Molecular Mechanisms regulating membrane trafficking in salivary glands

调节唾液腺膜运输的分子机制

• 批准号: 8344136
• 负责人: Roberto Weigert
• 金额: $ 130.12万
• 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/8344136
• 关键词: Acinar Cell; Acinus organ component; Actins; Address; Adrenergic Agents; Adrenergic Receptor; Affect; Agonist; Animals; Apical; Area; Basement membrane; Blood Circulation; Bypass; Cell Culture Techniques; Cell membrane; Cells; Complex; Confocal Microscopy; Cytochalasin D; Cytoplasm; Cytoplasmic Granules; Cytoskeleton; DNA; Dextrans; Docking; Drug Delivery Systems; Duct (organ) structure; Ductal; Ductal Epithelial Cell; Endocytosis; Epithelial Cells; Epithelium; Event; Exocrine Glands; Exocytosis; Experimental Models; Extracellular Space; F-Actin; Functional disorder; G-Protein-Coupled Receptors; Gene Silencing; Gene Transfer; Genetic; Gland; Green Fluorescent Proteins; Image; Imagery; Impairment; In Vitro; Intercalated Duct; Knockout Mice; Label; Life; Liquid substance; Major salivary gland structure; Mandible; Membrane; Membrane Protein Traffic; Microscopy; Modality; Modeling; Molecular; Molecular Weight; Motor; Motor Activity; Movement; Mus; Muscarinic Acetylcholine Receptor; Myosin ATPase; Nature; Organ; Parotid Gland; Pathway interactions; Peptides; Phase; Physiological; Physiology; Play; Process; Protein Secretion; Proteins; Rattus; Recruitment Activity; Resolution; Rest; Rodent; Role; Saliva; Salivary Glands; Salivary Proteins; Secretory Vesicles; Series; Serum Proteins; Side; Sorting - Cell Movement; Stromal Cells; Surface; System; Takeda brand of pioglitazone hydrochloride; Techniques; Tomatoes; Transgenic Mice; Transgenic Organisms; Viral; Water; adrenergic; base; basolateral membrane; design; dextran; gene therapy; immunocytochemistry; in vivo; in vivo Model; inhibitor/antagonist; latrunculin A; light microscopy; mouse model; novel; plasmid DNA; polarized cell; polypeptide; receptor; scaffold; small molecule; tool; two-photon; uptake

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 study the molecular machinery regulating the formation of the granules from the TGN and their fusion with the APM. We set up an experimental system aimed at imaging and tracking the secretory granules in the SGs of live animals and based on high-resolution intra-vital microscopy performed on a series of transgenic mouse models expressing selected fluorescently labeled molecules. Among them, a mouse expressing the soluble green fluorescent protein (GFP) in both the sub-mandibular and the parotid glands, enables a clear visualization of both the secretory granules and the APM. We estimated that in resting conditions, the major SGs contain approximately 2500-3000 granules per acinus, most of them accumulated in the sub apical area of the PM. Our analysis on the effect of various agonists of GPCRs has revealed two major differences between in vivo and ex-vivo models: 1) the stimulation of the beta-adrenergic but not the muscarinic receptors, enhances the mobility of the SCGs promoting their docking and subsequent fusion at the APM and 2) muscarinic receptors do not play any synergistic role with the adrenergic receptor during exocytosis. Furthermore, by using another mouse model, which expresses the Tomato fluorescent protein fused with a di-palmitoylated peptide, a well-established marker for the plasma membrane, we discovered that the SCGs after fusing with the plasma membrane completely collapse within 30-40 seconds. This result underscores another major differences between in vivo and ex-vivo models in which compound exocytosis (i.e. the sequential fusion of strings of SCGs), has been described as the primary modality of fusion. Notably, we also showed that the granules in close proximity of the APM recruit a series of cytosolic proteins such as actin, suggesting a role for the cytoskeleton during granule exocytosis. To address this issue we transduced the SGs of live rats with the small peptide Lifeact fused with GFP, a novel tool to label dynamically F-actin. Strikingly, we determined that actin is recruited onto the surface of the granules only after fusion has occurred, and it is released in to the cytoplasm only after their complete collapse. The impairment of the dynamics of the actin cytoskeleton, using pharmacological agents such as cytochalasin D (cyto D) or latrunculin A (lat A), did not affect the fusion of the SCGs with the APM, but it blocked substantially their collapse leading to the accumulation of fused granules which often expanded in size. Finally, we found that myosin IIa and IIb, two actin-based motor proteins are recruited on the fused SCGs and that their motor activity is required to drive the gradual collapse of the granules. These results suggest that the acto-myosin complex provides a contractile scaffold around the SCGs that facilitates the completion of the fusion at the APM. This machinery is utilized by other exocrine glands in which the gradual collapse of the SCGs is not energetically favored due to geometrical constrains. 2) Molecular machinery regulating endocytosis in salivary glands of live animals The role of endocytosis in the physiology of the SGs has never been elucidated. Uptake of proteins from either the apical or the basolateral domain of the ductal system have been described but never thoroughly investigated due to the lack of an appropriate experimental model. The presence under physiological conditions of salivary proteins in the bloodstream and of serum proteins in the saliva, argues strongly in favor of a constant and bi-directional transcytotic movement of proteins across the salivary gland epithelium. Our aim is to first define the endocytic pathways operating in SGs in vivo, and then to investigate the contribution of the endocytic events in the patho-physiology of the glands and especially during secretion. We set up various experimental systems in the SGs of live rodents aimed at imaging and studying endocytic events, which occur at either the apical or the basolateral membrane of the SGs epithelium, and in stromal cells. Interestingly, endocytosis in the SGs epithelium of a live animal is significantly reduced when compared with cell cultures, whereas endocytosis in stromal cells appears to occur at a faster rate than in vitro systems. Furthermore, we found that stimulation of the secretory activity of the SGs enhances endocytosis from the apical pole whereas does not have any effect on basolateral endocytosis or on uptake from stromal cells. Using both fluid-phase markers, such as fluorescently-labeled dextrans or smaller molecules, and probes that selectively label the PM, we found that in resting conditions the endocytic activity at the APM is extremely low, whereas stimulation of protein but not water secretion enhances internalization via compensatory endocytosis. Since the SGs are target organs for gene therapy we sought to investigate the endocytosis of plasmid DNA. By using a combination of pharmacological inhibitors, immunocytochemistry and IVM, we found that DNA is internalized in all the components of the SGs epithelium (intercalated ducts, large ducts and acini) without utilizing any of the canonical endocytic pathways described so far. Moreover, only a small proportion of internalized DNA is localized in the early endosomal compartments suggesting that lysosomal degradation is bypassed via endosomal escape. Finally, we observed that stimulation of protein secretion enhances the uptake of the DNA in acinar cells by the same compensatory endocytic pathway observed before. These results revealed unconventional endocytic pathways in live animals that may be exploited to better design non viral-based gene therapy. Endocytosis from the basolateral membrane of the SG epithelium was assessed by two main strategies by injecting fluorescently-labeled probes either systemically or directly into the SGs (fluid-phase endocytosis and non-selective membrane internalization. To characterize the nature of the endocytic pathways, we delivered to the SGs probes of different molecular weights (from 0.3 kDa up to 230 kDa) including dextrans, selected polypeptides, and hydrophobic molecules. Although at a slow rate, only small hydrophobic molecules, such as FM1-43 and DiI were endocytosed in the epithelium. All the other molecules were retained in the extracellular space without any evidence for endocytosis in acinar or ductal cells under both resting and stimulated conditions. These results suggest that the basement membrane surrounding the epithelial cells may act as a major barrier to control the access to the basolateral side of the epithelium. Indeed, under conditions in which the integrity of the basement membrane is compromised, most of the delivered molecules were internalized in the epithelium and accumulated in the endosomal compartment. These results highlight a major role of the basement membrane in controlling the accessibility of molecules to the basolateral side of the epithelium, a factor that needs to be taken in consideration when designing drugs that target plasma membrane receptors

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。我们的目标是研究调节 TGN 颗粒形成及其与 APM 融合的分子机制。我们建立了一个实验系统，旨在对活体动物 SG 中的分泌颗粒进行成像和跟踪，并基于在一系列表达选定荧光标记分子的转基因小鼠模型上进行的高分辨率活体显微镜检查。其中，在下颌下腺和腮腺中表达可溶性绿色荧光蛋白（GFP）的小鼠能够清晰地观察分泌颗粒和 APM。我们估计，在静息条件下，每个腺泡的主要 SG 含有大约 2500-3000 个颗粒，其中大多数聚集在 PM 的亚顶端区域。我们对各种 GPCR 激动剂作用的分析揭示了体内和离体模型之间的两个主要差异：1）刺激 β-肾上腺素受体而不是毒蕈碱受体，增强了 SCG 的移动性，促进其对接和随后的连接。 APM 处的融合和 2) 毒蕈碱受体在胞吐作用期间不与肾上腺素能受体发挥任何协同作用。此外，通过使用另一种小鼠模型，该模型表达与二棕榈酰化肽（一种成熟的质膜标记物）融合的番茄荧光蛋白，我们发现SCG与质膜融合后在30-40秒内完全崩溃。这一结果强调了体内和离体模型之间的另一个主要差异，其中复合胞吐作用（即 SCG 串的顺序融合）被描述为融合的主要方式。值得注意的是，我们还表明，靠近 APM 的颗粒会募集一系列胞质蛋白，例如肌动蛋白，这表明细胞骨架在颗粒胞吐作用过程中发挥着作用。为了解决这个问题，我们用与 GFP 融合的小肽 Lifeact 转导了活体大鼠的 SG，这是一种动态标记 F-肌动蛋白的新工具。引人注目的是，我们确定肌动蛋白仅在融合发生后才被募集到颗粒表面，并且仅在颗粒完全塌陷后才释放到细胞质中。使用细胞松弛素 D (cyto D) 或 latrunculin A (lat A) 等药物破坏肌动蛋白细胞骨架的动力学，不会影响 SCG 与 APM 的融合，但它基本上阻止了它们的崩溃，导致融合颗粒的堆积，其尺寸通常会扩大。最后，我们发现肌球蛋白 IIa 和 IIb（两种基于肌动蛋白的运动蛋白）在融合的 SCG 上被募集，并且它们的运动活动是驱动颗粒逐渐塌陷所必需的。这些结果表明，肌动球蛋白复合物在 SCG 周围提供了一个收缩支架，有助于 APM 融合的完成。这种机制被其他外分泌腺所利用，在这些外分泌腺中，由于几何限制，SCG 的逐渐塌陷在能量上并不有利。 2）调节活体动物唾液腺内吞作用的分子机制 内吞作用在 SG 生理学中的作用从未被阐明。已经描述了从导管系统的顶端或基底外侧域摄取蛋白质，但由于缺乏适当的实验模型，从未进行过彻底研究。在生理条件下，血流中唾液蛋白和唾液中血清蛋白的存在强烈支持蛋白质在唾液腺上皮上进行持续的双向转胞吞运动。我们的目标是首先定义在体内 SG 中运行的内吞途径，然后研究内吞事件在腺体病理生理学中的贡献，特别是在分泌过程中。我们在活体啮齿动物的 SG 中建立了各种实验系统，旨在成像和研究内吞事件，这些事件发生在 SG 上皮的顶膜或基底外侧膜以及基质细胞中。有趣的是，与细胞培养物相比，活体动物 SG 上皮的内吞作用显着减少，而基质细胞的内吞作用似乎比体外系统更快。此外，我们发现刺激 SG 的分泌活性会增强顶极的内吞作用，而对基底外侧内吞作用或基质细胞的摄取没有任何影响。使用流体相标记物（例如荧光标记的葡聚糖或较小的分子）和选择性标记 PM 的探针，我们发现在静息条件下，APM 的内吞活性极低，而刺激蛋白质但不刺激水的分泌会增强 APM 的内吞活性。通过补偿性内吞作用内化。由于 SG 是基因治疗的靶器官，我们试图研究质粒 DNA 的内吞作用。通过结合使用药物抑制剂、免疫细胞化学和 IVM，我们发现 DNA 内化到 SG 上皮的所有成分（闰管、大管和腺泡）中，而无需利用迄今为止描述的任何典型的内吞途径。此外，只有一小部分内化 DNA 位于早期内体区室中，这表明溶酶体降解是通过内体逃逸绕过的。最后，我们观察到刺激蛋白质分泌会通过之前观察到的相同补偿内吞途径增强腺泡细胞对 DNA 的摄取。这些结果揭示了活体动物中非常规的内吞途径，可用于更好地设计基于非病毒的基因疗法。 SG 上皮基底外侧膜的内吞作用通过两种主要策略进行评估：将荧光标记探针全身或直接注射到 SG 中（流体相内吞作用和非选择性膜内化）。为了表征内吞途径的性质，我们递送至不同分子量（从 0.3 kDa 至 230 kDa）的 SG 探针，包括葡聚糖、选定的多肽和疏水性尽管速度较慢，但​​只有小的疏水性分子（如 FM1-43 和 DiI）被内吞到上皮细胞中，所有其他分子都保留在细胞外空间，没有任何证据表明腺泡或导管细胞在静息和导管细胞中被内吞。这些结果表明，在上皮细胞完整性的条件下，上皮细胞周围的基底膜可能充当控制进入上皮细胞基底外侧的主要屏障。基底膜受到损害，大部分递送的分子被内化到上皮细胞中并积累在内体室中。这些结果强调了基底膜在控制分子到达上皮基底外侧的可及性方面的主要作用，这是设计靶向质膜受体的药物时需要考虑的因素