A protein interaction map of the mitotic spindle

Abstract

The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities.

Figure 1.

Comparison of intra-Dam1 complex interactions detected from genome-wide and focused two-hybrid screens. Protein interaction networks of subunits within the Dam1 complex derived from yeast two-hybrid studies and an in vitro expression experiment were generated with Cytoscape network visualization software. The bait construct for Ask1p (shown in red) was lethal to yeast and could not be screened. (A) The interactions reported from previous comprehensive two-hybrid screens identified seven of the 10 Dam1 complex subunits. (B) The network of interactions found by this study identified all 10 subunits of the Dam1 complex. The very high number of interactions detected between subunits is consistent with their association as a protein complex.

Figure 2.

A simplified spindle protein interaction network. This simplified network includes proteins with demonstrated spindle or chromosome functions as well as uncharacterized proteins that interact with multiple spindle proteins. Proteins that belong to the same complex or functional process are grouped into single nodes. cmplx, complex; APC, anaphase-promoting complex; CK1, casein kinase I; CKII, casein kinase II; CAF-I, chromatin assembly factor I; CRC, chromatin remodeling complex; CDK, cyclin-dependent kinase; HATs, histone acetyltransferases; HDACs, histone deacetylases; MAPs, microtubule-associated proteins; PP1, protein phosphatase I; PP2A, protein phosphatase 2A.

Figure 3.

She1-3GFP localizes to the mitotic spindle and the bud neck. (A) Localization of She1-3GFP during metaphase and anaphase. (B) She1-3GFP (green) colocalizes with Duo1-RFP (red) on the mitotic spindle. (C) She1-3GFP (green) colocalizes with Sli15-RFP (red) on microtubules. Bar, 4 μm.

Figure 4.

Comparison of protein interaction maps of Ndc80p phospho-mutants. The protein interaction maps shown here summarize the results of yeast two-hybrid screens performed using wild-type Ndc80p and phospho-mutants that mimic the phosphorylation and dephosphorylation of the four N-terminal Ipl1/Aurora B consensus sites, as baits. The color of the nodes corresponds with their GO Process classification. The interactions are classified as being common to all three alleles (blue lines), exclusive of the ndc80-4A allele (green lines), or exclusive of the ndc80-4D allele (orange lines). The protein interaction maps shown here were generated with OSPREY (http://biodata.mshri.on.ca/osprey; Breitkreutz et al., 2003).