Title

Using the green fluorescence protein and an inducible expression system to study the dynamic changes of the actin cortex in Dictyostelium

Date of Completion

January 1998

Keywords

Biology, Cell

Degree

Ph.D.

Abstract

Cell motility is a very important life phenomenon. It involves dynamic and coordinated changes of the actin cortex in cells. Actin binding proteins play a critical role in modulating the actin cytoskeleton which in turn leads to cell motility. In this study, the dynamic changes of the actin cortex in Dictyostelium cell were studied by molecular and cell biology approaches.^ A 240 kD actin binding protein, which can cross-link actin filaments into a three dimensional meshwork in vitro, has been isolated form the Dictyostelium. In order to understand the in vivo function of the protein, I sought to clone the gene so that mutants can be generated for further study. A 2.7 kB fragment was cloned by PCR using degenerate primers designed from peptide sequences. Surprisingly, the sequence showed high homology to fatty acid synthase. This is intriguing because the protein may act as a link between lipid synthesis and the cytoskeleton. During this study, a new method, named, partial inverse PCR (PIPCR) was developed for cloning flanking sequences. Cloning the rest of the gene is still in progress.^ The application of an inducible expression system to regulate the expression of the myosin heavy chain (MHCII) and RacB protein was tested. The inducible system showed very good induction kinetics and dose-response for a reporter gene, $\beta$-galactosidase. The induction profiles for MHCII and RacB protein were not as good as the reporter gene. However, the system was sufficient to partially revert the phenotype of the MHCII mutant. Also, when RacB was conditionally over-expressed, the cells developed an aberrant phenotype.^ A green fluorescent protein-actin binding domain (GFP-ABD) probe was developed to visualize the dynamic change of the actin cortex in live cells. Using this probe, new dynamic F-actin containing structures were observed. They included some actin rings and puntate structures on the ventral surface, lobular structures on the ventral side and some rocketing motility in the cytoplasm. Also, the cortical flow of actin filaments could be observed directly. The process is probably involved in generating traction during cell motility and it depends on myosin(s) other than myosin II. ^