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.</p

Effects of sub-lethal single, simultaneous, and sequential abiotic stresses on phenotypic traits of Arabidopsis thaliana

Plant responses to abiotic stresses are complex and dynamic, and involve changes in different traits, either as the direct consequence of the stress, or as an active acclimatory response. Abiotic stresses frequently occur simultaneously or in succession, rather than in isolation. Despite this, most studies have focused on a single stress and single or few plant traits. To address this gap, our study comprehensively and categorically quantified the individual and combined effects of three major abiotic stresses associated with climate change (flooding, progressive drought and high temperature) on 12 phenotypic traits related to morphology, development, growth and fitness, at different developmental stages in four Arabidopsis thaliana accessions. Combined sub-lethal stresses were applied either simultaneously (high temperature and drought) or sequentially (flooding followed by drought). In total, we analyzed the phenotypic responses of 1782 individuals across these stresses and different developmental stages. Overall, abiotic stresses and their combinations resulted in distinct patterns of effects across the traits analyzed, with both quantitative and qualitative differences across accessions. Stress combinations had additive effects on some traits, whereas clear positive and negative interactions were observed for other traits: 9 out of 12 traits for high temperature and drought, 6 out of 12 traits for post-submergence and drought showed significant interactions. In many cases where the stresses interacted, the strength of interactions varied across accessions. Hence, our results indicated a general pattern of response in most phenotypic traits to the different stresses and stress combinations, but it also indicated a natural genetic variation in the strength of these responses. Overall, our study provides a rich characterization of trait responses of Arabidopsis plants to sub-lethal abiotic stresses at the phenotypic level and can serve as starting point for further in-depth physiological research and plant modelling efforts

Effects of sublethal single, simultaneous and sequential abiotic stresses on phenotypic traits of Arabidopsis thaliana

Plant responses to abiotic stresses are complex and dynamic, and involve changes in different traits, either as the direct consequence of the stress, or as an active acclimatory response. Abiotic stresses frequently occur simultaneously or in succession, rather than in isolation. Despite this, most studies have focused on a single stress and single or few plant traits. To address this gap, our study comprehensively and categorically quantified the individual and combined effects of three major abiotic stresses associated with climate change (flooding, progressive drought and high temperature) on 12 phenotypic traits related to morphology, development, growth and fitness, at different developmental stages in four Arabidopsis thaliana accessions. Combined sublethal stresses were applied either simultaneously (high temperature and drought) or sequentially (flooding followed by drought). In total, we analysed the phenotypic responses of 1782 individuals across these stresses and different developmental stages. Overall, abiotic stresses and their combinations resulted in distinct patterns of effects across the traits analysed, with both quantitative and qualitative differences across accessions. Stress combinations had additive effects on some traits, whereas clear positive and negative interactions were observed for other traits: 9 out of 12 traits for high temperature and drought, 6 out of 12 traits for post-submergence and drought showed significant interactions. In many cases where the stresses interacted, the strength of interactions varied across accessions. Hence, our results indicated a general pattern of response in most phenotypic traits to the different stresses and stress combinations, but it also indicated a natural genetic variation in the strength of these responses. This includes novel results regarding the lack of a response to drought after submergence and a decoupling between leaf number and flowering time after submergence. Overall, our study provides a rich characterization of trait responses of Arabidopsis plants to sublethal abiotic stresses at the phenotypic level and can serve as starting point for further in-depth physiological research and plant modelling efforts

Forced apart : a microtubule-based mechanism for equidistant positioning of multiple nuclei in single cells

Cells can position multiple copies of components like carboxysomes, nucleoids, and nuclei at regular intervals. By controlling positions, cells, for example, ensure equal partitioning of organelles over daughter cells and, in the case of nuclei, control cell sizes during cellularization. Mechanisms that generate regular patterns are as yet poorly understood. We used fission yeast cell cycle mutants to investigate the dispersion of multiple nuclei by microtubule-generated forces in single cells. After removing internuclear attractive forces by microtubule-based molecular motors, we observed the establishment of regular patterns of nuclei. Based on live-cell imaging, we hypothesized that microtubule growth within internuclear spaces pushes neighbouring nuclei apart. In the proposed mechanism, which was validated by stochastic simulations, the repulsive force weakens with increasing separation because stochastic shortening events limit the extent over which microtubules generate forces. Our results, therefore, demonstrate how cells can exploit the dynamics of microtubule growth for the equidistant positioning of organelles

C. elegans Runx/CBFβ suppresses POP-1 TCF to convert asymmetric to proliferative division of stem cell-like seam cells

A correct balance between proliferative and asymmetric cell divisions underlies normal development, stem cell maintenance and tissue homeostasis. What determines whether cells undergo symmetric or asymmetric cell division is poorly understood. To gain insight into the mechanisms involved, we studied the stem cell-like seam cells in the Caenorhabditis elegans epidermis. Seam cells go through a reproducible pattern of asymmetric divisions, instructed by divergent canonical Wnt/β-catenin signaling, and symmetric divisions that increase the seam cell number. Using time-lapse fluorescence microscopy we observed that symmetric cell divisions maintain asymmetric localization of Wnt/β-catenin pathway components. Our observations, based on lineage-specific knockout and GFP-tagging of endogenous pop-1, support the model that POP-1TCF induces differentiation at a high nuclear level, whereas low nuclear POP-1 promotes seam cell self-renewal. Before symmetric division, the transcriptional regulator RNT-1Runx and cofactor BRO-1CBFβ temporarily bypass Wnt/β-catenin asymmetry by downregulating pop-1 expression. Thereby, RNT-1/BRO-1 appears to render POP-1 below the level required for its repressor function, which converts differentiation into self-renewal. Thus, we found that conserved Runx/CBFβ-type stem cell regulators switch asymmetric to proliferative cell division by opposing TCF-related transcriptional repression

A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry

Background: Seed size and number are important plant traits from an ecological and horticultural/agronomic perspective. However, in small-seeded species such as Arabidopsis thaliana, research on seed size and number is limited by the absence of suitable high throughput phenotyping methods. Results: We report on the development of a high throughput method for counting seeds and measuring individual seed sizes. The method uses a large-particle flow cytometer to count individual seeds and sort them according to size, allowing an average of 12,000 seeds/hour to be processed. To achieve this high throughput, post harvested seeds are first separated from remaining plant material (dust and chaff) using a rapid sedimentation-based method. Then, classification algorithms are used to refine the separation process in silico. Accurate identification of all seeds in the samples was achieved, with relative errors below 2%. Conclusion: The tests performed reveal that there is no single classification algorithm that performs best for all samples, so the recommended strategy is to train and use multiple algorithms and use the median predictions of seed size and number across all algorithms. To facilitate the use of this method, an R package (SeedSorter) that implements the methodology has been developed and made freely available. The method was validated with seed samples from several natural accessions of Arabidopsis thaliana, but our analysis pipeline is applicable to any species with seed sizes smaller than 1.5 mm.</p

A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry

Seed size and number are important plant traits from an ecological and horticultural/agronomic perspective. However, in small-seeded species such as Arabidopsis thaliana, research on seed size and number is limited by the absence of suitable high throughput phenotyping methods. Results: We report on the development of a high throughput method for counting seeds and measuring individual seed sizes. The method uses a large-particle flow cytometer to count individual seeds and sort them according to size, allowing an average of 12,000 seeds/hour to be processed. To achieve this high throughput, post harvested seeds are first separated from remaining plant material (dust and chaff) using a rapid sedimentation-based method. Then, classification algorithms are used to refine the separation process in silico. Accurate identification of all seeds in the samples was achieved, with relative errors below 2%. Conclusion: The tests performed reveal that there is no single classification algorithm that performs best for all samples, so the recommended strategy is to train and use multiple algorithms and use the median predictions of seed size and number across all algorithms. To facilitate the use of this method, an R package (SeedSorter) that implements the methodology has been developed and made freely available. The method was validated with seed samples from several natural accessions of Arabidopsis thaliana, but our analysis pipeline is applicable to any species with seed sizes smaller than 1.5 mm

A high throughput method for quantifying number and size distribution of Arabidopsis seeds using large particle flow cytometry

Seed size and number are important plant traits from an ecological and horticultural/agronomic perspective. However, in small-seeded species such as Arabidopsis thaliana, research on seed size and number is limited by the absence of suitable high throughput phenotyping methods. Results: We report on the development of a high throughput method for counting seeds and measuring individual seed sizes. The method uses a large-particle flow cytometer to count individual seeds and sort them according to size, allowing an average of 12,000 seeds/hour to be processed. To achieve this high throughput, post harvested seeds are first separated from remaining plant material (dust and chaff) using a rapid sedimentation-based method. Then, classification algorithms are used to refine the separation process in silico. Accurate identification of all seeds in the samples was achieved, with relative errors below 2%. Conclusion: The tests performed reveal that there is no single classification algorithm that performs best for all samples, so the recommended strategy is to train and use multiple algorithms and use the median predictions of seed size and number across all algorithms. To facilitate the use of this method, an R package (SeedSorter) that implements the methodology has been developed and made freely available. The method was validated with seed samples from several natural accessions of Arabidopsis thaliana, but our analysis pipeline is applicable to any species with seed sizes smaller than 1.5 mm