Periodic Reporting for period 4 - NewSpindleForce (A new class of microtubules in the spindle exerting forces on kinetochores)
Periodo di rendicontazione: 2019-04-01 al 2020-03-31
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. The key hypothesis of this project was that a microtubule bundle termed bridging fiber connects two sister kinetochore fibers like a bridge and regulates the forces acting on chromosomes. 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 developed an optogenetic approach to acutely disassemble bridging fibers and showed that they promote chromosome alignment at the metaphase plate by forces that depend on the microtubule overlap within the bridging fiber. By combining a theoretical model with imaging of the bridging fibers by superresolution microscopy, we discovered that spindle bundles have a shape of a left-handed helix, making the spindle a chiral object. 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.
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 and uncovered a strong link between the bridging fiber and sister kinetochore fibers by laser-cutting of a kinetochore fiber, which resulted in joint movement of the bridging and kinetochore fibers away from the spindle center (Kajtez et al., Nat Commun 2016). By investigating the localization of PRC1, a microtubule crosslinker that binds to antiparallel overlaps, we showed that nearly all overlap bundles link sister kinetochore fibers, acting as a bridge between them (Polak et al., EMBO Rep 2017). To study the bridging fiber at the superresolution level, we performed STED microscopy of human spindles, showing that microtubule bundles extend almost from pole to pole and acquire complex curved shapes (Novak et al., Nat Commun 2018). Moreover, we developed an Expansion microscopy protocol for mitotic spindles and used it to characterize bridging fibers in a side-view and end-on view of the spindle (Ponjavic et al., Methods Cell Biol 2020). In collaboration with Prof. Nenad Pavin from the University of Zagreb, we developed a theoretical model that includes a bridging fiber as a link between sister kinetochore fibers (Kajtez et al., Nat Commun 2016). By combining this model with experiments, we concluded that the bridging fiber withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.
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.
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.
This project yielded results that changed the view of the mitotic spindle architecture and functioning. Our discovery that bridging fibers link sister kinetochore fibers like a bridge and regulate the tension between sister kinetochores challenged the prevailing view that the tension on kinetochores is generated by molecular events occurring only at the end of the kinetochore fiber. The forces exerted by the bridging fiber play an important role not only in balancing the tension on kinetochores in metaphase, but also in the segregation of sister chromatids in anaphase. Our discovery that motor-driven sliding of microtubules in the bridging fiber in anaphase pushes the attached sister kinetochore fibers and kinetochores poleward was unexpected because according to the current view, the processes that contribute to kinetochore separation require kinetochores to be linked with the spindle poles. Our surprising finding that the spindle is chiral changed the view of spindle structure and force balance, showing that in addition to pulling and pushing forces, rotational forces act in the spindle and thus also on chromosomes.
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.
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.