Molecular Mechanisms regulating membrane trafficking in salivary glands

调节唾液腺膜运输的分子机制

• 批准号: 8929686
• 负责人: Roberto Weigert
• 金额: $ 145.11万
• 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/8929686
• 关键词: Acinar Cell; Acinus organ component; Actins; Actomyosin; Adrenergic Agents; Adrenergic Receptor; Affect; Agonist; Animals; Apical; Appearance; Binding Sites; Bioenergetics; Cell membrane; Cell physiology; Cells; Complex; Confocal Microscopy; Coupled; Cytokinesis; Cytoplasmic Granules; Cytoskeleton; Electron Transport; Endocytic Vesicle; Epithelium; Event; Exocrine Glands; Exocytosis; F-Actin; Filament; Functional disorder; Gap Junctions; Gene Silencing; Gene Transfer; Genetic; Homeostasis; Image; In Vitro; Individual; Knockout Mice; Life; Link; Maintenance; Mediating; Membrane; Membrane Protein Traffic; Metabolic; Metabolism; Methods; Microscopy; Mitochondria; Mitochondrial Proton-Translocating ATPases; Molecular; Monomeric GTP-Binding Proteins; Myosin ATPase; Myosin Light Chain Kinase; Myosin Type II; NADH; Organ; Pathway interactions; Phosphorylation; Physiology; Play; Process; Production; Protein Isoforms; Proteins; Rattus; Reactive Oxygen Species; Recruitment Activity; Regulation; Reporting; Rodent; Role; Salivary Glands; Secretory Vesicles; Series; Signal Transduction; Sorting - Cell Movement; Staging; System; Techniques; Transgenic Organisms; Water; Work; adrenergic; base; human EMS1 protein; in vivo; light microscopy; neurotransmission; non-muscle myosin; novel; polarized cell; prevent; scaffold; small molecule; tool; trans-Golgi Network; two-photon

1)Molecular basis of the actomyosin-driven membrane remodeling during regulated exocytosis in salivary glands. The major secretory units of the salivary glands (SGs) are the acini that are formed by pyramidal polarized cells in which the apical plasma membrane (APM) forms small canaliculi where proteins and water are released. Proteins destined to secretion are packed in secretory granules at the trans-Golgi network (TGN) and transported to the cell periphery where they fuse with the APM upon stimulation of GPCRs. Our aim is to elucidate the molecular machinery regulating the fusion and integration of the secretory granules with the APM and the maintenance of APM homeostasis. To this end, 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 have established that upon stimulation of the β-adrenergic receptor, the granules fuse with the APM, releasing their content into the lumen of the canaliculi. In addition, we found that a complex composed of F-actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB) is recruited onto the secretory granules after the fusion step. We showed that the actomyosin contractile activity is required for the integration of the granular membranes into the APM and the completion of exocytosis. In the last year our work has been focusing on elucidating two aspects of the regulation of the actomyosin complex during regulated exocytosis, and specifically: the machinery regulating F-actin assembly onto the secretory granules, and the mechanisms of recruitment and regulation of NMII. Our work has shown that F-actin is assembled around the secretory granules after their fusion with the APM, and plays three distinct roles during regulated exocytosis: first, stabilizes the granular membranes, second, prevents compound exocytosis, and finally, facilitates their gradual collapse. We have hypothesized that the F-actin scaffold is progressively assembled around the granules in two steps: the first requires the formation of linear filaments and provides the stabilization of the membranes, and the second requires the formation of branched filaments, and regulates the collapse of the granules. Consistent with this hypothesis, we found that Profilin1 and mDia2, two components of the machinery initiating the formation of linear filaments, are recruited onto the secretory granules right after fusion, whereas the actin branching factors Arp2/3, cortactin, WASP, and Wave2 are recruited at a later stage. Moreover, by using a pharmacological approach we found that blocking mDia2 resulted in the expansion of the secretory granules, whereas blocking the activity of Arp2/3 significantly delayed their gradual collapse. In addition, we have shown that β-adrenergic stimulation results in the appearance of specific phosphorylated forms of Arp2 (T237 and T238) and cortactin (Y421 and Y466) onto the secretory granules, thus establishing a link between signaling and cytoskeleton. We have also shown that NMIIA and NMIIB are recruited onto the secretory granules after their fusion with the APM, and by using a pharmacological approach we have determined that their contractile activity drives the gradual integration of the secretory granules into the APM. This contrasts with other cellular processes where actomyosin-based contractions employ only one isoform of NMII. Notably, by using conditional knock-out mice we determined that NMIIA regulates the gradual collapse of the secretory granules, whereas NMIIB is required for stabilizing the granular membranes. Since both NMIIA and NMIIB are recruited after the formation of the F-actin scaffold, we had assumed that this process would be mediated by their well-characterized actin-binding site. Unexpectedly, we found that both NMII isoforms are recruited in an actin-independent fashion, and that the main role of F-actin is to facilitate the proper assembly of the myosin filaments. We determined that this step is regulated by the phosphorylation of the regulatory chain of NMII that is catalyzed by the myosin light chain kinase (MLCK), which is also recruited onto the secretory granules. Moreover, we discovered that the phosphorylation of NMII is controlled by the recruitment of filaments composed of septin2 and septin6, two small GTPase that regulate the actin cytoskeleton during cytokinesis. Interestingly, blocking the ability of septins to form filaments severely impairs exocytosis, by slowing the gradual integration of the granular membranes. 2) Bioenergetics of regulated exocytosis. The integration of the membranes of the secretory granules and the APM is an energetically unfavorable process. A single exocytic event consumes significant energy to bring together and fuse a single secretory granule and the plasma membrane, as estimated for neurotransmission. We found that 150-200 secretory granules undergo exocytosis after β-adrenergic stimulation of an acinar cell and that each granule is retrieved by 50-75 endocytic vesicles. Therefore, a central question is: how is cell metabolism regulated during exocytosis? Our hypothesis is that mitochondrial function is increased during β-adrenergic stimulation and is tightly coupled to the exo-endocytic events. We have developed a method to follow the dynamics of the mitochondrial metabolic activity in the SGs of live rats. Since there are no reliable tools to quantitatively image the levels of cellular ATP in vivo, we used two-photon microscopy to determine NADH levels (the main substrate of the electron transport chain, ETC) and mitochondrial potential. Unexpectedly, we discovered that mitochondrial metabolism undergoes rapid and spontaneous oscillations in SGs under basal conditions (period: 10-15 sec). This finding contrast with what reported in exocrine glands in vitro where transient metabolic oscillations were observed only after agonist stimulation. These oscillations are regulated by reactive oxygen species but are insensitive to increase in the levels of intracellular Ca2+, indicating a novel regulation completely different than what described in ex vivo organs. Most notably, we found that mitochondrial oscillations are highly coordinated throughout the SG epithelium via the activity of gap junctions, which may transport a not yet identified small molecule that regulates the synchronization of the oscillations. Finally, our work has shown that β-adrenergic stimulation transiently halts NADH oscillations and increases mitochondrial potential, the latter indicating an increased production of ATP, as would be required to drive regulated exocytosis. Consistently, blocking the mitochondrial ATP-synthase inhibits the assembly of the actomyosin complex (a major energy requiring step). Interestingly, under β-adrenergic stimulations blocking the activity of complex I does not affect exocytosis, suggesting that a different pathway is used to regulate mitochondrial metabolism during stimulated secretion.

1）在调节唾液腺胞吐作用期间，肌动蛋白驱动的膜重塑的分子基础。 唾液腺（SGS）的主要分泌单位是由金字塔极化细胞形成的ACINI，其中顶端质膜（APM）形成小管，其中释放蛋白质和水。蛋白质的蛋白质在跨加利基网络（TGN）的分泌颗粒中包装在分泌颗粒中，并在刺激GPCR时将其与APM融合到细胞外围。我们的目的是阐明调节分泌颗粒与APM的融合和整合的分子机械以及APM稳态的维持。为此，我们在活啮齿动物的SGS中开发了一个实验系统，该系统旨在成像和跟踪单个分泌颗粒，并可视化APM的动力学。 我们已经确定，在刺激β-肾上腺素受体时，颗粒将与APM融合，将其含量释放到运河的腔内。此外，我们发现，融合步骤后，由F-肌动蛋白和两个非肌肉肌球蛋白II（NMIIA和NMIIB）组成的复合物被募集到分泌颗粒上。我们表明，肌球蛋白收缩活性是将颗粒膜整合到APM中并完成胞吐作用所必需的。在去年，我们的工作一直集中在调节的胞吐作用期间，尤其是：调节F-肌动蛋白组装到分泌颗粒上的机械以及NMII的募集和调节机制。 我们的工作表明，F-肌动蛋白与APM融合后围绕分泌颗粒组装，并在调节的胞吐作用过程中扮演三个不同的作用：首先，稳定颗粒状膜，其次，其次防止复合胞吞作用，最后促进了它们的逐渐崩溃。我们假设F-肌动蛋白支架是在颗粒周围逐渐组装的，分为两个步骤：第一个需要形成线性细丝，并提供膜的稳定化，第二个需要形成分支的细丝，并调节颗粒的塌陷。与这一假设一致，我们发现profilin1和mdia2是启动线性细丝形成的机械的两个组成部分，在融合后立即募集到分泌颗粒上，而肌动蛋白分支分支因子ARP2/3，ARP2/3，Cortactin，Wasp和Wave2在以后的阶段招募。此外，通过使用药理学方法，我们发现阻断MDIA2导致分泌颗粒的扩展，而阻断ARP2/3的活性显着延迟了它们的逐渐塌陷。此外，我们已经表明，β-肾上腺素能刺激导致ARP2（T237和T238）（T237和T238）和Cortactin（Y421和Y466）的特定磷酸化形式出现在分泌颗粒上，从而在信号和细胞骨架之间建立了联系。 我们还表明，NMIIA和NMIIB与APM融合后被招募到分泌颗粒上，并且通过使用药理方法，我们确定它们的收缩活性将分泌颗粒逐渐整合到APM中。这与其他细胞过程形成鲜明对比，基于肌动蛋白的收缩仅采用一种NMII的同工型。值得注意的是，通过使用条件敲除小鼠，我们确定NMIIA调节分泌颗粒的逐渐崩溃，而NMIIB稳定颗粒状膜需要NMIIB。由于F-肌动蛋白支架形成后，NMIIA和NMIIB均被招募，因此我们假设该过程将由其特征良好的肌动蛋白结合位点介导。出乎意料的是，我们发现这两种NMII同工型都是以肌动蛋白独立的方式募集的，F-肌动蛋白的主要作用是促进肌球蛋白丝的适当组装。我们确定该步骤是由肌球蛋白轻链激酶（MLCK）催化的NMII调节链的磷酸化调节，该链也被募集到分泌颗粒上。此外，我们发现NMII的磷酸化受到由Septin2和Septin6组成的细丝的募集，这是两个小的GTPase，它们调节细胞因子过程中肌动蛋白细胞骨架。有趣的是，通过减慢颗粒状膜的逐步整合，阻止隔蛋白形成细丝的能力严重损害了胞吞作用。 2）调节胞吐作用的生物能学。分泌颗粒和APM的膜的整合是一个充满活力的过程。估计，一个单一的外囊肿会消耗大量能量，以将单个分泌颗粒和质膜融合在一起，并融合一个神经传递的颗粒。我们发现150-200分泌颗粒在β-肾上腺素能刺激腺泡细胞后发生胞吐作用，并且每个颗粒都通过50-75个内吞囊泡检索。因此，一个核心问题是：在胞吐作用期间如何调节细胞代谢？我们的假设是，在β-肾上腺素能刺激过程中，线粒体功能增加，并与外胞吞事件紧密耦合。我们已经开发了一种遵循活大鼠SGS线粒体代谢活性动力学的方法。由于没有可靠的工具可以定量地对体内细胞ATP的水平进行定量图像，因此我们使用了两光子显微镜来确定NADH水平（电子传输链的主要基板等）和线粒体电位。出乎意料的是，我们发现线粒体代谢在基础条件下（周期：10-15秒）在SGS中经历了快速和自发的振荡。这一发现与在体外报道的体外报道的形成鲜明对比，在体外报告的情况仅在激动剂刺激后才观察到瞬时代谢振荡。这些振荡受活性氧的调节，但对细胞内Ca2+水平的增加不敏感，表明新的调节与离体器官中所述的完全不同。最值得注意的是，我们发现线粒体振荡通过间隙连接的活性在整个SG上皮上高度协调，该间隙连接的活性可能运输尚未确定的小分子，该分子调节了振荡的同步。最后，我们的工作表明，β-肾上腺素能刺激会暂时停止NADH振荡并增加线粒体电位，后者表明ATP的产生增加，这是驱动调节的胞吐作用所必需的。一致地，阻塞线粒体ATP合成酶会抑制肌动球蛋白络合物的组装（需要步骤的主要能量）。有趣的是，在阻断复合物I活性的β-肾上腺素能刺激下不会影响胞吐作用，这表明在刺激分泌过程中使用了不同的途径来调节线粒体代谢。