Conference Name: American Society for Cell Biology (ASCB) Annual Meeting
Ashley DeWitt, Elizabeth Kranz, Jennifer Phelan, Brandon Nader, Kathleen L. Gould and Dawn M. Clifford Hart
Grand Valley State University, Department of Cell and Molecular Biology, Allendale, MI 49401. Howard Hughes Medical Institute, Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
During cytokinesis, physical separation of one cell into two identical cells occurs through constriction of a protein-rich ring structure, called the contractile ring. 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. To identify specific sites of phosphorylation, a combination of site-directed mutagenesis (serine to non-phosphorylatable alanine) with mass spectroscopy and two-dimensional phosphopeptide mapping were used. Both methods identified multiple Sid2 phosphorylation sites within Mid1. To analyze the significance of Sid2 phosphorylation in vivo, phospho-site mutants were generated at the endogenous mid1 locus and examined for cell division defects. 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 contractile 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.