The role of microtubule pivoting in formation of mitotic spindles



During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome. The central question is how microtubules find kinetochores. By combining experiments with theory, we have discovered a new search mechanism: Microtubules swipe through the cell and thus explore the space in search for kinetochores, until they approach a kinetochore and capture it. This search strategy is cost-effective for the cell, because it requires only a small number of microtubules and does not need energy from ATP. Results were published in Kalinina et al., Nat. Cell Biol. (2013). Now we investigate the role of microtubule pivoting in reassembly of the mitotic spindle.

This project is funded by the Unity Through Knowledge Fund.




Dynein, microtubule and cargo: a ménage à trois



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. Results were published in Ananthanarayanan et al., Cell (2013). Now we develope a model for dynein redistribution. The model includes properties of dynein described above, in addition to properties obtained from in vitro experiments.





Positioning of microtubule asters



Dynamic microtubules interact with the cell cortex to generate pushing and/or pulling forces that position microtubule organizing centers correctly with respect to the confining geometry of living cells. In particular, pulling forces mediated by cortex-linked dynein have been implicated in centering processes. However, the mechanism by which pulling forces may contribute remain poorly understood. We address this question in an in vitro experiment, where microtubule asters are grown in microfabricated chambers, and pulling forces arise from interactions between microtubule ends and dynein attached to the chamber walls. Surprisingly, we find that microtubule asters center more reliably by a combination of pulling and pushing forces than by pushing forces alone: slipping of pushing microtubules along the chamber walls generates an anisotropic distribution of microtubules that leads to reliable aster centering once microtubule ends are captured by dynein. Results obtained for various geometries show that this positioning strategy should fail for strongly elongated cells. Results were published in Laan et al., Cell (2012) and Pavin et al., New J. Phys. (2012). Now we investigate centering of the centrosome in HeLa cells.







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