Molecular mechanisms of membrane remodeling

膜重塑的分子机制

• 批准号: 9556671
• 负责人: Roberto Weigert
• 金额: $ 147.69万
• 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/9556671
• 关键词: 5' -AMP-activated protein kinase; Acinar Cell; Actins; Actomyosin; Albumins; Apical; Behavior monitoring; Bile fluid; Biliary; Binding Sites; Biological Models; Biophysics; Blood; Cell Culture Techniques; Cell membrane; Cell physiology; Cells; Cellular Membrane; Cellular Metabolic Process; Characteristics; Complement; Complex; Confocal Microscopy; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; Development; Dextrans; Endocytosis; Energy Metabolism; Ensure; Environment; Epithelium; Equilibrium; Event; Exocytosis; F-Actin; Face; Family; Filament; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Hepatocyte; Homeostasis; Hour; Image; Immunofluorescence Immunologic; Individual; Injectable; Intravenous; Investigation; Kinetics; Knockout Mice; Lactation; Lipids; Liver; Maintenance; Mammary gland; Mediating; Membrane; Metabolism; Methods; Microscopy; Molecular; Molecular Genetics; Molecular Weight; Movement; Mus; Myosin Light Chain Kinase; Myosin Type II; Neonatal; Neoplasm Metastasis; Organ Culture Techniques; Oxytocin; Pathologic; Permeability; Pharmacology; Phosphorylation; Physiological; Play; Process; Protein Isoforms; Proteins; Recruitment Activity; Regulation; Resolution; Rodent; Role; STK11 gene; Salivary Glands; Scanning Electron Microscopy; Secretory Vesicles; Series; Shapes; Signal Transduction; Stream; Structure; Surface; System; Techniques; Testing; Tight Junctions; Time; Tissues; Transgenic Mice; Transmission Electron Microscopy; Wild Type Mouse; apical membrane; base; beta-adrenergic receptor; bile canaliculus structure; breast imaging; carboxyfluorescein; cellular imaging; cellular microvillus; expectation; in vivo; intravital imaging; intravital microscopy; light microscopy; member; membrane flux; milk supply; non-muscle myosin; protein biomarkers; scaffold; secretory protein; spatiotemporal; time use; tool; trafficking; trans-Golgi Network; tumor progression; two-photon

Molecular basis of membrane remodeling during secretion at the plasma membrane. Secretory epithelia such as salivary glands (SGs), mammary glands (MGs) and the liver represent three robust model systems to study various aspects of the remodeling of membranes during intracellular trafficking processes, such as constitutive and regulated protein- and lipid-secretion, and plasma membrane homeostasis. 1) Regulated exocytosis in salivary glands In SG 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 APM upon stimulation of GPCRs, 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. Last year, we focused on elucidating the mechanisms of recruitment and regulation of NMII. We showed that NMIIA and NMIIB are recruited onto the SGs after their fusion with the APM, and 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. 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 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 NMII filaments. Indeed, F-actin facilitates the recruitment of the myosin light chain kinase (MLCK), which in turn activates both NMII isoforms via the phosphorylation of two residues (S19 and T18) which initiate the formation of contractile filaments. Finally, we discovered that three members of the Septin family of GTPases, Septin 2, 6, and 7, are recruited on the SGs after their fusion with the APM, and control the activation of MLCK. These results provide a springboard to begin investigating the biophysical basis underlying the process of membrane integration. 2) Lipid droplets secretion in mammary glands In MGs, the lipid droplet (LD) fraction of milk supplies preformed lipids for neonatal development, and the assembled LDs are secreted by a unique apocrine mechanism, that has never been investigated in vivo. To this end, we developed a method for the intravital imaging of mammary cells in transgenic mice that express fluorescently tagged marker proteins. For the first time, we described the kinetic analysis of LD growth and secretion at peak lactation in real time. LD transit from basal to apical regions was slow (0-2 um/min) and frequently intermittent. Droplets grew by the fusion of preexisting droplets, with no restriction on the size of fusogenic partners. Most droplet expansion took several hours and occurred in APM nucleation centers, either close to or in association with the apical surface. Interestingly, droplets were coated with F-actin and NMIIA, although their function is, at the moment, poorly understood. Large droplets gradually pushed the APM, inducing its extensive deformation, which resulted in the budding of the droplets into the apical lumen. Droplets continued to expand as they were emerging from the cell. Contrary to expectations, LDs attached to the APM, but still associated with the cytoplasm were released after oxytocin-mediated contraction of the myoepithelium. This initial investigation will serve as a basis to unravel the machinery regulating the deformation and the budding of the apical plasma membrane. Plasma membrane homeostasis in the liver The bile canaliculi network in the liver is formed by the apical domains of the hepatocytes. Its homeostasis is the result of a balance between endo- and exocytic processes, which tightly regulate the flux of membranes and the maintenance of functional tight junctions. Our aim is to investigate the mechanisms that regulate the homeostasis of the bile canaliculi and ensure their proper functionality. As a first step, we determined 1) a high-resolution structure of the bile network in vivo, by using a combination of high-resolution confocal and serial block-face scanning electron microscopy; and 2) its dynamic behavior by monitoring changes in the canaliculi structure and in bile transport, by using time-lapse intravital microscopy. Interestingly, we observed that bile canaliculi undergo continuous peristaltic contractions and expansions, which facilitate bile secretion. We found that this process is mediated by the actomyosin cytoskeleton, as inhibition of F-actin assembly or a RhoKinase controlling NMIIA dramatically blocks the peristaltic movements and delays biliary flow. In addition, we investigated the role of the liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK), which play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. To this end, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-). We found that LKB1 plays a fundamental role in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. These studies have unraveled a key role for both the actomyosin cytoskeleton and molecules implicated in membrane trafficking and cell metabolism.

质膜分泌过程中膜重塑的分子基础。唾液腺 (SG)、乳腺 (MG) 和肝脏等分泌上皮细胞代表了三个强大的模型系统，用于研究细胞内运输过程中细胞膜重塑的各个方面，例如组成型和调节性蛋白质和脂质分泌，以及质膜稳态。 1) 唾液腺中受调节的胞吐作用 在 SG 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 GPCR 刺激下与 APM 融合，从而将其内容物释放到腺泡小管。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。去年，我们重点阐述了NMII的招募和监管机制。我们表明，NMIIA 和 NMIIB 在与 APM 融合后被招募到 SG 上，并且它们的收缩活动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。通过使用条件敲除小鼠，我们确定 NMIIB 需要通过稳定 F-肌动蛋白支架并提供将膜推向 APM 的连续收缩活动来控制颗粒膜整合的初始步骤。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 NMII 丝的正确组装。事实上，F-肌动蛋白促进肌球蛋白轻链激酶 (MLCK) 的募集，进而通过两个残基（S19 和 T18）的磷酸化激活两种 NMII 亚型，从而启动收缩丝的形成。最后，我们发现 GTPases Septin 家族的三个成员 Septin 2、6 和 7 在与 APM 融合后被招募到 SG 上，并控制 MLCK 的激活。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) 乳腺中的脂滴分泌 在 MG 中，牛奶中的脂滴 (LD) 部分为新生儿发育提供预先形成的脂质，并且组装的 LD 通过独特的顶浆分泌机制分泌，这种机制从未在体内研究过。为此，我们开发了一种对表达荧光标记标记蛋白的转基因小鼠乳腺细胞进行活体成像的方法。我们首次实时描述了泌乳高峰期 LD 生长和分泌的动力学分析。 LD 从基底区域到顶端区域的传输很慢（0-2 um/min）并且经常是间歇性的。液滴通过先前存在的液滴融合而生长，融合伙伴的大小没有限制。大多数液滴膨胀需要几个小时，并且发生在 APM 成核中心，靠近或与顶面相关。有趣的是，液滴上涂有 F-肌动蛋白和 NMIIA，尽管目前人们对它们的功能知之甚少。大液滴逐渐推动 APM，引起其广泛变形，导致液滴萌芽进入顶腔。液滴在从细胞中出现时继续膨胀。与预期相反，在催产素介导的肌上皮收缩后释放出附着在 APM 上但仍与细胞质相关的 LD。这项初步研究将作为解开调节顶端质膜变形和出芽的机制的基础。肝脏中的质膜稳态肝脏中的胆小管网络由肝细胞的顶端区域形成。其稳态是内吞和外吐过程之间平衡的结果，该过程严格调节膜的通量和功能性紧密连接的维持。我们的目的是研究调节胆小管稳态的机制并确保其正常功能。作为第一步，我们确定了 1) 体内胆汁网络的高分辨率结构，通过使用高分辨率共焦和串行块面扫描电子显微镜的组合； 2）通过使用延时活体显微镜监测胆管结构和胆汁运输的变化来观察其动态行为。有趣的是，我们观察到胆小管经历持续的蠕动收缩和扩张，从而促进胆汁分泌。我们发现这个过程是由肌动球蛋白细胞骨架介导的，因为抑制 F-肌动蛋白组装或控制 NMIIA 的 RhoKinase 会显着阻止蠕动运动并延迟胆汁流动。此外，我们还研究了肝激酶 B1 (LKB1) 及其下游效应器 AMP 激活蛋白激酶 (AMPK) 的作用，它们通过调节膜运输和能量代谢在极性建立中发挥关键作用。为此，我们使用了肝脏特异性（白蛋白-Cre）LKB1基因敲除小鼠（LKB1(-/-)）。我们发现LKB1在体内维持功能性紧密连接（TJ）中发挥着基础性作用。透射电镜检查免疫荧光研究表明，具有微绒毛的肝细胞顶膜基本上延伸到 LKB1(-/-) 肝脏的基底外侧区域，LKB1 的缺失导致了更长更宽的小管结构。与连接蛋白 cingulin 的错误定位相关 为了测试连接功能，我们使用活体显微镜来量化 6-羧基荧光素二乙酸酯 (6-CFDA) 的转运动力学，该物质在肝细胞中加工成其荧光衍生物 6-羧基荧光素 (6-CFDA)。 CF)并分泌到小管中，在LKB1(-/-)小鼠中，6-CF大部分保留在肝细胞中，小管分泌。延迟，并且 6-CF 出现在血液中 为了测试 6-CF 是否通过可渗透的 TJ 转运，我们静脉注射低分子量（3 kDa）右旋糖酐与 6-CFDA 组合。在野生型小鼠中，3 kDa 葡聚糖保留在脉管系统中，而在 LKB1(-/-) 小鼠中，它迅速出现在异常胆小管中，证实连接破坏导致血流和胆小管之间的细胞旁交换。这些研究揭示了肌动球蛋白细胞骨架和参与膜运输和细胞代谢的分子的关键作用。