Dictyostelium discoideum Paxillin Regulates Actin-Based Processes

Abstract

Paxillin is a key player in integrating the actin cytoskeleton with adhesion, and thus is essential to numerous cellular processes including proliferation, differentiation and migration in animal cells. PaxB, the Dictyostelium discoideum orthologue of paxillin, has been shown to be important for adhesion and development, much like its mammalian counterpart. Here, we use the overproduction of PaxB to gain better insight into its role in regulating the actin cytoskeleton and adhesion. We find that PaxB overexpressing (PaxBOE) cells can aggregate and form mounds normally, but are blocked in subsequent development. This arrest can be rescued by addition of wild-type cells, indicating a non-cell autonomous role for PaxB. PaxBOE cells also have alterations in several actin-based processes, including adhesion, endocytosis, motility and chemotaxis. PaxBOE cells exhibit an EDTA-sensitive increase in cell-cell cohesion, suggesting that PaxB-mediated adhesion is Ca2+ or Mg2+ dependent. Interestingly, cells overexpressing paxB are less adhesive to the substratum. In addition, PaxBOE cells display decreased motility under starved conditions, decreased endocytosis, and are unable to efficiently chemotax up a folate gradient. Taken together, the data suggest that proper expression of PaxB is vital for the regulation of development and actin-dependent processes.

Paxillin and Phospholipase D Interact To Regulate Actin-Based Processes in Dictyostelium discoideum

“…Cells were washed twice with PBM and then starved on an 8-well chambered slide for 6 h. Cells were fixed (3.7% formaldehyde solution in phosphate-buffered saline [PBS] for 5 min) and permeabilized (0.2% Triton X-100 solution in PBS for 5 min). PaxB and PldB were detected with peptide-purified anti-PaxB and anti-PldB antibodies (10,15), respectively, and with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG. F-actin was labeled with 8 U/ml rhodamine-phalloidin (Invitrogen, Eugene, OR) for 1 h in the dark.…”

“…A total of 1 ϫ 10 7 cells were collected and boiled for 3 min in SDS-PAGE sample buffer in a final volume of 500 l. Proteins were separated by SDS-PAGE, transferred to Hybond-P membranes (Amersham Bioscience, Piscataway, NJ), immunoblotted with a peptide-purified anti-PaxB or anti-PldB antibody (10,15), and visualized by use of an enhanced chemiluminescence substrate for horseradish peroxidase (HRP) detection (Pierce, Rockford, IL).…”

“…Beads were then removed from suspension with a magnetic rack. The samples were separated electrophoretically in SDS-10% polyacrylamide gels and immunoblotted with a peptide-purified anti-PaxB or anti-PldB antibody (10,15).…”

“…Like mammalian cells, Dictyostelium discoideum has paxillin and PLD. Dictyostelium discoideum paxillin, PaxB, is required for proper cell sorting and development past the mound stage (6) and is involved in regulating actin-required processes, including adhesion, endocytosis, and cell migration (15). One of three Dictyostelium discoideum PLD's, PldB, is implicated in multicellular development and cell migration (10,37).…”

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes

“…Paxillins are also known to localize to focal adhesions in mammalian cells and act as a complex adaptor proteins at the plasma membrane, leading to the reorganization of the actin cytoskeleton [306,307]. PaxB in Dictyostelium has been shown to be important for maintaining directionality during chemotaxis and localize to punctate regions on the basal surface of Dictyostelium cells (see Box 2) [308]. …”