One of the most important events during the cell cycle is chromosome segregation and errors in this process will lead to chromosome missegregation. To separate sister chromatids into daughter cells, sister kinetochores must be attached to the microtubules emanating from opposite spindle poles in order to establish bipolar attachment. Even though this process is highly regulated, incorrect attachment takes place occasionally. Syntelic attachment occurs when both sister kinetochores are connected to microtubules from the same spindle pole. In monotelic attachment, only one of the sister kinetochores connects to the microtubules from a spindle pole 
. It is also possible for both sister kinetochores to be detached. These incorrect attachments have to be corrected before anaphase entry, or chromosome missegregation will occur.
The kinetochore is a multi-protein complex that connects chromosomes to microtubules. More than 60 kinetochore proteins have been identified in budding yeast. The CBF3 (centromere binding factor) complex associates directly with centromeric DNA, while the DASH/Dam1 complex residues at the kinetochore-microtubule interface. As a ten-protein complex including Dam1 and Ask1, the DASH can form a ring structure around a single microtubule and mediate the kinetochore-microtubule interaction 
. Ndc80 (Ndc80, Nuf2, Spc24, Spc25), COMA (Ctf19-Okp1-Mcm21-Ame1), and MIND (Mtw1p including Nnf1-Nsl1-Dsn1) complexes bridge the gap between centromere-bound CBF3 and microtubule-associated DASH 
Chromosome attachment is monitored by the spindle checkpoint which includes Bub1, Bub3, Mad1, Mad2, Mad3, and Mps1 
. Detached kinetochores activate the checkpoint by allowing the formation of a Mad2-Mad3/BubR1-Bub3-Cdc20 complex. Because Cdc20 is an essential activator of the anaphase-promoting complex (APC), the binding of Cdc20 by the spindle checkpoint components blocks APCCdc20
mediates the ubiquitination and the subsequent degradation of the anaphase inhibitor securin, known as Pds1 in budding yeast 
. Pds1 protein inhibits anaphase by binding to separase Esp1 and preventing Esp1-dependent cleavage of cohesin, a protein complex that holds sister chromatids together 
. Therefore, the activation of the spindle checkpoint prevents anaphase entry by blocking Pds1 degradation, and stabilized Pds1 protein indicates the activation of the spindle checkpoint.
Chromosome bipolar attachment generates tension on sister kinetochores. The observation that the application of tension on an improperly attached chromosome in grasshopper cells abolishes the anaphase entry delay directly demonstrates the role of tension in cell cycle regulation 
. To analyze the response to the absence of tension in yeast cells, tension defects can be induced by the block of DNA synthesis or by the abrogation of sister chromatid cohesion 
. In both situations, the lack of tension prevents anaphase entry as indicated by the stabilized Pds1 protein levels. Ipl1 and Sgo1 were found to be required to sense tension defects and prevent anaphase entry, but they are dispensable for cell cycle arrest induced by the disruption of the spindle structure 
. In addition to its checkpoint function, Ipl1 kinase also promotes the turnover of kinetochore-microtubule interaction when tension is absent 
. Therefore, it is speculated that Ipl1 may activate the checkpoint by generating detached chromosomes when tension is absent. In contrast, Sgo1 does not play a role in destabilizing kinetochore attachment and its checkpoint function remains unclear at the molecular level 
As one of the six kinesin-related proteins in budding yeast, Kar3 was identified as being essential for yeast nuclear fusion during mating 
. Unlike other kinesins, Kar3 protein contains a motor domain at its carboxy terminus that possesses minus-end-directed motility 
. Recent evidence indicates that Kar3 localizes at the spindle midzone and may also function as an interpolar-microtubule cross-linker to prevent spindle collapse 
. Moreover, Kar3 protein promotes the poleward transport of chromosomes along astral microtubules 
. Two proteins, Cik1 and Vik1, associate with Kar3 through coiled-coil domains to form Cik1/Kar3 or Vik1/Kar3 heterodimers. Both kar3Δ
mutants show defects in mating, spindle morphogenesis, and chromosome segregation 
, but their direct role in mitosis remains unclear.
We previously showed that cik1Δ
mutants are sensitive to hydroxyurea (HU), a DNA synthesis inhibitor, and these mutants exhibit chromosome bipolar attachment defects after HU treatment 
. We recently found that cik1Δ
mutants are synthetically lethal with tension checkpoint mutants ipl1-321
, indicating a role for Cik1/Kar3 in chromosome segregation. To further study the function of Cik1/Kar3, we constructed a plasmid PGALCIK1-CC
that contains the coiled-coil domain of Cik1. Our results indicate that overexpression of CIK1-CC
can competitively disrupt the Cik1-Kar3 interaction, which allows us to conditionally abolish Cik1/Kar3 function. With this method, we show that dysfunctional Cik1/Kar3 results in significant co-segregation of sister chromatids in the absence of the spindle checkpoint. Strikingly, dysfunctional Cik1/Kar3 also causes co-segregation of sister chromatids in ipl1-321
cells. Given the role of Ipl1 and Sgo1 in sensing chromosomes that lack tension, these data suggest that the loss of function of Cik1/Kar3 results in an increased frequency of syntelic attachment. Results with live-cell imaging and cohesin mutants further support this conclusion. Therefore, syntelic attachments can be induced in budding yeast by inactivating Cik1/Kar3 complex and this method will be a very useful tool for studying the response to tension defects.