To carry out chromosome segregation faithfully during mitosis, the mitotic spindle must undergo precise changes at appropriate cell cycle stages (for a review see Botstein et al. 1997
). In budding yeast, the microtubule organizing center, or spindle pole body (SPB), duplicates after passage through G1
. A bipolar spindle then assembles and establishes attachments to each pair of sister chromatids. At this stage, cohesion between sister chromatids prevents the spindle from elongating past a metaphase length of ~1–2 μm (Michaelis et al. 1997
). At the onset of anaphase, chromatid cohesion is eliminated by action of the anaphase promoting complex (APC), a ubiquitin ligase that targets several proteins for degradation during mitosis (King et al. 1995
). With the loss of cohesion, sister chromatid separation and spindle elongation can occur to segregate the chromosomes. Finally, after spindle elongation, the spindle breaks down as the cells exit mitosis.
The mitotic spindle is primarily composed of microtubules. However, several other proteins are required for spindle assembly, function, and regulation (for a review see Sobel 1997
; Winsor and Schiebel 1997
). In yeast, spindle proteins include the kinesin-related motors Cin8p, Kip1p, and Kar3p (Roof et al. 1992
; Saunders and Hoyt 1992
; Saunders et al. 1997
), which provide the forces and structure required to assemble a bipolar spindle and to facilitate spindle elongation. There are also nonmotor microtubule-associated proteins such as Stu1p (Pasqualone and Huffaker 1994
), Ase1p, and Bim1p that are associated with mitotic spindles. Ase1p plays a structural role by cross-linking microtubules in the spindle midzone (Pellman et al. 1995
). Bim1p has an incompletely understood role in spindle function (Schwartz et al. 1997
; Tirnauer et al. 1999
), as well as a role in cytoplasmic microtubule–mediated spindle migration and orientation (Korinek et al. 2000
; Lee et al. 2000
In addition to the structural and mechanical components required to form a functional spindle, other factors are needed to attach the chromosomes to spindle microtubules. This attachment occurs at kinetochores, multiprotein complexes associated with centromeric DNA. Although the organization of the DNA-binding components of the yeast kinetochore has been characterized (Sorger et al. 1994
; Espelin et al. 1997
; Meluh and Koshland 1997
), complete elucidation of kinetochore activities requires the identification of all of the proteins involved. In addition, the mechanism by which the kinetochore attaches to spindle microtubules, and how this attachment is regulated, remain to be determined.
Changes in the mitotic spindle, particularly the transition to anaphase spindle elongation, are under tight cell cycle control. In addition to the regulation by the APC and the cyclin-dependent kinase, Cdc28p, spindle function is also monitored by the spindle assembly checkpoint. This checkpoint regulation is mediated by at least seven proteins: Mad1p, Mad2p, Mad3p, Bub1p, Bub2p, Bub3p, and Mps1p (Hoyt et al. 1991
; Li and Murray 1991
; Weiss and Winey 1996
). In response to spindle damage or an unattached kinetochore, the spindle assembly checkpoint directly inhibits the Cdc20p-bound form of the APC resulting in a metaphase arrest (Hwang et al. 1998
). In yeast, this arrest results in a large-budded cell with a short mitotic spindle and undivided DNA.
Previous work suggested that the S
proteins Duo1p and Dam1p are required for spindle function (Hofmann et al. 1998
; Jones et al. 1999
). Two-hybrid analysis and in vitro binding studies indicated that these proteins interact with each other physically. Both Duo1p and Dam1p localize along the length of the mitotic spindle, and Dam1p binds directly to microtubules in vitro. Moreover, temperature-sensitive mutants of both genes show spindle defects. Here, we characterize a collection of novel temperature-sensitive alleles of duo1
. Our analyses show that Duo1p and Dam1p form a conserved protein complex in vivo that is required for diverse aspects of spindle integrity and for kinetochore function.