Analysis of the regulation of intermediate filaments and desmosomes.

1.角蛋白丝是构成细胞骨架的重要中间丝之一。利用酵母双杂交技术，我们鉴定了一种新的蛋白质Trichoplein，它是一种角蛋白8的结合蛋白。Trichoplein显示与透明质酸和果胶的同源性。针对Trichoplein产生的抗体特异性识别细胞裂解物中相对分子质量为61 kDa的多肽。为了研究哺乳动物ERBIN [leucine-rich repeats and PSD-95/dlg-A/ZO-1（PDZ）domains]蛋白的功能，我们利用ERBIN和p0071在Madin-Darby犬肾（MDCK）细胞粘附连接和桥粒上的共定位，筛选酵母双杂交文库。ERBIN的亚细胞定位受Rho家族小GTP酶的调节。我们鉴定了Densin-180的结合蛋白δ-连环蛋白/神经斑嗜蛋白相关的犰狳重复蛋白（NPRAP），并表明Densin-180与突触处的δ-连环蛋白/NPRAP和N-钙粘蛋白相关。我们还分离出MAGUIN-1作为Densin-180结合蛋白，并提出二聚化或多聚化的潜力允许MAGUIN-1同时结合Densin-180和PSD-95，导致这些蛋白在突触后膜上的三元复合物组装。这些观察结果将为进一步研究细胞极性和细胞粘附以及理解癌症的病理机制铺平道路。

1.Keratin filament is one of intermediate filaments (IFs), which are major components of the cytoskeleton. By use of the yeast two-hybrid technique, we identified a new protein, Trichoplein, as a keratin 8-binding protein. Trichoplein shows homology to trichohyalin and plectin. Antibodies raised against Trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was co-immunoprecipitated with keratin 8/18, and co-localized with keratin 8/18 in cells.2.To elucidate the functions of mammalian LAP [leucine-rich repeats and PSD-95/dlg-A/ZO-1 (PDZ) domains] proteins, we screened yeast two-hybrid libraries, using ERBIN co-localized with p0071 at adherens junctions and desmosomes of Madin-Darby canine kidney (MDCK) cells. The subcellular localization of ERBIN could be regulated by Rho family small GTPases. We identified delta-catenin/neural plakophilin-related armadillo-repeat protein (NPRAP) as a binding protein for Densin-180, and showed that Densin-180 is associated with delta-catenin/NPRAP and N-cadherin at synapses. We also isolated MAGUIN-1 as a Densin-180-binding protein, and suggested that the potential to dimerize or multimerize allows MAGUIN-1 to bind simultaneously to both Densin-180 and PSD-95, leading to the ternary complex assembly of these proteins at the postsynaptic membrane. These observations will pave the way toward further research on cell polarity and cell adhesions as well as understanding of pathological mechanisms of cancer.