Analysis of the regulation of intermediate filaments and desmosomes.

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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.