The Role of Self-association in Fin1 Function on the Mitotic Spindle

Abstract

Stabilization of spindle microtubules during anaphase is essential for proper chromosome segregation. Fin1 is a budding yeast protein that localizes to the poles and microtubules of the spindle during anaphase and contributes to spindle stability. The N-terminal half of Fin1 is phosphorylated at multiple sites by the cyclin-dependent kinase Clb5-Cdk1, and dephosphorylation in anaphase triggers its localization to the spindle. The C-terminal half of Fin1 contains coiled-coil motifs that are required for its self-association. Here we investigated the functional importance of the two regions of Fin1. Fin1 mutants lacking the C-terminal coiled-coil domains localized to spindle pole bodies but not along spindle microtubules. These mutants failed to self-associate and displayed reduced binding to microtubules in vitro but were functional in vivo and stabilized anaphase spindles when dephosphorylated. Deletion of the Fin1 C terminus suppressed the lethal phenotypes of the phospho-mutant Fin1 5A . Our findings suggest that the N-terminal region of Fin1 is sufficient for its regulated function as a spindle-stabilizing factor and that this function involves association with the spindle pole body. The ability of the C-terminal region to promote Fin1 self-association and microtubule binding may underlie the lethal effects of the deregulated Fin1 5A mutant.The faithful segregation of chromosomes during mitosis requires precise regulatory control of spindle microtubule dynamics. During metaphase, microtubules exhibit high dynamic instability, which facilitates the capture of chromosomes at their kinetochores. At the onset of anaphase, microtubule dynamics are silenced, and the spindle is thereby stabilized (1-3). The change in microtubule dynamics at the onset of anaphase depends on regulated changes in the activity of proteins that influence microtubule behavior (4).The budding yeast protein Fin1 is a microtubule-stabilizing factor that associates with the spindle during anaphase. The activity of Fin1 is carefully regulated during the cell cycle (5). Phosphorylation in early mitosis by the cyclin-dependent kinase Clb5-Cdk1 inhibits Fin1 function and prevents its association with the spindle. Dephosphorylation in anaphase promotes its localization to the spindle, where it acts as a stability factor. A Fin1 phospho-mutant (Fin1 5A ) localizes prematurely to the spindle, alters metaphase spindle structure, and causes lethal errors in chromosome segregation (5).Fin1 purified from yeast self-associates to form a 10-nm-diameter filament (6). Fin1-Fin1 interactions have also been demonstrated in vivo (7,8). Oligomerization of Fin1 is mediated by sequences in the C-terminal portion of the protein, which are predicted to form coiled-coils (9). It has been suggested that Fin1 filaments provide structural support to the mitotic spindle, but the importance of Fin1 self-association for spindle stability is unknown (10, 11).We tested the importance of Fin1 self-association by analyzing the properties of a series of Fin1 truncations. We fo...

Fin1-PP1 Helps Clear Spindle Assembly Checkpoint Protein Bub1 from Kinetochores in Anaphase

“…7B). As ase1 Δ is synthetically lethal with fin1 Δ (Tong et al, 2004; Woodbury and Morgan, 2007b), we also examined Fin1 localization in cdc15-2 ase1 Δ mutant, which was the same as cdc15-2 single mutants cells (Fig. 7B).…”

The Toxoplasma gondii centrosome is the platform for internal daughter budding as revealed by a Nek1 kinase mutant

“…Since Nek1 orthologs have been associated with defects in mitotic progression (e.g. Chen et al, 2002;O'Regan et al, 2007;Smith et al, 2012;Woodbury and Morgan, 2007), we used MORN1 as a marker for the spindle, spindle poles and basal complex (the growing end of the daughter bud's cytoskeleton) to monitor the progression of mitosis and cytokinesis (Gubbels et al, 2006;Hu et al, 2006). As shown in the pre-mitotic panel of Fig.…”

An overview of Cdk1-controlled targets and processes

“…During this stage, the mitotic spindle is thought to be stabilized by Fin1, a self-associating coiled-coil protein that can form filaments between SPBs [390,391]. Fin1 is phosphorylated by Clb5-Cdk1 from S phase through metaphase [391,392], which inhibits the association of Fin1 with the spindle until Fin1 is dephosphorylated in anaphase due to degradation of Clb5 and activation of the phosphatase Cdc14 [392]. Fin1 dephosphorylation targets it to the poles and microtubules of the elongating spindle, where it contributes to spindle integrity and contributes to efficient chromosome segregation [392].…”