Background image mask

LSSS 2017-2018

2017LSSS2018

Life Sciences Seminar Series

 

Back to seminar list

Iva Tolic

Ruđer Bošković Institute, Zagreb, Croatia

Mysteries of the mitotic spindle: chirality, torques and twist

Talk abstract

During cell division, microtubules of the mitotic spindle segregate chromosomes by exerting forces on kinetochores, protein complexes on the chromosomes. The central question is what forces drive chromosome segregation. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. By combining laser ablation, photoactivation and theoretical modeling, we show that kinetochores can separate without any attachment to one spindle pole (Vukušić et al., Dev Cell 2017). This separation requires the bridging fiber, which connects sister kinetochore fibers. Bridging microtubules in intact spindles slide apart together with kinetochore fibers, indicating strong crosslinks between them. Thus, sliding of microtubules in the bridging fibers drives pole separation and pushes kinetochore fibers apart to segregate chromosomes. In addition to forces, torques may also exist in the spindle, yet they have not been investigated. We show that the spindle is chiral, based on our finding that microtubule bundles follow a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles (Novak et al., bioRxiv 167437). STED super-resolution microscopy, as well as confocal microscopy, of human spindles shows that the bundles have complex curved shapes. Kinesin-5 (Kif11/Eg5) inactivation abolished spindle chirality. Our theoretical model predicts that bending and twisting moments generate curved shapes of bundles. We find that bundles turn by about -2°/µm around the spindle axis, which we explain by a twisting moment of -10 pNµm. We conclude that torques, in addition to forces, exist in the spindle and determine its chiral architecture.

Selected Publications

Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes.Vukušić K, Buđa R, Bosilj A, Milas A, Pavin N, Tolić IM
Dev Cell 2017 Oct 9; 43(1):11-23.e6

Abstract

During cell division, mitotic spindle microtubules segregate chromosomes by exerting forces on kinetochores. What forces drive chromosome segregation in anaphase remains a central question. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. Here we show, by combining laser ablation, photoactivation, and theoretical modeling, that kinetochores can separate without any attachment to one spindle pole. This separation requires the bridging fiber, a microtubule bundle that connects sister kinetochore fibers. Bridging fiber microtubules in intact spindles slide apart with kinetochore fibers, indicating strong crosslinks between them. We conclude that sliding of microtubules within the bridging fibers drives pole separation and pushes kinetochore fibers poleward by the friction of passive crosslinks between these fibers. Thus, sliding within the bridging fiber works together with the shortening of kinetochore fibers to segregate chromosomes.

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores.Kajtez J, Solomatina A, Novak M, Polak B, Vukušić K, Rüdiger J, Cojoc G, Milas A, Šumanovac Šestak I, Risteski P, Tavano F, Klemm AH, Roscioli E, Welburn J, Cimini D, Glunčić M, Pavin N, Tolić IM
Nat Commun 2016 Jan 5; 7:10298

Abstract

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term 'bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.

Fusion of protein aggregates facilitates asymmetric damage segregation.Coelho M, Lade SJ, Alberti S, Gross T, Tolić IM
PLoS Biol 2014 Jun; 12(6):e1001886

Abstract

Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division.

Dynein motion switches from diffusive to directed upon cortical anchoring.Ananthanarayanan V, Schattat M, Vogel SK, Krull A, Pavin N, Tolić-Nørrelykke IM
Cell 2013 Jun 20; 153(7):1526-36

Abstract

Cytoplasmic dynein is a motor protein that exerts force on microtubules. To generate force for the movement of large organelles, dynein needs to be anchored, with the anchoring sites being typically located at the cell cortex. However, the mechanism by which dyneins target sites where they can generate large collective forces is unknown. Here, we directly observe single dyneins during meiotic nuclear oscillations in fission yeast and identify the steps of the dynein binding process: from the cytoplasm to the microtubule and from the microtubule to cortical anchors. We observed that dyneins on the microtubule move either in a diffusive or directed manner, with the switch from diffusion to directed movement occurring upon binding of dynein to cortical anchors. This dual behavior of dynein on the microtubule, together with the two steps of binding, enables dyneins to self-organize into a spatial pattern needed for them to generate large collective forces.

Pivoting of microtubules around the spindle pole accelerates kinetochore capture.Kalinina I, Nandi A, Delivani P, Chacón MR, Klemm AH, Ramunno-Johnson D, Krull A, Lindner B, Pavin N, Tolić-Nørrelykke IM
Nat Cell Biol 2013 Jan; 15(1):82-7

Abstract

During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome. The central question is how microtubules find kinetochores. According to the pioneering idea termed search-and-capture, numerous microtubules grow from a centrosome in all directions and by chance capture kinetochores. The efficiency of search-and-capture can be improved by a bias in microtubule growth towards the kinetochores, by nucleation of microtubules at the kinetochores and at spindle microtubules, by kinetochore movement, or by a combination of these processes. Here we show in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole, instead of growing towards the kinetochores. This pivoting motion of microtubules is random and independent of ATP-driven motor activity. By introducing a theoretical model, we show that the measured random movement of microtubules and kinetochores is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of capture depends mainly on how fast microtubules pivot, which was confirmed experimentally by speeding up and slowing down microtubule pivoting. Thus, pivoting motion allows microtubules to explore space laterally, as they search for targets such as kinetochores.