Functional characterization of AbpG (Actin-binding protein G) – Its role in regulating the chemotactic migration of Dictyostelium cells

• Main Authors: Wei-Chi Lin, 林韋齊
• Other Authors: Mei-Yu Chen
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/2bt7ej

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 103 === Chemotactic cell migration is an important process involved in various physiological and pathological events, including inflammatory and immune responses and cancer metastasis, and the underlying mechanisms are not fully understood. The cell behavior and mechanisms of chemotactic migration in the simple eukaryote Dictyoselium discoideum are similar to those in higher organisms. Therefore, we used Dictyoselium discoideum as a model system to study the molecular mechanisms and regulation of chemotactic cell migration. The advantages of this model system include the many available genetic tools, and the existence of a haploid state that facilitates genetic screening. Aiming to uncover novel molecular players in the complex network underlying chemotactic cell migration, members in our laboratory had performed a restriction enzyme-mediated integration (REMI) screening in Dictyostelium for mutants defective in the chemotactic response to cAMP. In this thesis study, through analyzing one of the collected chemotaxis-defective mutants, i.e., T6#16, we have identified a previously uncharacterized gene that is involved in the regulation of cell motility, and named it acting-binding protein G (abpG) after the functional characterization of its product. Consistent with the importance of cell migration in Dictyostelium development, abpG- cells showed abnormal developmental morphology, forming smooth plaques on the bacteria lawn and fruiting bodies that were smaller than wild-type ones on the non-nutrient agar. Using micropipette chemotaxis assays, we found that abpG- cells could sense and move towards cAMP, yet they migrated more slowly than wild-type cells did. We expressed the Flag-tagged full-length AbpG in abpG- cells and tested its effects on development and cell migration, and the results showed that Flag-AbpG expression rescued the defects of abpG- cells, indicating that the loss of abpG function was responsible for these defects. Using immune-fluorescence cell staining or time-lapse microscopy, we found that, whether in fixed or live migrating cells, AbpG displayed a cortical/lamellipodial distribution that colocalized with F-actin signals. Sequence analysis on AbpG showed that AbpG contains a single type 2 calpoin homology (CH2) domain in its N-terminal region followed by two large coiled-coil regions. Domain mapping results showed that the aa501-550 region but not the CH2 domain of AbpG was required for the cortical/lamellipodial distribution. In vitro F-actin sedimentation assays demonstrated that AbpG(501-550) could directly interact with F-actin. Deleting the aa501-550 region from AbpG resulted in the loss of cortical/lamellipodial distribution, inability to interact with F-actin, and failure to rescue the developmental defect of abpG- cells, indicating the importance of the F-actin-interacting region, aa501-550, for the distinct subcellular distribution and cellular function of AbpG. To explore the effect of AbpG on actin dynamics, we performed in vitro F-actin polymerization and depolymerization assays, and the results showed that AbpG(401-600) could suppress actin polymerization and AbpG(501-550) and AbpG(401-600) could enhance F-actin depolymerization. Deleting the AbpG aa501-550 region from AbpG(401-600) disrupted its effects on actin dynamics. Consistent with the in vitro actin assay results, we found that abpG- cells contained higher levels of F-actin. To sum up, we have identified a novel actin-binding protein that can directly interact with F-actin, and provided evidence showing that it may modulate cell migration through regulating cellular actin dynamics.