You decide to spend your career elucidating the mechanism(s) by which cells divide their cytoplasm by physically separating into two (cytokinesis) following separation of their chromosomes (M phase). You take advantage of reductionism, which is a general 'strategy/agenda' to investigate complicated cellular processes whereby understanding the behavior of the parts allows you to understand the behavior of the whole. To identify the genes whose gene products (proteins) are required for cytokinesis, you conduct a temperature sensitive genetic screen in the model system budding yeast and identify three mutants that you name cytoplasm separation mutant csm1, csm2, and csm3, all of which fail to properly split off a daughter bud cell.
a) In one or two sentences, explain why you conducted a temperature sensitive genetic screen to identify csm mutants.
b) Using a biochemical affinity chromatography strategy for proteins that bind to Csm2, you identify two more potential cytoplasm separation proteins that you name Csm4 and Csm5. You hypothesize that all five Csm proteins (1-5) localize to the division site between the mother cell and the daughter bud cell. In one or two sentences describe an experimental strategy to test this hypothesis and determine the temporal order (timing) of their localization to the division site in dividing budding yeast cells. Describe how you could test the functional role of Csm4 and Csm5 in chromosome separation.
