Phospho-Regulation of the Mid1 Scaffold in Contractile Ring Function
Cell & Molecular Biology
College of Liberal Arts and Sciences
The contractile ring is composed of F-actin, type II myosin and more than 100 cytokinetic ring proteins. In fission yeast, the anillin-related protein Mid1 plays a critical role in organizing the early steps of contractile ring formation and functions as a scaffold to bridge the cell cortex with the contractile ring. Cells lacking mid1 form off-centered, highly disorganized ring structures and exhibit severe cytokinesis defects. Coincident with its cortical accumulation, Mid1 becomes hyper-phosphorylated. Our previous research demonstrates that cyclin-dependent kinase, Cdc2, and the polo-like kinase, Plo1, directly phosphorylate Mid1. In addition to consensus phosphorylation motifs for Plo1 and Cdc2, Mid1 contains several RXXS motifs, which fit the phosphorylation consensus sequence for Sid2 kinase. Sid2 is the most downstream kinase in the Septation Initiation Network signaling cascade, which signals from the spindle pole body to trigger constriction of the contractile ring. A combination of site-directed mutagenesis with mass spectroscopy and two-dimensional phosphopeptide mapping identified multiple Sid2 phosphorylation sites within Mid1. Interestingly, phospho-site mutants displayed cell division defects, including sensitivity to low dose latrunculin A and disorganized actin localization. While phospho-site mutants maintained the mitotic spindle checkpoint, the majority of cells exhibited severe polarity phenotypes and Mid1 protein levels increased when compared to checkpoint activated cells expressing wild-type Mid1. Given that Mid1 departure from the ring coincides with Sid2 relocalization to the division site, Sid2 may temporally regulate the interaction of Mid1 with the membrane or other contractile ring components; our initial experiments favor Sid2 regulating Mid1 dissociation from the cell cortex.
Chicago Cytoskeleton Meeting
Hart, Dawn; DeWitt, Ashley; Kranz, Elizabeth; Phelan, Jennifer; Nader, Brandon; Gould, Kathleen L.; and Clifford Hart, Dawn M., "Phospho-Regulation of the Mid1 Scaffold in Contractile Ring Function" (2011). Faculty Scholarly Dissemination Grants. 407.