rodent salivary glands, siRNA-mediated downregulation of septin2 resulted in a severe impairment of regulated exocytosis of mucin-containing granules and highlighted an additional role for septin2 in pre-fusion events. Finally, we determined whether the requirement for a functional actomyosin complex is unique to the rodent salivary glands or is a general mechanism shared by other secretory systems. We found that this machinery is conserved in the mouse exocrine pancreas, as shown by imaging regulated exocytosis in the acini of live mice expressing Lifeact and mTomato or Lifeact and either Myosin IIa or IIb. In animals that were starved for 24 hours, no exocytosis was detected. On the other hand, administration of food elicited exocytosis of secretory granules, which underwent a gradual collapse and, upon fusion with the APM, recruited sequentially actin and myosin II. Based on our findings we proposed that the assembly of the actomyosin complex is required in exocrine glands and in those secretory systems that possess a unique geometry in which the secretory vesicles have a lower membrane tension (diameters > 1 m) than the target membrane (0.3-0.4 m) making the gradual collapse of the secretory granules energetically unfavorable. The contractile activity of the actomyosin complex may work to overcome this energy barrier by i) contributing to the expansion of the fusion pore, and ii) facilitating the movement of membranes from the granules toward the APM.

1) 调节唾液腺腺泡细胞蛋白质分泌的分子机制。在 SG 中，主要的分泌单位是由锥体极化细胞形成的腺泡，腺泡在顶端质膜 (APM) 处形成小小管，分泌唾液蛋白和水。注定要分泌的蛋白质被包装在分泌颗粒 (SCG) 中，释放到细胞质中，并转运到细胞外周。在这里，在刺激适当的 G 蛋白偶联受体 (GPCR) 后，颗粒与 APM 融合，将其内容物释放到泪管管腔中。我们的目标是阐明调节它们与 APM 融合的分子机制以及如何维持 APM 的稳态。我们在活体啮齿动物的 SG 中开发了一个实验系统，旨在成像和跟踪单个分泌颗粒，并可视化 APM 的动态。我们使用高分辨率活体显微镜在一系列转基因小鼠模型上进行，这些模型普遍表达：i）可溶性绿色荧光蛋白（GFP-小鼠），或ii）与膜靶向肽融合的串联番茄荧光蛋白（ m-Tomato 小鼠），iii）或两种分子（GFP/m-Tomato 小鼠）。 GFP 小鼠提供了分泌颗粒和 APM 的清晰可视化，并能够对胞吐事件进行准确的定量分析。另一方面，m-Tomato 小鼠能够在融合后对 APM 和分泌颗粒进行成像。尽管通过透射和扫描电子显微镜对腺泡细胞的形态进行了分析，但这些细胞结构及其三维排列的几个关键方面尚未完全阐明。通过进行 z 扫描并生成单个腺泡的体积渲染，我们确定每个腺泡细胞平均包含 250-300 个直径在 1.0-1.5 m 之间的分泌颗粒。此外，我们发现每个腺泡细胞有助于形成至少两个长度为10-15 m、直径为0.3-0.4 m的小管。 我们已经证明，刺激 β-肾上腺素能受体（而非毒蕈碱受体）可增强分泌颗粒的活动性，并促进其与 APM 的对接和随后的融合。此外，我们确定融合孔打开后，分泌颗粒在40-60秒内完全塌陷，并且它们的膜整合到泪小管中。总体而言，这些结果揭示了体内观察和离体模型之间的两个主要差异，其中，i）毒蕈碱受体已被证明可引发胞吐作用，ii）已描述了复合胞吐作用（即分泌颗粒串的顺序融合）作为融合的主要方式。 通过使用表达 m-Tomato 和与 GFP（一种动态标记 F-肌动蛋白的新工具）融合的小肽 lifeact 的小鼠，我们确定，在融合孔打开时，APM 扩散到颗粒的限制膜中触发 F-肌动蛋白募集到其表面。 F-肌动蛋白的组装启动并促进分泌颗粒的逐渐崩溃，如通过细胞松弛素 D (cyto D) 或 latrunculin A (lat A) 等药理学试剂损害肌动蛋白细胞骨架的动力学所示。事实上，在这些条件下，融合颗粒在融合孔打开后被捕获在 APM 处，并且尺寸相对于其原始直径膨胀 2-3 倍。这种扩张是两个过程的结果：i) 由于液体分泌的刺激而导致腺泡小管中的静水压力增加，以及 ii) 相邻分泌颗粒的顺序融合。这些观察结果强烈表明，肌动蛋白在受调节的胞吐作用过程中发挥三个主要作用：i) 促进 APM 处分泌颗粒逐渐塌陷的完成，ii) 保护颗粒免受静水压力的影响，以及 iii) 防止分泌颗粒不受调节的融合。相邻的颗粒。我们假设，为了产生驱动分泌颗粒逐渐崩溃所需的收缩活动，还需要肌球蛋白运动。事实上，我们发现肌球蛋白 II 肌动蛋白马达的三种亚型中的两种（IIa 和 IIb）在刺激受调节的胞吐作用后被募集到分泌颗粒上。有趣的是，用肌球蛋白II阻断肌球蛋白II的运动活性导致分泌颗粒逐渐崩溃的延迟。我们试图确定肌球蛋白 II 的两种亚型如何被募集到分泌颗粒的膜上。我们首先询问肌球蛋白 II 是否是通过 F-肌动蛋白招募的。为此，我们将表达 GFP-肌球蛋白 IIa 或 GFP-肌球蛋白 IIb 的敲入小鼠与表达 RFP-lifeact 的小鼠杂交。我们发现肌球蛋白 IIa 和 IIb 在 F-肌动蛋白组装到颗粒上后都会被募集。然而，我们观察到，i) 即使在 F-肌动蛋白分解后，两种肌球蛋白仍继续积极地募集到膜上，并且 ii) 两种肌球蛋白的募集仅受到肌动蛋白解聚剂的轻微影响。这些结果表明肌球蛋白 II 募集和保留可能需要其他分子，为了识别它们，我们对从大鼠颌下腺纯化的分泌颗粒进行了蛋白质组分析。在与颗粒相关的蛋白质中，我们鉴定出了 Septin2，这是一种小型 GTP 酶，之前认为它可以作为支架蛋白，通过招募肌球蛋白轻链激酶 (MLCK) 促进肌球蛋白 II 的磷酸化。 Septin2确实在SG中表达，并在分泌颗粒与质膜融合后被募集到分泌颗粒上。此外，用特异性抑制剂氯吡脲 (FCF) 抑制 septin2 活性，会导致受调节的胞吐作用期间肌动蛋白细胞骨架的组装受损。有趣的是，我们发现，在果蝇幼虫的 SG（与啮齿动物唾液腺相似的系统）中，siRNA 介导的 septin2 下调导致含粘蛋白颗粒的受调节胞吐作用严重受损，并强调了 septin2 在融合前事件。最后，我们确定了对功能性肌动球蛋白复合物的需求是否是啮齿动物唾液腺所独有的，还是其他分泌系统所共有的一般机制。我们发现这种机制在小鼠外分泌胰腺中是保守的，如表达 Lifeact 和 mTomato 或 Lifeact 以及肌球蛋白 IIa 或 IIb 的活体小鼠腺泡中调节的胞吐作用的成像所示。在饥饿 24 小时的动物中，没有检测到胞吐作用。另一方面，进食会引起分泌颗粒的胞吐作用，分泌颗粒逐渐崩溃，并在与 APM 融合后，依次募集肌动蛋白和肌球蛋白 II。根据我们的发现，我们提出，外分泌腺和具有独特几何形状的分泌系统需要肌动球蛋白复合物的组装，其中分泌囊泡的膜张力（直径 > 1 m）低于目标膜（0.3 -0.4 m）使分泌颗粒逐渐崩溃，在能量上不利。肌动球蛋白复合物的收缩活性可能通过 i) 促进融合孔的扩张，以及 ii) 促进膜从颗粒向 APM 的移动来克服这种能量障碍。