At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.

No description available.

Thesis advisor: David R. Burgess / Centration of the nucleus after fertilization is an essential step for setting-up cell division and proper embryonic development in many proliferating cells such as the sea urchin. The sperm aster must capture the female pronucleus for fusion as well as the nucleus becoming positioned at the center of the cell. Microtubules (MTs) are known to play a role in this centration but the exact mechanism remains unknown. This begins to investigate current models of nuclear centration and the role of various interactions. Three phases of migration were observed as the male aster migrated with support in independent movements of the male and female pronuclei. Dimpling affects present that altered the morphology of the cell were observed when engagement occurred between the male and female pronuclei. It was discovered that this dimpling effect was a result of an interaction between MTs and the cortex, as confirmed by visualization of sheared cells in which only the cortex remained. Stemming from previous and current research in the lab, the role of post-translational modifications (PMTs) in nuclear centration was investigated for the different forces exerted due to various factors. Tyrosinated and detyrosinated populations were observed with and without the presence of parthenolide (PTL), an agent that inhibits detyrosination. PTL was observed to not only prevent the proper migration, but also that it expanded tyrosination of tubulin – which would further disrupt the force vectors created through the PMTs promotion of dyneins and kinesins. The results have lead to a new hypothesis to be furthered in order to gain an in-depth understanding in the mechanism(s) for pronuclear migration. / Thesis (BS) — Boston College, 2018. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Factor Quinolinone Inhibitor 1 (FQI1), discovered as a small molecule inhibitor of the transcription factor LSF, causes cell death in many cancer cell lines and inhibits tumor growth in tumor xenografts and an endogenous hepatocellular carcinoma model in mice. Significantly, multiple animal studies have shown minimal to no toxicity after FQI1 treatment, making it a promising potential lead chemotherapeutic for multiple cancer types. In determining how FQI1 causes cancer cell death, it was previously shown that FQI1 treatment, like knockdown of LSF expression by siRNA, produced a mitotic arrest with condensed but unaligned chromosomes, but with no clearly observable transcriptional dysregulation. In this thesis, I establish that introducing FQI1 to cells already in mitosis induces a mitotic arrest in colorectal cancer cells, demonstrating that FQI1 inhibits mitotic processes directly while these processes are occurring. This mitotic arrest is characterized by defects in the mitotic spindle and limited connections of mitotic spindles to the kinetochores, as indicated by a dramatic decrease cold-stable microtubules in mitosis. Additionally, in a dose-dependent manner, FQI1 treatment resulted in supernumerary γ-tubulin-containing mitotic centrosomes and γ-tubulin-deficient aster-like bodies, indicating a defect in centrosome stability. As FQI1 is known to be a specific inhibitor of LSF, with its dose dependence for LSF inhibition directly proportional to its ability to inhibit cell proliferation, these findings suggested the novel hypothesis that LSF regulates mitosis through non-transcriptional mechanisms by interacting with key mitotic proteins required for proper spindle formation and metaphase alignment. By mass spectrometry, multiple proteins were identified that interact with biotinylated LSF in mitosis in a FQI1-sensitive manner, with several related to the formation and stability of the mitotic spindle. Proximity ligation assays validated endogenous LSF interactions with CKAP5, a processive microtubule polymerase that protects kinetochore microtubules from depolymerization, and MISP, a requirement for proper mitotic spindle positioning. However, in this assay these interactions were not demonstrably FQI1-sensitive. In conclusion, FQI1 treatment results in defects in kinetochore-microtubule attachment and centrosome stability, triggering a mitotic arrest. Combined with the target specificity of FQI1, this suggests the hypothesis that LSF is required for proper mitotic spindle formation through its protein interactions in mitosis. / 2022-10-16T00:00:00Z

Using statistical physics principles to solve problems in biology is one of the most promising directions due to the complexity and non-equilibrium fluctuations in biological systems. In this work, we try to describe the dynamics at both cellular and molecular levels. Microtubule dynamics and dynamic disorder of enzyme proteins are two of the examples we investigated. The dynamics of microtubules and the mechanical properties of these polymers are essential for many key cellular processes. However, critical discrepancies between experimental observations and existing models need to be resolved before further progress towards a complete model can be made. We carried out computational studies to compare the mechanical properties of two alternative models, one corresponding to the existing, conventional model, and the other considering an additional type of tubulin lateral interaction described in a cryo-EM structure of a proposed trapped intermediate in the microtubule assembly process. Our work indicates that a class of sheet structures is transiently trapped as an intermediate during the assembly process in physiological conditions. In the second part of the work, we analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can serve as a basic functional motif with properties normally discussed with larger networks in the field of systems biology. The work on slow enzyme dynamics fills the missing gap between studies on intramolecular and network dynamics. We also showed that enzyme fluctuations could be amplified into fluctuations in phosphorylation networks. This can be used as a novel biochemical "reporter" for measuring single enzyme conformational fluctuation rates. / Ph. D.

Kinesin superfamily motors have a well-characterized ability to move along microtubules and transport cargo. However, some members of the kinesin superfamily can also remodel microtubule networks by controlling tubulin polymerization dynamics and by organizing microtubule structures. The kinesin-8 family of motors play a central role in cellular microtubule length control and in the regulation of spindle size. These motors move in a highly processive manner along the microtubule lattice towards plus ends. Once at the microtubule plus end, these motors have complex effects on polymerization dynamics: kinesin-8s can either destabilize or stabilize microtubules, depending upon the context. My thesis work identified a tethering mechanism that facilitates the processivity and plus end-binding activity of Kip3 (kinesin-8 in budding yeast), which is essential for the destabilizing activity of kinesin-8 in cells. A concentration-dependent model was proposed to explain the divergent effects of Kip3 on microtubule dynamics. Moreover, a novel activity of Kip3 in organizing microtubules was discovered: Kip3 can slide anti-parallel microtubules apart. The sliding activity of Kip3 counteracts the depolymerizing activity of Kip3 in controlling spindle length and stability. A lack of sliding activity causes fragile spindles during the process of chromosome segregation in anaphase. The tail domain of Kip3, which binds both microtubules and tubulin dimers, plays a critical role in all these activities. Together, my work defined multiple mechanisms by which Kip3 remodels the microtubule cytoskeleton. The physiological importance of these regulatory mechanisms will be discussed.

This thesis uses methods and models from non-equilibrium statistical physics to describe intracellular processes. Bidirectional microtubule-based transport within axons is modeled as a quasi-one-dimensional stochastic lattice gas with two particle species moving in opposite directions under mutual exclusion interaction. Generically occurring clusters of particles in current models for intracellular transport can be dissolved by additionally considering the dynamics of the transport lattice, i.e., the microtubule. An idealized model for the lattice dynamics is used to create a phase transition toward a homogenous state with efficient transport in both directions. In the thermodynamic limit, a steady state property of the dynamic lattice limits the maximal size of clusters. Lane formation mechanisms which are due to specific particle-particle interactions turn out to be very sensitive to the model assumptions. Furthermore, even if some particle-particle interaction is considered, taking the lattice dynamics into account almost always improves transport. Thus the lattice dynamics seems to be the key aspect in understanding how nature regulates intracellular traffic. The last part introduces a model for the dynamics of a microtubule which is limited in its growth by the cell boundary. The action of a rescue-enhancing protein which is added to the growing tip of a microtubule and then slowly dissociates leads to interesting aging effects which should be experimentally observable.

Les microtubules-qui définissent la forme des axones, des cils et des flagelles, et qui servent de rails pour le transport intracellulaire-subissent de fortes contraintes exercées par les forces intracellulaires. La structure des microtubules et leur rigiditépeuvent en théorie être affectées par des contraintes physiques. Cependant, il reste à établir comment les microtubules tolèrent de telles forces et quelles sont les conséquences de ces forces sur la structure des microtubules. En utilisant un dispositif demicrofluidique, j’ai pu montrer que la rigidité des microtubules diminue progressivementà chaque cycle de courbure induit par des contraintes hydrodynamiques.Comme dans d'autres exemples de fatigue des matériaux, l'application de contraintes mécaniques sur des défauts pré-existants le long des microtubules est responsable de la génération de dommages plus étendus. Ce processus rend les microtubules moins rigides.J’ai pu aussi montrer que les microtubules endommagés peuvent se réparer en intégrant de nouveaux dimères de tubuline à leur surface et de récupérer ainsi leur rigidité initiale. Nos résultats démontrent que les microtubules sont des matériaux biologiquesayant des propriétés d’auto-réparation, et que la dynamique des microtubules ne se produit pas exclusivement à leurs extrémités. La mise en évidence de ces nouvelles propriétés permet de montrer comment les microtubules peuvent s’adapter à des contraintesmécaniques. / Microtubules—which define the shape of axons, cilia and flagella, and provide tracks for intracellular transport—can be highly bent by intracellular forces, and microtubule structure and stiffness are thought to be affected by physical constraints. Yet how microtubules tolerate the vast forces exerted on them remains unknown. Here, by using a microfluidic device, we show that microtubule stiffness decreases incrementally with each cycle of bending and release. Similar to other cases of material fatigue, the concentration of mechanical stresses on pre-existing defects in the microtubule lattice is responsible for the generation of more extensive damage, which further decreases microtubule stiffness. Strikingly, damaged microtubules were able to incorporate new tubulin dimers into their lattice and recover their initial stiffness. Our findings demonstrate that microtubules are ductile materials with self-healing properties, that their dynamics does not exclusively occur at their ends, and that their lattice plasticity enables the microtubules’ adaptation to mechanical stresses.

At the onset of cell division microtubules growing from spindle pole bodies (SPB) interact with each other to form the mitotic spindle enabling proper chromosome positioning and segregation. However, the exact mechanism of microtubule dynamics and microtubule associated proteins (MAPs) underlying spindle assembly is still not well understood. We developed an in vivo method to observe spindle assembly in the fission yeast Schizosaccharomyces pombe by inducing depolymerization of already formed and grown spindles by subjecting the cells to low temperatures, followed by subsequent repolymerization at a permissive temperature. We observed that microtubules pivot, i.e., perform angular movement around the SPB in a random manner, exploring the intranuclear space. Eventually microtubules extending from opposite SPBs come into contact and establish an antiparallel connection thus reassembling the spindle. Mutant approaches revealed that deletion of ase1 and klp5 did not prevent spindle reassembly, however introduced aberrations during the spindle formation. Amazingly, cut7p showed direct colocalization with microtubule overlap during spindle reassembly. Abrogation of cut7p led to inability to form a functional spindle. Thus, cut7p is the main regulator of spindle formation in fission yeast. None of the mutant strains affected microtubule pivoting, confirming that microtubule pivoting is a random movement unrelated to MAPs.

Microtubules (MTs) are highly dynamic structures essential for the spatio-temporal intracellular organization and transport, signal propagation, cell differentiation, motility and division. To perform these roles, MTs create arrangements capable of fast and precise adaptation to various signals. MTs are under the control of many factors regulating MT nucleation, stability and dynamics. Bone marrow-derived mast cells (BMMCs) are important immune system cells, which can cause serious diseases if their functions are deregulated. Although MT reorganization during BMMC activation is well established, the molecular mechanisms that control their remodelling are largely unknown. In the presented thesis we functionally characterised GIT1/βPIX signalling proteins, PAK1 kinase, and Ca2+ signalling in the regulation of MT nucleation in BMMCs and other cell types. We also elucidated the function of miltefosine (hexadecylphosphocholine), a promising candidate for the treatment of mast cell-driven diseases. We found that GIT1/βPIX signalling proteins are γ-tubulin-interacting proteins associating with centrosomes in BMMCs. MT nucleation is positively regulated by GIT1 and Ca2+ , whereas βPIX is a negative regulator of MT nucleation in BMMCs. Cytosolic Ca2+ affects γ-tubulin properties and stimulates the...