Microtubule-based positioning mechanisms

A brief look at the animal and plant kingdom shows a large variety of spatial patterns like the periodic stripes of zebras and the arrangement of flower petals. Interestingly, a microscopic look at tissues and single cells reveals very well structured organisations at smaller length scales as well. In this thesis I provide mechanistic insights into the organization of such patterns. To build complex structures, cells require mechanism to set a length scale. Apart from mechanisms based on the reaction and diffusion of interacting molecules, mechanical processes like the growth of cytoskeletal filaments can set length scales at the subcellular level. The cytoskeleton mechanically supports cells but more importantly for our purpose generates forces that can change cellular architecture. In eukaryotic cells the contribution is based on three filaments (microtubules, actin and intermediate filaments), but in this thesis the focus is set on microtubules. Microtubules are stiff and dynamic filaments that enable them to play a prominent role in cellular organization. Microtubules are composed of tubulin heterodimers that arrange longitudinally and laterally to form a slender hollow tube with high rigidity. Microtubules in cells grow away from specialized nucleation sites and have a certain probability to undergo a transition to a state of shortening. This switching mechanism, termed dynamic instability, determines how far microtubules grow away from their nucleation site. This length regulating mechanism aids the positioning of cellular components in cooperation with molecular motors that transport material along microtubules and forces generated by growing and shrinking microtubules in contact with cellular objects (organelles, membrane). The role of microtubules in intercellular positioning mechanisms is reviewed in chapter 1. In chapter 2 and 3 we investigated the role of microtubules in positioning nuclei in cells. compartments throughout the cellular space. The spacing between compartments is in many cases Eukaryotic but also prokaryotic cells disperse organelles and micro- regulated and equidistant patterns have been described in particular for the case of nuclei in multinucleated cells. The spacing between nuclei is regulated to control the patterning of cells in developing embryos but the occurrence of irregular patterns in large multinucleated muscle cells also correlates with muscle diseases. In chapter 2 we used fission yeast cells with a cytokinetic defect to generate a model multinucleated cell. Fission yeast cells are easy to genetically modify and the organization of their microtubule network is well understood.   Cells had a cluster of nuclei at their centre but in absence of the minus-end directed motor klp2p the pattern changed to an arrangement in which the nuclei were well dispersed and positioned at equidistant intervals. Patterning depended on the presence of microtubules and we observed the growth of microtubules away from the envelope of nuclei towards neighbouring nuclei. We hypothesized that impingement of microtubules onto neighbouring nuclei generates nuclear repulsive forces.  The net effect of microtubule interactions with cell walls and nuclei may be a force field in which nuclei are stably positioned at equidistant positions. However dominant forces generated by klp2p cause sliding between microtubules originating from sister nuclei that pull nuclei together. Our studies thus suggest a mechanism for equidistant positioning of organelles and a way to switch between patterns. Switching behaviour is observed in biology for example during light induced redistribution of chloroplasts in plant cells. An increasing number of biological findings are now supported by computer models, as it allows to deduce whether a limited set of interacting components can explain a biological phenomenon. To evaluate whether repulsive pushing forces by dynamic unstable microtubules in between nuclei are sufficient to pattern nuclei we developed a simple 1D stochastic model of microtubule growth and nuclear motion in a tetranucleated cell. Our model demonstrated that the dynamics and accuracy of nuclear positioning in fission yeast cells is in agreement with the measured parameters of dynamic instability of microtubules. For this we compared nuclear oscillations and nuclear redistribution after pattern perturbation in experiments and simulations. An overestimation of the force generation between nuclei in our model caused a larger internuclear distance then experimentally observed. This discrepancy disappeared when we took into account that force generation at cell walls is more efficient than at nuclear envelopes. The model in chapter 3 thus clearly revealed that equidistant nuclear positioning can be explained by force generation of microtubules undergoing dynamic instability. In chapter 4 we investigated the role of microtubule dynamics in the regulation of spindle elongation. During mitosis, microtubules form the mitotic spindle that segregates chromosomes to different cell halves. Microtubules from two spindle halves interdigitate at the spindle centre and template a multi protein assembly called the spindle midzone. Microtubules within the midzone grow and slide relative to each other causing spindle elongation. Moreover, the midzone regulates cytokinesis and as such mechanism that control midzone assembly are of interest to identify new targets for cancer therapy. In chapter 4 we investigated, using fission yeast as a model, the mechanism that regulates the length of the midzone during spindle elongation. We demonstrate that spindle elongation velocity is limited by the speed at which motors push overlapping microtubules apart. However, under conditions of reduced microtubule growth, the elongation is being limited by microtubule growth. These results show that sliding and microtubule growth are coupled to prevent that spindle halves can separate from each other by sliding alone. This insight will help to reveal the function of a myriad of protein interactions that take place at the midzone.   This thesis reveals mechanically insights into pattern formation based on microtubule dynamics. In chapter 5 we discuss the relevance of our findings for the patterning of nuclei in larger eukaryotic cells. Preliminary results on the binding of the major midzone protein ase1p show that the binding affinity of ase1p microtubule crosslinkers depends on the number of microtubules that it can potentially bind to. These results suggest how ase1p may be recruited to the centre of mitotic spindles, where microtubule interdigitation is strongest