Core--Cytoskeletal Facility

创伤引起大量生物活性介质的释放，如脂质、激素和细胞因子（项目I-A）。这些配体引起功能和代谢的直接变化，但也改变细胞对随后刺激的反应（破坏性或建设性）。这些适应性反应的机制（分别称为启动或预处理）仍然是我们研究的重点（项目II、III、IV、VII和IX）。配体激活的第二信使信号、离子通量和蛋白磷酸化继续得到广泛的研究。但配体结合也会引发受体内化。使用转化模型，我们（和其他人）现在接受了几种生物活性介质（如儿茶酚、脂质和TNFa）的活化受体被网格蛋白包被的囊泡内化。包覆网格蛋白的核内体沿着微管轨道在细胞内运输。令人惊讶的是，最近的研究表明，受体内化对于启动一个新的信号传导阶段是必要的。内化g蛋白偶联受体（GPCR）发出的信号影响转录导向的蛋白激酶级联反应（包括PKC和MAPK模块），导致细胞反应性的持续变化（与项目V）。然而，在哺乳动物，特别是人类细胞中，先前的信号事件如何影响内化受体、内吞机制和微管细胞骨架之间的相互作用尚不清楚。此外，目前尚不清楚巨噬细胞等促炎细胞是否对内皮细胞或平滑肌细胞等易损靶点有类似的反应。我们假设创伤后释放的循环介质（肾上腺素能、溶脂和细胞因子）（项目V、VII和IX）增强了膜、受体和随后的信号传输的内化。我们最初专注于选择的配体：1 .与创伤的相关性；第三，激活PKC和MAPK模块。我们将研究这些测试配体如何影响人类和啮齿类动物髓系和非髓系细胞的组成膜/受体运输和内化依赖信号。这些研究将为评估目前可获得的治疗策略是否可以针对网格蛋白包被的内吞作用和基于微管的贩运形成基础。

Trauma provokes the release of a large number of bioactive mediators, such as lipids, hormones and cytokines (Project I-A). These ligands elicit immediate changes in function and metabolism, but also modify the cellular response(s) to subsequent stimuli (both destructively or constructively). The mechanism(s) of these adapted responses (termed priming or preconditioning, respectively) remain the focus of our investigations (with Projects II, III, IV, VII, and IX). Deservedly, ligand activated signaling by second messengers, ion-flux and protein phosphorylation continue to receive extensive study. But ligand binding also triggers receptor internalization. Using transformed models we (and others) now accept that activated receptors for several classes of bioactive mediators (such as catechols, lipids and TNFa) are internalized by clathrin coated vesicles. Clathrin coated endosomes are transported about the cell on microtubular tracks. Surprisingly, recent studies indicate that receptor internalization is necessary to initiate a novel phase of signaling. Signals emanating from internalized G-protein coupled receptors (GPCR) affect transcriptionally directed protein kinase cascades (including PKC and the MAPK modules) causing sustained changes in cell responsiveness (with Project V). However it is unclear how previous signaling events influence the interactions between internalized receptors, the endocytotic machinery and the microtubular cytoskeleton, in mammalian, especially human cells. Moreover it is unclear whether pro-inflammatory cells such as macrophages respond similarly to vulnerable targets such as endothelial or smooth muscle cells (with Project VI). We hypothesize that circulating mediators (adrenergic, lysolipid and cytokines ) (with Projects V, VII and IX) released after trauma augment internalization for membranes, receptors and subsequent signal traffic. We initially focus on ligands selected for i. pertinence to trauma, ii. mediation by GPCR, and iii, activation of PKC and MAPK modules. We will examine how these test ligands influence a. constitutive membrane/receptor trafficking and b. internalization dependent signaling in a set of myeloid and non myeloid cells from humans and rodents. These studies will form the basis for evaluating whether currently accessible therapeutic strategies can be directed against clathrin coated endocytosis and microtubule based trafficking.