Periodic Reporting for period 4 - NewSpindleForce (A new class of microtubules in the spindle exerting forces on kinetochores)
Berichtszeitraum: 2019-04-01 bis 2020-03-31
To study the biological role of bridging fibers, we developed an optogenetic approach for acute removal of PRC1 to disassemble bridging fibers (Milas et al., Methods Cell Biol 2018), and discovered that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers (Jagric et al., bioRxiv 2019).
To explore the dynamics and role of bridging fibers in anaphase, we developed an assay for kinetochore dynamics based on laser ablation (Buda et al., Methods Cell Biol 2017), which allowed us to demonstrate that kinetochores can separate without attachment to one spindle pole. The observed separation requires the bridging fiber. Photoactivation experiments showed that bridging microtubules slide apart, indicating that this sliding pushes the attached kinetochore fibers poleward to segregate chromosomes (Vukusic et al., Dev Cell 2017). By using combined depletion and inactivation of candidate proteins together with CRISPR technology, we revealed that a surprising cooperation of mitotic motors, the PRC1-dependent motor KIF4A/kinesin-4 together with EG5/kinesin-5, drive the sliding of bridging microtubules. Thus, two mechanistically distinct sliding modules, one based on a self-sustained and the other on a crosslinker-assisted motor, power the mechanism that drives spindle elongation and kinetochore segregation in human cells (Vukusic et al., bioRxiv 2019).
During the work on bridging fibers in metaphase, we had an unexpected finding that the spindle is chiral. Chirality is evident from our observation that microtubule bundles twist along a left-handed helical path (Novak et al., Nat Commun 2018). We conclude that rotational forces, in addition to pushing and pulling forces, exist in the spindle and determine its chiral architecture.
In this project we developed new methods to observe and perturb spindles, including expansion microscopy of the spindle, laser-cutting of spindle microtubules to detach them from the pole, and acute removal of spindle proteins by optogenetics. These technological developments will be valuable resources for the community.
In summary, the new concepts and methods resulting from this project are transforming the field of spindle mechanobiology. We expect our findings to be important not only for the understanding of a well-functioning spindle, but also of errors in chromosome segregation. As such errors are characteristic of several serious diseases, revealing their origins is of general interest because of prospective medical applications.