Biology, myosin light chain, dictyostelium discoideum, mutants
Biology | Cell and Developmental Biology | Chemistry | Physical Sciences and Mathematics
Myosin is a molecular motor that in conjunction with actin filaments produces contraction. In muscle and non-muscle cells, myosin is activated by the phosphorylation of its regulatory light chain subunits (RMLC) by myosin light chain kinase (MLCK). In Dictyostelium, a genetically manipulable, unicellular slime mold, the identified MLCK is MLCK-A. MLCK-A varies from most MLCKs in that it is not calcium/calmodulin dependent and it exhibits a low specific activity. MLCK-A is regulated by autophosphorylation and external stimuli.
MLCK-A has been shown to respond to concavalin A (con A) by increasing RMLC phosphorylation. However, in MLCK-A null cells, there is a similar amount of RMLC phosphorylation as in untreated wild type cells, indicating the presence of another kinase. cAMP has also been shown to increase RMLC phosphorylation and cGMP. The link between RMLC phosphorylation and cGMP has been further strengthened by results that mutants that are unable to increase levels of cGMP do not increase RMLC phosphorylation when stimulated by cAMP. Also, when cGMP is added to fresh lysates of cells, MLCK-A activity is shown to increase.
In this research, experimentation was done to further understand the regulation of this kinase, MLCK-A, and RMLC. cGMP mutants were con A treated in order to test whether an increase in RMLC phosphorylation via MLCK-A can be activated without an increase in cGMP. No results were obtained for the mutants and the treatment of the wild types produced varied and unexpected results. I also attempted to introduce truncated versions of the gene for MLCK-A (mlkA) into mlkA null cells in order to understand which regions of the kinase are necessary for RMLC phosphorylation. However, the cells never survived far enough beyond the plasmid insertion for con A treatment. Finally, wild type cells were treated with thiabendazole, a drug that depolymerizes microtubules, in order to see if the loss of one cytoskeletal component will be compensated for by actin and myosin, as shown by an increase in RMLC phosphorylation. The results do not provide an indication as to whether or not this compensation occurs.
Department 1 Awarding Honors Status
Peasley, E. (1995). Experimentation in the Pursuit of the Regulation of the Regulatory Myosin Light Chain in Dictyostelium discoideum (Undergraduate honors thesis, University of Redlands). Retrieved from https://inspire.redlands.edu/cas_honors/668