The role of chromosome arms and kinetochore architecture in the formation and amphitelic attachments - implications for mitotic fidelity

Proper chromosome segregation and mitotic fidelity depends on previous chromosome bi-orientation. For bi-orientation to occur chromosome have to attach to the microtubules coming from opposite poles, which will create the tension required for attachment stabilization. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. Moreover, what is the role of kinetochore architecture in proper chromosome congression and segregation stays unknown. Here, taking advantage of two model systems, Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs) and Indian muntjac fibroblast we addressed the question of chromosome bi-orientation and mitotic fidelity, focusing on processes that play an important role in initial attachment stabilization, chromosome congression and segregation. To address this we generated Drosophila S2 cells undergoing SMUGs. SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. The advantage of this system when compared with MUGs generated in mammalian cells is the preservation of chromatid morphology. Over time, laterally attached kinetochores converted into end-on attachments, experienced intra-kinetochore stretch or structural deformation, and SMUGs eventually exited a delayed mitosis after satisfying the spindle-assembly checkpoint (SAC). Here we show that conversion from lateral to end-on kinetochore-microtubule attachments was promoted by polar ejection forces (PEFs) generated by Chromokinesins ultimately leading to SAC satisfaction in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochoremicrotubule attachments, independently of bi-orientation. Besides initial chromosome positioning around the newly formed spindle, changes in kinetochore structure during mitosis increase the probability of formation of amphitelic attachments. During prometaphase, kinetochores increase in size forming wider crescent structures, which facilitate their capture by microtubules. Upon initial attachments, kinetochore structure decreases, becoming less prone to error formation. However, the physiological relevance of kinetochore size in error formation remains unknown. To investigate whether kinetochore size influences chromosome segregation fidelity we took advantage of immortalized fibroblasts from the Indian muntjac, the mammalian with the lowest known chromosome number (n=3). Indian muntjac chromosomes are morphologically distinct and can be easily tracked by live-cell fluorescence microscopy. One remarkable feature of the Indian muntjac cells is the unusually large kinetochore of chromosome 3+X that can bind up to 100 microtubules. Here we show that large kinetochores are more prompt to establish erroneous attachments leading to chromosome missegregation. Moreover, our results indicate that kinetochore size affects the pathway of chromosome congression and bi-orientation during mitosis. Overall, these findings provide direct evidence that kinetochore size is an important determinant of chromosome segregation fidelity in mammals.