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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - NewSpindleForce (A new class of microtubules in the spindle exerting forces on kinetochores)

Teaser

Summary

At the onset of division the cell forms a spindle, a micro-machine made of microtubules, which divide the chromosomes by pulling on kinetochores, protein complexes on the chromosome. The central question in the field is how accurate chromosome segregation results from the interactions between kinetochores, microtubules and the associated proteins. According to the current paradigm, the forces on kinetochores are produced by kinetochore fibers, bundles of microtubules extending between the spindle pole and the kinetochore. The central hypothesis of this project is that interpolar microtubules, termed bridging fibers, connect two sister kinetochore fibers like a bridge. We started the project by testing this hypothesis. We uncovered a strong connection between the bridging fiber and sister kinetochore fibers by cutting a kinetochore fiber with a laser, after which the bridging fiber moved together with the kinetochore fibers and kinetochores. We found that in a metaphase spindle almost all interpolar bundles are linked to sister kinetochore fibers as bridges. By exploring the role of bridging fibers in anaphase, we discovered that sliding of microtubules within the bridging fiber pushes the attached kinetochore fibers and their kinetochores poleward to segregate chromosomes. Understanding the role of bridging microtubules in force generation and chromosome movements not only sheds light on the mechanism of chromosome segregation, but may also increase the potential of mitotic anticancer strategies, as the spindle is a major target for chemotherapy.

Work performed

The central hypothesis of this project is that interpolar microtubules, termed bridging fibers, connect two sister kinetochore fibers like a bridge. We started the project by testing this hypothesis. We uncovered a strong connection between the bridging fiber and sister kinetochore fibers by cutting a kinetochore fiber with a laser, after which the bridging fiber moved together with the kinetochore fibers and kinetochores. We characterized the bridging fibers (Objective 1) and explored the relationship between overlap microtubules and kinetochore fibers throughout the spindle by investigating the localization of PRC1 (part of Objective 2). We showed that in a metaphase spindle almost all overlap bundles are linked to sister kinetochore fibers, and vice versa. In collaboration with Prof. Nenad Pavin from the University of Zagreb, we developed a theoretical model that includes the bridging fiber as a link between sister kinetochore fibers (Objective 5). Our theory and experiments show that the bridging fiber withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape. Next, we explored the role of bridging fibers in anaphase (Objective 4). We found that sliding of microtubules in the bridging fiber pushes the attached sister kinetochore fibers and their kinetochores poleward through the friction of crosslinks between these fibers. We found that kinesin-6 (MKLP1/KIF23) contributes to the stabilization of the midzone and kinetochore separation in early anaphase (related to Objective 2). During the work on bridging fibers in metaphase, we had an unexpected finding: We discovered that the spindle is chiral. Chirality is evident from our observation that microtubule bundles twist along a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles. Inactivation of kinesin-5 (Kif11/Eg5) abolished the chirality of the spindle (related to Objective 3). In summary, we completed Objectives 1, 4, and 5, and performed part of the research under Objectives 2 and 3. We published 9 papers in the following journals: Nat Commun, Dev Cell, EMBO Rep, Trends Biochem Sci, Annu Rev Biophys, Eur Biophys J, Methods Cell Biol, Cell Cycle, Matters Select.

Final results

This project yielded results that go beyond the state of the art, changing the current view of the mitotic spindle architecture and functioning: (i) Interpolar microtubules termed bridging fibers link sister kinetochore fibers like a bridge. Almost all interpolar bundles in a metaphase spindle are linked to sister kinetochore fibers, and vice versa. The bridging fiber withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape (Kajtez, Solomatina, Novak et al., Nat Commun 2016; Polak, Risteski et al., EMBO Rep 2017); (ii) During anaphase, sliding within the bridging fiber pushes the attached sister kinetochore fibers and their kinetochores poleward, thereby segregating chromosomes (Vukusic, Buda et al., Dev Cell 2017); (iii) The spindle is chiral, which is evident from our finding that microtubule bundles twist along a left-handed helical path. Thus, in addition to forces, torques exist in the spindle and determine its chiral architecture (Novak, Polak, Simunic, Boban et al., bioRxiv 167437, 2017).
With respect to development of new techniques, we have established a method to sever microtubule bundles in the mitotic spindle by using femtosecond laser ablation, and to characterize them by image analysis (Buda, Vukusic and Tolic, Methods Cell Biol 2017). By using this method, we were able to sever a single kinetochore fiber on the outer side of the spindle and observe the movement of the kinetochore fiber stub together with the bridging fiber and the sister kinetochore fiber away from the spindle. Compared to previous spindle ablations from several labs (e.g., Khodjakov, Dumont), in which the kinetochore fiber stub becomes quickly reconnected with the spindle and pulled towards the spindle pole, in our assay the stub stays for several minutes or longer away from the rest of the spindle. This is an important feature because it allows for the characterization of the shape changes of the cut fibers and changes in interkinetochore distance due to laser cutting, in order to extract information about the forces that were acting in the system before the cutting. Moreover, because of this feature, our method is unique in that it allows for studies of anaphase dynamics of a single kinetochore pair displaced from the spindle.
Until the end of the project, we expect to dissect the role of mitotic motor proteins and non-motor microtubule crosslinkers in (i) crosslinking of bridging microtubules with kinetochore microtubules, (ii) generation and maintenance of the helical shape of bridging fibers, (iii) sliding apart of bridging microtubules during chromosome segregation.