Role of Molecular Motors and Maps in Spindle Dynamics and Chromosome Segregation in the Fission Yeast Schizosaccharomyces Pombe

Mitosis is a key event in the life of a cell, where duplicated chromosomes are separated into the daughter cells. Defects associated with chromosome segregation can lead to aneuploidy, a hallmark of cancer. Chromosome segregation is achieved by the mitotic spindle, which is composed of microtubules (MTs), motors and microtubule associated protein (MAPs). Motors such as kinesins generate forces within the spindle while MAPs perform functions such as organize the spindle pole and maintain the bipolar spindle. Both motors and MAPs contribute to spindle mechanics. Here I used the relatively simple fission yeast to address how defects in spindle mechanics affect chromosome segregation. The metaphase spindle is maintained at a constant length by an antagonistic force-balance model yet how the regulation of metaphase spindle length contribute to subsequent chromosome segregation remains unexplored. To test the force-balance model, I applied gene deletion and fast microfluidic temperature-control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. I show that kinesin-5 cut7p and MT bundler ase1p contribute to outward pushing forces, and kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Motors and MAPs cooperate to focus MTs at the spindle pole. Defects in MT focusing lead to defects in chromosome segregation, resulting in aneuploidy. The mechanism behind these observations is not well understood. Here I identified a new mechanism for aneuploidy in fission yeast. Kinesin-14 pkl1p and MAP msd1p localize to the spindle poles and focus the MT minus ends. Their absence leads to pole and MT defocusing, resulting in protrusion of MT minus ends due to cut7p-dependent pushing forces at the spindle midzone. Infrequent long MT minus end protrusions can push the already separated chromosome mass back to the cell center, where cytokinesis will `cut\u27 the chromosome mass, creating two daughter cells with unequal chromosome content

Links of cytoskeletal integrity with disease and aging

Aging is a complex feature and involves loss of multiple functions and nonreversible phenotypes. However, several studies suggest it is possible to protect against aging and promote rejuvenation. Aging is associated with many factors, such as telomere shortening, DNA damage, mitochondrial dysfunction, and loss of homeostasis. The integrity of the cytoskeleton is associated with several cellular functions, such as migration, proliferation, degeneration, and mitochondrial bioenergy production, and chronic disorders, including neuronal degeneration and premature aging. Cytoskeletal integrity is closely related with several functional activities of cells, such as aging, proliferation, degeneration, and mitochondrial bioenergy production. Therefore, regulation of cytoskeletal integrity may be useful to elicit antiaging effects and to treat degenerative diseases, such as dementia. The actin cytoskeleton is dynamic because its assembly and disassembly change depending on the cellular status. Aged cells exhibit loss of cytoskeletal stability and decline in functional activities linked to longevity. Several studies reported that improvement of cytoskeletal stability can recover functional activities. In particular, microtubule stabilizers can be used to treat dementia. Furthermore, studies of the quality of aged oocytes and embryos revealed a relationship between cytoskeletal integrity and mitochondrial activity. This review summarizes the links of cytoskeletal properties with aging and degenerative diseases and how cytoskeletal integrity can be modulated to elicit antiaging and therapeutic effects

Cytoskeletal scaffolds in neuronal development

Neuronal polarization is one of the most studied topics in neuroscience. In less than seven days neurites sprout out from the neuron, explore the surrounding environment and mature in axon or dendrites. This process is possible because the neuronal cytoskeleton can rapidly modify its architecture changing neuronal shape and length. Among all, two main proteins are involved: tubulin that supports the neurite elongation and builds a solid frame, while actin supports pathfinding. In this period of important cytoskeletal changes, it is possible to observe the actin waves (AW) that are highly dynamic structures emerging at the neurite base which move up to its tip, causing a transient retraction of the growth cone (GC). Since their discovery in 1988, there have been only few studies about AWs, usually linked to the neurite outgrowth and axon elongation. In the present work, I used long term live cell imaging to investigate alternative roles of such cytoskeletal phenomena. I examined in details AWs and I concluded that they do not promote the neurite outgrowth and that neurites can elongate for hundreds of microns without the AWs. Super resolution nanoscopy indicates that myosin II shapes the GC like AWs structure. The highly concentrated myosin inside the wave can bend the tubulin that support the neurite provoking twists and kinks in the microtubular cytoskeleton. These tubulin twists (TT) cause the GC retraction and are completely abolished with the inhibition of myosin II, that compromises the AW morphology. My results indicate that myosin II has an important role in the AWs dynamics and can bend the tubulin in a way that was not previously observed. Finally, we suggested a role for AWs and TTs in GC exploration and in neurite maturation. Part of these results have already been published in Frontiers in Cellular Neuroscience in the article: Actin waves do not boost neurite outgrowth in the early stages of neuron maturation. We have a second manuscript in preparation entitled \u201cTubulin twists drive Growth Cone retraction and promote tubulin mixed polarity\u201d

Microtubule Post-Translational Detyrosination Coordinates Network Stability And Mechanics In The Cardiomyocyte

The microtubule network of the cardiomyocyte exhibits specialized architecture, stability, and mechanical behavior that accommodates the demands of a working muscle cell. Post-translationally detyrosinated microtubules are physically coupled to the sarcomere, the contractile unit of the muscle, and resist both the contraction and relaxation of the muscle. The cumulative impact of the microtubule network on myocyte mechanics and the enzyme responsible for detyrosinating tubulin are unknown. Further, control of microtubule growth and shrinkage dynamics represents a potential intermediate in the formation of the stable, physically coupled microtubule network, yet the molecular determinates that govern dynamics have not been studied in the cardiomyocyte. I hypothesize that depolymerization of the microtubule network or knockdown of the vasohibin/small vasohibin binding protein complex, a putative tubulin carboxypeptidase in cardiomyocytes, will improve the contractile kinetics of cardiomyocytes isolated healthy or failing human hearts. Additionally, I hypothesize that desmin intermediate filaments may stabilize growing microtubules at the sarcomere Z-disk in a detyrosination-dependent manner. Using a combination of biochemical assays in tandem with direct observation of myocyte mechanics and microtubule dynamics in primary adult cardiomyocytes I find the following: 1) depolymerization of the microtubule network improves contraction and relaxation kinetics in cardiomyocytes isolated from failing human hearts; 2) knockdown of either vasohibin 1 or small vasohibin binding protein reduced levels of microtubule detyrosination resulting in improvements in contractile kinetics and a reduction in cellular stiffness; and 3) tyrosination increases renders the microtubule more dynamic while desmin intermediate filaments stabilize the growing microtubule. In summation, this dissertation establishes a mechanism for the formation of the post-translationally detyrosinated microtubule network, and further underscores the potential of detyrosination as a therapeutic target for the treatment of heart disease

Structural and biophysical investigation of +TIPs in yeast and -TIPs in higher eukaryotes

In eukaryotic cells, microtubules represent a highly dynamic protein filament system that is involved in cellular processes as cell division or transport of cargo. Microtubules oscillate between growth and shrinking, and the switch between these states is caused by catastrophe and rescue events. The building block of microtubules is the heterodimer tubulin, which polymerizes into tubular structures and switches from a curved state in the soluble form to a straight state in microtubules. Due to the polarity of tubulin, microtubules feature a plus-end and a minus-end. The highly dynamic plus-end is regulated by the plus-end tracking proteins (+TIPs). Certain +TIPs can function as a microtubule polymerase or rescue shrinking microtubules. Since budding yeast contains only a small number of microtubules, this organism is predestinated to study +TIPs and microtubule dynamics by microscopy on the system level. The exact function and mechanism of yeast +TIPs such as Bik1 remain unresolved. In addition, it is unexplained how kinesins such as Kip2 or Kip3 can act as a microtubule polymerase or rescue factor. In my thesis, the budding yeast +TIPs Bik1, Kip2 and Kip3 were investigated to understand the role of these proteins in the formation of the +TIP network and how these proteins are capable of influencing microtubule dynamics. Recently, it has been discovered that minus-end tracking proteins (-TIPs) recognize the minus-end in cells such as neuronal cells. However, it is enigmatic how -TIPs target the microtubule minus-end. In order to elucidate the mechanism how -TIPs track the minus-end, my work focused on the discovered first -TIP class of CAMSAPs. In all projects, biophysical methods were applied, and besides for Kip2 crystal structures were determined to unravel mechanistic details of the proteins. In budding yeast, Bik1 plays an important role especially in the dynein pathway, which is one of two major pathways for spindle positioning. Bim1 localizes Bik1 to the microtubule plus-end because Bik1 cannot autonomously track the plus-end. Here, we biophysically and structurally describe the interaction of the Bik1 CAP-Gly domain with the C-terminal tail of the +TIP Bim1. The crystal structure of the complex showed that Bik1 CAP-Gly binds specifically to C-terminal phenylalanine residues with a different binding mode compared to CAP-Gly domains of higher eukaryotes. Based on the structure, two different mutants were conceived to perturb the Bik1-Bim1 interaction. Then, the effect of this perturbation on Bik1 localization, microtubule length and Kar9 function was analyzed in yeast cells. Besides, we proved that the coiled-coil of Bik1 interacts with the C-terminal tail of microtubule polymerase Stu2, establishing Bik1 as an adaptor protein between Bim1 and Stu2. Apart from Bim1, the budding yeast kinesin Kip2 also has the ability to transport Bik1 to the plusend. We biophysically characterized the interaction of the Bik1 coiled-coil with the Kip2 coiledcoil. The C-terminal unstructured part of Kip2 turned out to be essential for the Bik1-Kip2 interaction, allowing an elegant way to disrupt this interaction without removing the Kip2 coiledcoil. In addition, Kip2 functions as a microtubule polymerase. By studying the interaction of the Kip2 motor domain with soluble tubulin, we were able to postulate a mechanism how Kip2 can polymerize microtubules. Furthermore, we identified the importance of the Bik1-Kip2 interaction for the polymerase activity. The budding yeast kinesin Kip3 can depolymerize microtubules but exhibits the ability to rescue them as well. The N-terminal motor domain of Kip3 is responsible for the depolymerization activity. We discovered that Kip3 possesses a C-terminal tubulin-binding domain (TBD), followed by a weak microtubule-binding domain. The crystal structure of the Kip3 TBD was solved, and a sophisticated assembly of alpha-helices was revealed. Furthermore, the combination of the Kip3 motor domain together with the Kip3 TBD was identified as the minimal construct that can rescue microtubules. Therefore, we proposed that the Kip3 motor domain can also act as an anchor at the microtubule plus-end so that the Kip3 TBD can fulfill its rescue function by either increasing the tubulin concentration or facilitating the exchange of tubulin. Most microtubules minus-ends are attached to the centrosome. However, some microtubules can occur with free minus-ends because not all microtubules are attached to the centrosome or cells such as neuronal cells entirely lack the centrosome. Thus, -TIPs like CAMSAPs can stabilize these free minus-ends. CAMSAP proteins have a CKK domain that can autonomously track the microtubule minus-end. In this study, we determined the crystal structure of this CKK domain. Our collaborator used this structure for fitting into a cryo-EM map of microtubules decorated by the CKK domain. Combined with other experimental results, we found that the CKK domain recognizes a unique curved state of tubulin that only occurs at the microtubule minus-end. Overall, important insights into the mechanisms of Bik1 Kip2, Kip3 and CAMSAP were obtained. In the +TIP network, the understanding of Bik1 as a critical adaptor protein was considerably increased. Furthermore, we revealed new insights into the function of Kip2 as a microtubule polymerase. For Kip3, a mechanism for its microtubule rescue function was postulated. In the case of CAMSAP, it was discovered how this protein can recognize the microtubule minus-end. This represents the first described mechanism of a -TIP

Mechanics and kinetics of dynamic instability.

During dynamic instability, self-assembling microtubules (MTs) stochastically alternate between phases of growth and shrinkage. This process is driven by the presence of two distinct states of MT subunits, GTP- and GDP-bound tubulin dimers, that have different structural properties. Here, we use a combination of analysis and computer simulations to study the mechanical and kinetic regulation of dynamic instability in three-dimensional (3D) self-assembling MTs. Our model quantifies how the 3D structure and kinetics of the distinct states of tubulin dimers determine the mechanical stability of MTs. We further show that dynamic instability is influenced by the presence of quenched disorder in the state of the tubulin subunit as reflected in the fraction of non-hydrolysed tubulin. Our results connect the 3D geometry, kinetics and statistical mechanics of these tubular assemblies within a single framework, and may be applicable to other self-assembled systems where these same processes are at play

Computational study of T cell repolarization during target elimination

T Cells are one of the most important players of the immune system. They are responsible for the elimination of the pathogen-infected or tumorigenic cells (target cells). When a target cell is recognized, the T Cell establishes a contact zone called the immunological synapse (IS). Subsequently, the cytoskeleton rotates and the MTOC relocates to the IS. The cytoskeleton rotation is correlated with a movement of organelles attached to microtubules (MT). The MTOC repositioning results from an interplay between MTs and dyneins in the IS pulling MTs via two mechanisms: cortical sliding and capture-shrinkage. Since many aspects of the process remain unknown, we designed a theoretical model for the molecular-motor-driven motion of the MT cytoskeleton in the cell with one or two IS. The model offers explanations of several experimental results including the biphasic nature of the MTOC movement. We also compared the two mechanisms in different cell configurations and found that the T Cell performs one of the most important immune reactions with stunning efficiency by the advantageous placement of dyneins and by employing two mechanisms acting in synergy. We also analyzed Ca2+ diffusion in the T Cell following the MTOC repositioning. We provided the evidence that mitochondria relocate towards the IS with the MTOC and their placement together with their ability of absorption and redistribution significantly increase the Ca2+ concentration.T Zellen sind einer der wichtigsten Spieler des Immunsystems. Sie sind verantwortlich für die Beseitigung von infizierten-oder tumorösen Zellen (Zielzellen). Wenn eine Zielzelle erkannt ist, schafft die T-Zelle eine Immunologische Synapse (IS) genannte Kontaktzone. Dann rotiert das Zytoskelett und das MTOC zieht zur IS. Die Rotation ist mit einer Bewegung von an Mikrotubuli (MT) angehefteten Organellen korreliert. Die MOTC Umpositionierung ergibt sich aus dem Zusammenspiel zwischen MT und Dyneinen in der IS wobei MTs über zwei Mechanismen gezogen werden: ”cortical slidingünd ”captureshrinkage”. Da viele Aspekte des Prozesses unbekannt bleiben entwarfen wir ein theoretisches Modell für die durch molekulare Dyneinen Bewegung des MT Zytoskeletts in der Zelle mit einer oder zwei IS. Das Modell bietet Erklärungen mehrerer experimenteller Ergebnisse einschließlich der biphasischen Natur der MTOC Bewebung. Ebenso verglichen wir die beiden Mechanismen unter verschiedenen Konfigurationen und fanden, dass die T-Zelle eine der wichtigsten Immunreaktionen durch nutzbar Anordnung von Dyneinen und Einsatzes zweier in Synergie arbeitenden Mechanismen mit erstaunlicher Effizienz durchführt. Wir analysierten auch folgenden Ca2+ Diffusion in der T-Zelle. Wir liefern den Nachweis, dass Mitochondrien mit das MTOC zu der IS ziehen und ihre Plazierung, zusammen mit der Fähigkeit der Absorption und Umverteilung, die global Ca2+ Konzentration signifikant steiger

Microtubule mechanics and the implications for their assembly

Microtubules are cytoskeletal protein polymers relevant to a wide range of cell functions. In order to polymerize, the constituent tubulin subunits need to bind the nucleotide GTP, but its subsequent hydrolysis to GDP in the microtubule lattice induces depolymerization. The resulting behaviour of stochastic switching between growth and shrinkage is called dynamic instability. Both dynamic instability and microtubule mechanical properties are integral to many cell functions, yet are poorly understood. The present study uses thermal fluctuation measurements of grafted microtubules with different nucleotide contents to extract stiffnesses, relaxation times, and drag coefficients with an unprecedented precision. Both the stiffness and the relaxation time data indicate that stiffness is a function of length for GDP microtubules stabilized with the chemotherapy drug taxol. By contrast, measurements on microtubules polymerized with the non-hydrolizable GTP-analogue GMPCPP show a significantly higher, but constant, stiffness. The addition of taxol is shown to not significantly affect the properties of these microtubules, but a lowering of the GMPCPP content restores the length-dependent stiffness seen for taxol microtubules. The data are interpreted on the basis of a recent biopolymer model that takes into account the anisotropic architecture of microtubules which consist of loosely coupled protofilaments arranged in a tube. Using taxol microtubules and GMPCPP microtubules as the respective analogues of the GDP and GTP state of microtubules, evidence is presented that shear coupling between neighbouring protofilaments is at least two orders of magnitude stiffer in the GTP state than in the GDP state. Previous studies of nucleotide effects on tubulin have focussed on protofilament bending, and the present study is the first to be able to show a dramatic effect on interprotofilament bonds. The finding’s profound implications for dynamic instability are discussed. In addition, internal friction is found to dominate over hydrodynamic drag for microtubules shorter than ∼ 4 μm and, like stiffness, to be affected by the bound nucleotide, but not by taxol. Furthermore, the thermal shape fluctuations of free microtubules are imaged, and the intrinsic curvatures of microtubules are shown for the first time to follow a spectrum reminiscent of thermal bending. Regarding the extraction of mechanical data, this assay, though previously described in the literature, is shown to suffer from systematic flaws