OSCITON – Croatian Science Foundation Project (HRZZ)

Oscillatory dynamics of the cytoskeleton

Oscillations in the cytoskeleton are of general importance in cell biology as a mean of spatial and temporal regulation of the cellular organization, cell division and motility. The project OSCITON focuses on two oscillatory processes. First, we will investigate the mechanism of microtubule-driven kinetochore oscillations during mitosis, and the role of kinesin-8 motors in this process. Second, we will study the oscillatory repolarization of Dictyostelium cells during random migration. This interdisciplinary project is designed to optimize the use of resources and bring together researchers with the expertise in a range of disciplines, including molecular and cellular biology, biochemistry, microscopy, and theoretical biophysics.

2015-2019 OSCITON
Research area: Cell biology
ResearcherProf Iva Tolić
Host Institution: Ruđer Bošković Institute, Zagreb
Project: Oscilatory dynamics of the cytoskeleton (OSCITON)
HRZZ funding: HRK 1.000.000,00


Research papers

The mitotic spindle is chiral due to torques within microtubule bundles.
Maja Novak, Bruno Polak, Juraj Simunić, Zvonimir Boban, Barbara Kuzmić, Andreas Thomae, Iva M. Tolić, Nenad Pavin. Nat Commun, 9(1): 3571 (2018). PDF | web

Screenshot 2019-05-07 at 13.53.18Mitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins. Forces are required for the congression of chromosomes to the metaphase plate and their separation in anaphase. However, besides forces, torques may exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles in human spindles follow a left-handed helical path, which cannot be explained by forces but rather by torques. Kinesin-5 (Kif11/Eg5) inactivation abolishes spindle chirality. Our theoretical model predicts that bending and twisting moments may generate curved shapes of bundles. We found that bundles turn by about −2 deg µm−1 around the spindle axis, which we explain by a twisting moment of roughly −10 pNµm. We conclude that torques, in addition to forces, exist in the spindle and determine its chiral architecture.


Metaphase kinetochore movements are regulated by kinesin-8 motors and microtubule dynamic instability.
Anna H. Klemm, Agneza Bosilj, Matko Glunčić, Nenad Pavin, Iva M. Tolić. Mol Biol Cell, 29(11): 1332-1345 (2018). PDF | web

During metaphase, sister chromatids are connected to microtubules extending from the opposite spindle poles via kinetochores to protein complexes on the chromosome. Kinetochores congress to the equatorial plane of the spindle and oscillate around it, Screenshot 2019-05-07 at 13.26.39with kinesin-8 motors restricting these movements. Yet, the physical mechanism underlying kinetochore movements is unclear.We show that kinetochore movements in the fission yeast Schizosaccharomyces pombe are regulated by kinesin-8-promoted microtubule catastrophe, force-induced rescue, and microtubule dynamic instability. A candidate screen showed that among the selected motors only kinesin-8 motors Klp5/Klp6 are required for kinetochore centering. Kinesin-8 accumulates at the end of microtubules, where it promotes catastrophe. Laser ablation of the spindle resulted in kinetochore movement toward the intact spindle pole in wild-type and klp5Δ cells, suggesting that kinetochore movement is driven by pulling forces. Our theoretical model with Langevin description of microtubule dynamic instability shows that kinesin-8 motors are required for kinetochore centering, whereas sensitivity of rescue to force is necessary for the generation of oscillations. We found that irregular kinetochore movements occur for a broader range of parameters than regular oscillations. Thus, our work provides an explanation for how regulation of microtubule dynamic instability contributes to kinetochore congression and the accompanying movements around the spindle center.


Reviews, commentaries and methods

Helical twist and rotational forces in the mitotic spindle.
Iva M.Tolić, Maja Novak, Nenad Pavin.  Biomolecules, 9(4): 132 (2019). PDF | web

biomolecules-09-00132-g005-550The mitotic spindle segregates chromosomes into two daughter cells during cell division. This process relies on the precise regulation of forces acting on chromosomes as the cell progresses through mitosis. The forces in the spindle are difficult to directly measure using the available experimental techniques. Here, we review the ideas and recent advances of how forces can be determined from the spindle shape. By using these approaches, it has been shown that tension and compression coexist along a single kinetochore fiber, which are balanced by a bridging fiber between sister kinetochore fibers. An extension of this approach to three dimensions revealed that microtubule bundles have rich shapes, and extend not simply like meridians on the Earth’s surface but, rather, twisted in a helical manner. Such complex shapes are due to rotational forces, which, in addition to linear forces, act in the spindle and may be generated by motor proteins such as kinesin-5. These findings open new questions for future studies, to understand the mechanisms of rotational forces and reveal their biological roles in cells.


Optogenetic reversible knocksideways, laser ablation and photoactivation on the mitotic spindle in human cells.
Ana Milas, Mihaela Jagrić, Jelena Martinčić, Iva M. Tolić. Methods Cell Biol, 145: 191-215 (2018). PDF | web

At the onset of mitosis, cells assemble the mitotic spindle, a dynamic micromachine made of microtubules and associated proteins. Although most of these proteins have been identified, it is still unknown how their collective behavior drives spindle formation and function. Over the last decade, RNA interference has been the main tool for revealing the role of spindle proteins. However, the effects of this method are evident only after a longer time period, leading to difficulties in the interpretation of phenotypes. Optogenetics is a novel technology that enables fast, reversible, and precise control of protein activity by utilization of light. In this chapter, we present an optogenetic knocksideways method for rapid and reversible translocation of proteins from the mitotic spindle to mitochondria using blue light. Furthermore, we discuss other optical approaches, such as laser ablation of microtubule bundles in the spindle and creation of reference marks on the bundles by photoactivation of photoactivatable GFP. Finally, we show how different optical perturbations can be combined in order to acquire deeper understanding of the mechanics of mitosis.


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