Molecular Mechanisms regulating membrane trafficking in salivary glands

调节唾液腺膜运输的分子机制

基本信息

批准号：
9155527
负责人：
Roberto Weigert
金额：
$ 141.35万
依托单位：
NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
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/9155527
关键词：
Acinar Cell Acinus organ component Actins Actomyosin Adrenergic Agents Affect Agonist Animals Apical Appearance Behavior Binding Sites Bioenergetics Cell membrane Cell physiology Cells Complex Confocal Microscopy Coupled Cytokinesis Cytoplasm Cytoplasmic Granules Cytoskeleton Electron Transport Endocytic Vesicle Epithelium Event Exocytosis F-Actin Filament Functional disorder Gap Junctions Gene Silencing Gene Transfer Genetic Homeostasis Image Impairment 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 Population Process Production Protein Isoforms Proteins Rattus Reactive Oxygen Species Recruitment Activity Regulation Reporting Rodent Role Salivary Glands Secretory Vesicles Series Signal Transduction Site Sorting - Cell Movement Staging System Techniques Transgenic Organisms Water Work adrenergic base basolateral membrane beta-adrenergic receptor cell motility human EMS1 protein in vivo inhibitor/antagonist light microscopy neurotransmission non-muscle myosin novel polarized cell prevent scaffold small molecule tool trans-Golgi Network two-photon

项目摘要

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 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 live rodents aimed at imaging and tracking individual secretory granules, and visualizing the dynamics of the APM. We established that upon stimulation of the beta-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 granules after the fusion step. We showed that the actomyosin contractile activity regulates the integration of the granular membranes into the APM and the completion of exocytosis. In the last year we focused on elucidating two aspects of the regulation of the actomyosin complex during regulated exocytosis, and specifically: the machinery regulating F-actin assembly onto the granules, and the mechanisms of recruitment and regulation of NMII. F-actin is assembled around the secretory granules after their fusion with the APM, and plays three distinct roles: 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 mDia1 and mDia2, two components of the machinery initiating the formation of linear filaments, are recruited onto the secretory granules right after fusion. Impairment of the activity of mDia1/mDia2 by either pharmacological approaches or using selected conditional knock-out mice resulted in the expansion of the granules after fusion with the APM. In addition, we found that the actin branching factors Arp2/3, cortactin, WASP, and Wave2 are recruited after the F-actin linear filaments are assembled onto the granules. Impairing the activity of the Arp2/3 complex by a specific pharmacological inhibitor or by using an ARPC3 conditional knock-out mice, another component of the Arp2/3 complex, significantly delay the integration of the granules into the APM. In addition, we have shown that beta-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 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 NMIIB is required to control the initial steps of the integration of the granular membrane, by stabilizing the F-actin scaffold and providing a continuous contractile activity that pushes the membranes towards the APM. On the other hand, NMIIA is required at later stages of the process to control the expansion of the fusion pore. 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 granules. Moreover, we discovered that the phosphorylation of NMII is controlled by the recruitment of filaments composed of septin2, septin6, septin7, and septin9, that are small GTPase regulating the actin cytoskeleton during cytokinesis. This septin complex is required to recruit the MLCK onto the secretory granules. 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 beta-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 beta-adrenergic stimulation and is temporally and spatially 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. We discovered that mitochondrial metabolism undergoes spontaneous oscillations in SGs under basal conditions (period: 10-15 sec). This finding contrasts with what reported in vitro where transient metabolic oscillations were observed only after agonist stimulation. Moreover, these oscillations are regulated by reactive oxygen species but are insensitive to manipulation of intracellular Ca2+, indicating a completely novel regulation of this process. 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. Our work has also shown that in the acinar cells of the SGs vivo mitochondria are organized in two distinct populations: one localized beneath the basolateral membrane, the other, localized in the cytoplasm and extending toward the APM. We found that beta-adrenergic stimulation: 1) transiently halts NADH oscillations; 2) induces an initial increase in mitochondrial potential followed by a sustained depolarization, a behavior consistent with an increased production of ATP; and 3) increases the motility of the mitochondria at the site of exocytosis. These finding are consistent with a rearrangement of the mitochondrial dynamics and bioenergetics in order to sustain regulated exocytosis. Finally, blocking the mitochondrial ATP-synthase inhibits the assembly of the actomyosin complex (a major energy requiring step), whereas 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）唾液腺调节胞吐作用期间肌动球蛋白驱动的膜重塑的分子基础。唾液腺 (SG) 的主要分泌单位是由极化细胞形成的腺泡，其中顶端质膜 (APM) 形成小小管，在小管中释放蛋白质和水。注定要分泌的蛋白质被包装在跨高尔基体网络 (TGN) 的分泌颗粒中，并被运输到细胞外周，在 GPCR 刺激下它们与 APM 融合。我们的目标是阐明调节分泌颗粒与 APM 融合和整合以及维持 APM 稳态的分子机制。为此，我们在活体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，并可视化 APM 的动态。我们确定，在刺激 β-肾上腺素能受体时，颗粒与 APM 融合，将其内容物释放到小管管腔中。此外，我们发现由 F-肌动蛋白和两种非肌肉肌球蛋白 II 亚型（NMIIA 和 NMIIB）组成的复合物在融合步骤后被募集到颗粒上。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。去年，我们重点阐明了在受调节的胞吐作用过程中肌动球蛋白复合物的调节的两个方面，特别是：调节 F-肌动蛋白组装到颗粒上的机制，以及 NMII 的募集和调节机制。 F-肌动蛋白与 APM 融合后在分泌颗粒周围组装，并发挥三个不同的作用：首先，稳定颗粒膜，其次，防止复合胞吐作用，最后，促进其逐渐崩溃。我们假设 F-肌动蛋白支架分两步逐步围绕颗粒组装：第一步需要形成线性丝并提供膜的稳定性，第二步需要形成分支丝并调节膜的塌陷。颗粒。与这一假设一致，我们发现 mDia1 和 mDia2（启动线性丝形成机制的两个组成部分）在融合后立即被招募到分泌颗粒上。通过药理学方法或使用选定的条件敲除小鼠来损害 mDia1/mDia2 的活性，导致颗粒在与 APM 融合后膨胀。此外，我们发现肌动蛋白分支因子 Arp2/3、cortactin、WASP 和 Wave2 在 F-肌动蛋白线性丝组装到颗粒上后被招募。通过特定的药理学抑制剂或使用 ARPC3 条件敲除小鼠（Arp2/3 复合物的另一个组成部分）损害 Arp2/3 复合物的活性，可显着延迟颗粒与 APM 的整合。此外，我们还发现，β-肾上腺素能刺激会导致分泌颗粒上出现特定磷酸化形式的 Arp2（T237 和 T238）和皮质素（Y421 和 Y466），从而在信号传导和细胞骨架之间建立联系。我们还表明，NMIIA 和 NMIIB 在与 APM 融合后被招募到分泌颗粒上，并且通过使用药理学方法，我们确定它们的收缩活性驱动颗粒逐渐整合到 APM 中。这与其他细胞过程形成对比，在其他细胞过程中，基于肌动球蛋白的收缩仅使用一种 NMII 亚型。值得注意的是，通过使用条件敲除小鼠，我们确定需要 NMIIB 来控制颗粒膜整合的初始步骤，通过稳定 F-肌动蛋白支架并提供将膜推向 APM 的连续收缩活动。另一方面，在工艺的后期阶段需要NMIIA来控制熔合孔的扩张。由于 NMIIA 和 NMIIB 都是在 F-肌动蛋白支架形成后招募的，因此我们假设该过程将由其充分表征的肌动蛋白结合位点介导。出乎意料的是，我们发现两种 NMII 亚型都以肌动蛋白独立的方式招募，并且 F-肌动蛋白的主要作用是促进肌球蛋白丝的正确组装。我们确定该步骤是由 NMII 调节链的磷酸化调节的，而 NMII 调节链是由肌球蛋白轻链激酶 (MLCK) 催化的，MLCK 也被募集到颗粒上。此外，我们发现 NMII 的磷酸化是通过招募由 septin2、septin6、septin7 和 septin9 组成的细丝来控制的，这些细丝是在胞质分裂过程中调节肌动蛋白细胞骨架的小 GTP 酶。这种脓毒蛋白复合物需要将 MLCK 募集到分泌颗粒上。有趣的是，阻断脓蛋白形成细丝的能力会减慢颗粒膜的逐渐整合，从而严重损害胞吐作用。 2）受调节的胞吐作用的生物能学。分泌颗粒膜和 APM 的整合是一个能量上不利的过程。根据神经传递的估计，单个胞吐事件消耗大量能量来聚集并融合单个分泌颗粒和质膜。我们发现，在腺泡细胞受到β-肾上腺素能刺激后，150-200 个分泌颗粒进行胞吐作用，并且每个颗粒由 50-75 个内吞小泡回收。因此，一个中心问题是：胞吐过程中细胞代谢是如何调节的？我们的假设是，线粒体功能在β-肾上腺素能刺激期间增强，并且在时间和空间上与外吞事件紧密耦合。我们开发了一种方法来追踪活体大鼠 SG 中线粒体代谢活动的动态。由于没有可靠的工具对体内细胞 ATP 水平进行定量成像，我们使用双光子显微镜来确定 NADH 水平（电子传递链的主要底物，ETC）和线粒体电位。我们发现线粒体代谢在基础条件下（周期：10-15 秒）在 SG 中经历自发振荡。这一发现与体外报道的结果形成鲜明对比，体外报道仅在激动剂刺激后观察到短暂的代谢振荡。此外，这些振荡受活性氧调节，但对细胞内 Ca2+ 的操纵不敏感，表明该过程的全新调节。最值得注意的是，我们发现线粒体振荡通过间隙连接的活动在整个 SG 上皮中高度协调，间隙连接可能运输一种尚未识别的小分子来调节振荡的同步。我们的工作还表明，在 SG 体内的腺泡细胞中，线粒体被组织成两个不同的群体：一个位于基底外侧膜下方，另一个位于细胞质中并向 APM 延伸。我们发现β-肾上腺素能刺激：1) 暂时停止 NADH 振荡； 2) 诱导线粒体电位最初增加，随后持续去极化，这种行为与 ATP 产量增加一致； 3) 增加线粒体在胞吐作用位点的运动性。这些发现与线粒体动力学和生物能学的重新排列以维持受调节的胞吐作用一致。最后，阻断线粒体 ATP 合酶会抑制肌动球蛋白复合物的组装（一个主要的能量需求步骤），而阻断复合物 I 的活性不会影响胞吐作用，这表明在刺激分泌过程中使用不同的途径来调节线粒体代谢。

项目成果

期刊论文数量（23）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Polyethylenimine-mediated expression of transgenes in the acinar cells of rats salivary glands in vivo.

聚乙烯亚胺介导的大鼠体内唾液腺腺泡细胞中转基因的表达。

DOI：
发表时间：
2014
期刊：
影响因子：
0
作者：
Sramkova, Monika;Parente, Laura;Wigand, Timothy;Aye, Myo;Shitara, Akiko;Weigert, Roberto
通讯作者：
Weigert, Roberto

Altered endosome biogenesis in prostate cancer has biomarker potential.

前列腺癌中改变的内体生物发生具有生物标志物潜力。

DOI：
发表时间：
2014-12
期刊：
影响因子：
0
作者：
Johnson, Ian R D;Parkinson;Shandala, Tetyana;Weigert, Roberto;Butler, Lisa M;Brooks, Doug A
通讯作者：
Brooks, Doug A

Discovery of new cargo proteins that enter cells through clathrin-independent endocytosis.

发现通过网格蛋白独立的内吞作用进入细胞的新货物蛋白。

DOI：
发表时间：
2009-05
期刊：
影响因子：
0
作者：
Eyster, Craig A;Higginson, Jason D;Huebner, Robert;Porat;Weigert, Roberto;Wu, Wells W;Shen, Rong;Donaldson, Julie G
通讯作者：
Donaldson, Julie G

Expression of plasmid DNA in the salivary gland epithelium: novel approaches to study dynamic cellular processes in live animals.

唾液腺上皮中质粒 DNA 的表达：研究活体动物动态细胞过程的新方法。