Molecular Function of Palladin's Ig Domains in Cell Adhesion and Motility

描述(申请人提供)：细胞-细胞和细胞-基质粘连都需要肌动蛋白细胞骨架的参与。肌动蛋白细丝被交联成与细胞-细胞连接相关的环状环，以及锚定在细胞-基质连接处的收缩应激纤维。Palladin是最近发现的一种蛋白质，它定位于细胞-细胞和细胞-基质连接，也可以定位于应激纤维、Z盘和其他富含肌动蛋白的结构。除了细胞培养研究证明Palladin在肌动蛋白细胞骨架的组装中起关键作用外，另外两条证据表明Palladin在依赖肌动蛋白的细胞行为中发挥核心作用：(1)Palladin对于正常哺乳动物的发育至关重要，这从Palladin基因敲除小鼠的胚胎致命表型证明；(2)人类Palladin基因突变形成一种遗传性胰腺癌，这种疾病的特征是细胞间黏附丧失，以及细胞失去调节，肌动蛋白依赖的侵袭性细胞能动性丧失。这项建议的目标是了解Palladin在组织肌动蛋白阵列中的确切分子功能；具体地说，我们将在体外和体内检验Palladin作为肌动蛋白交联蛋白发挥作用的假设。Palladin以三种主要的亚型存在，含有三到五个拷贝的高度保守的Ig样结构域。已发表的对其他三种Ig结构域蛋白质的研究表明，Palladin的Ig结构域可能直接与f-肌动蛋白结合。我们已经产生的初步结果表明，纯化的全长Palladin与f-肌动蛋白结合，并在体外产生肌动蛋白束。此外，我们的结果表明，命名为ig3的结构域是与肌动蛋白结合的最小的Palladin片段，而ig4和Ig5不结合，并且包含ig3+ig4结构域的片段能够将肌动蛋白细丝交联成束。为了充分研究这种作用的分子基础，我们建议表达、纯化和鉴定Palladin的所有Ig结构域。我们的目标是(1)利用我们最近确定的IG3的溶液结构和合理的突变方法，最终绘制出palladin Ig结构域中包含的肌动蛋白结合位点的图谱，并研究肌动蛋白结合在palladin生物学活性中的作用；(2)检验palladin在体外和体内作为肌动蛋白交联蛋白的假设，并通过荧光和电子显微镜确定肌动蛋白产生的束的几何形状；以及(3)测试palladin形成同源二聚体的假设，绘制二聚体结构域，并确定二聚体形成是否对palladin的生物学功能至关重要。此外，我们还将确定Palladin的ig4结构域的结构，并研究该结构域中的一个新突变，该突变已在胰腺癌细胞系中发现。到目前为止，还没有关于Palladin或Palladin的近亲家族成员的Ig结构域的结构信息，因此我们提出的研究是第一次对Palladin的Ig结构域进行详细的结构/功能分析。 公共卫生相关性：肌动蛋白细丝形成复杂的亚细胞阵列，对于建立细胞形状、维持相邻细胞之间的黏附以及在细胞与其底层结缔组织之间产生连接至关重要。Palladin是最近被描述的一种蛋白质，它在组织这些肌动蛋白细丝阵列方面发挥了关键作用，并且Palladin的突变或过度表达与多种病理有关，包括致命的出生缺陷、皮肤、血管和神经系统的瘢痕形成，以及胰腺癌的侵袭。因此，了解Palladin的精确分子功能可能有助于深入了解控制正常细胞基本功能和导致病理细胞行为的功能障碍的细胞机制。

DESCRIPTION (provided by applicant): Both cell-cell and cell-matrix adhesions require the participation of the actin cytoskeleton. Actin filaments are crosslinked into circumferential rings that are associated with cell-cell junctions and contractile stress fibers that are anchored at cell-matrix junctions. Palladin is a recently identified protein that localizes to cell-cell and cell-matrix junctions, and also to stress fibers, Z-discs and other actin-rich structures. In addition to cell culture studies that demonstrate palladin's essential role in the assembly of the actin cytoskeleton, two other lines of evidence suggest that palladin plays a central role in actin-dependent cell behaviors: (1) palladin is critically important for normal mammalian development, as evidenced by the embryonic lethal phenotype of the palladin knockout mouse and (2) the human palladin gene is mutated in a form of inherited pancreatic cancer, which is a disease characterized by a loss of cell-cell adhesion and disregulated, actin-dependent, invasive cell motility. The goal of this proposal is to understand the precise molecular function of palladin in organizing actin arrays; specifically, we will test the hypothesis that palladin functions as an actin-crosslinking protein, both in vitro and in vivo. Palladin exists as three major isoforms, containing between three and five copies of a highly conserved Ig-like domain. Published studies of three other Ig-domain proteins suggest that palladin's Ig domains may bind directly to f-actin. We have generated preliminary results showing that purified, full-length palladin binds to f-actin and generates actin bundles in vitro. In addition, our results show that the domain designated Ig3 is the smallest palladin fragment that binds to actin, while Ig4 and Ig5 do not bind, and that a fragment containing the Ig3+Ig4 domains is able to cross-link actin filaments into bundles. To fully investigate the molecular basis for this effect, we propose to express, purify and characterize all of the Ig domains of palladin. Our aims are (1) to conclusively map the actin binding site contained within palladin's Ig domains, using our recently determined solution structure of Ig3 and rational mutagenesis approaches, and to investigate the role of actin-binding in palladin's biological activity, (2) to test the hypothesis that palladin functions as an actin- crosslinking protein in vitro and in vivo, and to determine the geometry of the actin-generated bundles by both fluorescence and electron microscopy, and (3) to test the hypothesis that palladin forms homodimers, map the dimerization domains, and establish if dimer formation is key to palladin's biological function. In addition, we will determine the structure of palladin's Ig4 domain and investigate a novel mutation in this domain that has been identified in a pancreatic cancer cell line. To date, no structural information has been published regarding the Ig domains of palladin or palladin's close family members, such that our proposed research represents the first detailed structure/function analysis of palladin's Ig domains. Public Health Relevance: Actin filaments form complex subcellular arrays that are essential for establishing cell shape, maintaining adhesion between neighboring cells, and generating connections between cells and their underlying connective tissue. Palladin is a recently described protein that plays a key role in organizing these actin filament arrays, and mutation or over-expression of palladin has been implicated in multiple pathologies, including lethal birth defects, scar formation in the skin, vasculature and nervous system, and the invasion of pancreatic cancer. Thus, understanding the precise molecular function of palladin is likely to yield insights into the cellular mechanisms that control both essential functions in normal cells, and dysfunctions that result in pathological cell behavior.