PgmNr Y525: Investigating the Role of Septin Phosphorylation in Controlling of Septin Organization at Cytokinesis.

Authors:
M. McQuilken 1 ; A. Grassetti 1 ; A. Verma 2 ; G. Harris 2 ; S. Gerber 1 ; S. Abrahamsson 3 ; R. Oldenbourg 2 ; A. Gladfelter 1


Institutes
1) Dartmouth College, Hanover, NH; 2) Marine Biological Laboratory, Woods Hole, MA; 3) The Rockefeller University, New York, NY.


Keyword: Cytoskeleton

Abstract:

     Septins are conserved filament-forming proteins that act in cytokinesis, membrane remodeling, cell polarization, and migration. They closely associate with membranes and, in some systems, components of the cytoskeleton.  Although septin function is critical for diverse cell events, it is not well understood how they assemble in vivo or how they are remodeled throughout the cell cycle.  In budding yeast, septins assemble initially as a patch and ring that then flips into a collar at the mother-bud neck as the bud emerges.  Late in the cell cycle the collar splits into two rings that generally will be disassembled prior to start of the next cell cycle.  The orientation of the dipole moment of GFP has been well established, and thus, constraining GFP to an endogenous septin allows for an assessment of septin organization in vivo by polarization microscopy.  Polarized fluorescence microscopy analysis has previously shown that septins are arranged in ordered, paired filaments and undergo a coordinated 90° reorientation during cytokinesis in vivo.  We hypothesized that the reason for this reorganization is the presence of two unequal and differentially arranged septin populations within a septin collar at the mother-bud neck and that over the course of the cytokinesis, the dominant population is no longer enriched in the higher order structure.  Data from our lab and others have confirmed a decrease in septin concentration in the collar at mother-bud neck during cytokinesis and we see the kinetics of the decrease depend on known septin regulators including the kinase Gin4.  Our goal is to understand the mechanisms regulating changes in septin abundance at septin ring splitting.  We have used polarization microscopy to screen mutant yeast strains with abnormally organized septins and have identified septin interactors important for distinct aspects of the assembly, stability, and reorganization of septins. We predicted that these identified regulators control post-translational modifications to trigger reorganization of a subset of septins during cytokinesis.  We therefore performed mass spectroscopy on septins synchronized at different stages of the cell cycle and found that there is a difference in the phosphorylation of the N- and C-terminal septin-interacting interfaces depending on whether septins are in collars or split rings.  These data suggest that selective phosphorylation of septins can control their polymerization and localization in a cell cycle dependent manner at cytokinesis.



Yeast Database Genetic Index
1. gene symbol: Gin4; systematic name: YDR507C
2. gene symbol: Shs1; systematic name: YDL225W
3. gene symbol: Cdc11; systematic name: YJR076C
4. gene symbol: Cdc10; systematic name: YCR002C
5. gene symbol: Cdc12; systematic name: YHR107C
6. gene symbol: Cdc3; systematic name: YLR314C
7. gene symbol: Rts1; systematic name: YOR014W
8. gene symbol: Cla4; systematic name: YNL298W
9. gene symbol: Bud4; systematic name: YJR092W