Phosphorylation of Spc110p by Cdc28p-Clb5p kinase contributes to correct spindle morphogenesis in S. cerevisiae

Abstract

Spindle morphogenesis is regulated by cyclin-dependent kinases and monitored by checkpoint pathways to accurately coordinate chromosomal segregation with other events in the cell cycle. We have previously dissected the contribution of individual B-type cyclins to spindle morphogenesis in Saccharomyces cerevisiae. We showed that the S-phase cyclin Clb5p is required for coupling spindle assembly and orientation. Loss of Clb5p-dependent kinase abolishes intrinsic asymmetry between the spindle poles resulting in lethal translocation of the spindle into the bud with high penetrance in diploid cells. This phenotype was exploited in a screen for high dosage suppressors that yielded spc110Δ13, encoding a truncation of the spindle pole body component Spc110p (the intranuclear receptor for the γ-tubulin complex). We found that Clb5p-GFP was localised to the spindle poles and intranuclear microtubules and that Clb5p-dependent kinase promoted cell cycle dependent phosphorylation of Spc110p contributing to spindle integrity. Two cyclin-dependent kinase consensus sites were required for this phosphorylation and were critical for the activity of spc110Δ13 as a suppressor. Together, our results point to the function of cyclin-dependent kinase phosphorylation of Spc110p and provide, in addition, support to a model for Clb5p control of spindle polarity at the level of astral microtubule organisation.

Dissecting the involvement of formins in Bud6p-mediated cortical capture of microtubules in S. cerevisiae

“…Time lapse recordings were carried out as previously described (Huisman et al, 2007) using a Nikon Eclipse E800 with a CFI Plan Apochromat 100ϫ, N.A. 1.4 objective, Chroma Technology filter sets and a Coolsnap-HQ CCD camera (Roper Scientific).…”

“…When indicated, still images were also obtained by compiling five-plane z-stacks. Images of cells co-expressing GFP and CFP-tagged constructs were obtained by a protocol that discriminates between CFP and GFP using a CFP/YFP filter set (Huisman et al, 2007). Images were processed using Metamorph software and digital overlays were used for scoring.…”

“…The old SPB is dynamically tethered to the emerging bud by its existing astral MTs. Astral MT organisation at the new SPB awaits spindle assembly (Shaw et al, 1997), a delay dependent upon Sphase cyclin-dependent kinase activity (Segal et al, 2000b;Huisman et al, 2007). Thus, the new SPB can move away from the bud neck as the spindle assembles.…”

Ase1p phosphorylation by cyclin-dependent kinase promotes correct spindle assembly inS. cerevisiae

“…For time-lapse recordings, logarithmic either in 3.7% formaldehyde for 30 min for scoring budding index and progression of the spindle pathway, or in 70% ethanol for analysis of DNA content by flow cytometry. 2 A second aliquot was processed for preparation of whole cell extracts and protein gel blot analysis. 47 Protein gel blots were probed with monoclonal antibody 12C5A (Roche) and anti-tubulin B512 (Sigma).…”

“…1 Using S. cerevisiae as a model, we have dissected a sequence of events underlying mitotic spindle morphogenesis under CDK control in order to demonstrate contributions by single B-type cyclins and determine the key targets involved. 2,3 The organization of a spindle to stage chromosomal segregation is a multistep process 4 that is orchestrated in yeast by the spindle pole body (SPB), the counterpart of the animal centrosome. 5 Following duplication, SPBs embedded in the nuclear envelope organize two sets of intranuclear microtubules (MTs) setting up a bipolar spindle: those attaching sister kinetochores to opposite poles to achieve biorientation (kinetochore MTs), and those creating an antiparallel bundle (interpolar MTs) Spindle morphogenesis and dynamics follow an orderly sequence of events coupled to the oscillatory activation of cyclindependent kinase (CDK).…”