A search for proteins interacting with Costars

• Main Authors: Pei-Hsuan Wu, 吳沛宣
• Other Authors: Mei-Yu Chen
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/72022992382249897241

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 98 === Chemotactic cell movement is a phenomenon observed in many physiological and pathogenetic processes. The complex signaling and molecular mechanisms underlying chemotaxis have not been fully elucidated. Costars has been discovered previously in the laboratory in a forward genetics search for novel molecular players involved in chemotaxis-related mechanisms using Dictyostelium discoideum as a model system. Costars is a conserved protein; Costars-like sequences exist among diverse species, ranging from Dictyostelium to Homo sapiens. Dictyostelium cells lacking Costars display reduced migration during chemotaxis, and exhibit aberrant F-actin distribution; these defects can be rescued by the expression of either Dictyostelium Costars (Dd.Costars) or the human Costars homolog (Hs.mCostars). However, how Costars proteins function to regulate cell migration and actin cytoskeleton is still unclear. In hope to understand the cellular function and action mechanism of Costars through the identification of its binding partners, this thesis study aimed at finding Costars-interacting proteins. Using Hs.mCostars as the bait, we carried out yeast two-hybrid screening in human liver, testis and fetal brain cDNA libraries, and found Chromosome 14 open reading frame 45 (C14orf45), Gametogenetin (GGN), ATG16 autophagy related 16-like 2 (ATG16L2), and KIFC2 (kinesin family member C2) as candidates for Costars-interacting proteins. Further analyses in HEK293T cells detected the interaction of Hs.mCostars with the 639-791 aa fragment but not the full-length protein of KIFC2. In addition, using in vitro GST pull-down combined with Proteomics analysis, we also identified Heat shock 70 kDa protein 9B (mortalin-2) as a candidate for Hs.mCostars-interacting protein. Furthermore, 3 by immunoprecipitation from lysates of FLAG-Costars-expressing Dictyostelium cells and subsequent Proteomics analysis, we identified SmlA (small aggregates) and Cap34 (actin capping protein) as candidate Dd.Costars-interacting proteins. Immunofluorescence cell staining experiments demonstrated that Dd.Costars could colocalize with SmlA and Cap34. Whether these identified candidate Costars-interacting proteins participate in Costars-mediated regulation of cell movement and actin dynamics requires further investigation.