Microtubule Behavior Research Articles

Impact of physiological loads of arterial wall on nucleus deformation in endothelial cells: A computational study

IntroductionComputational modeling can enhance the understanding of cell mechanics. To achieve this, finite element models of endothelial cells were proposed with shapes mimicking their natural state inside the endothelium within the cardiovascular system. Implementing the recently proposed bendo-tensegrity concept, these models consider flexural (buckling) as well as tensional/compressional behavior of microtubules and also incorporate the waviness of intermediate filaments. Materials and methodsFour different models were created (flat and domed hexagons, both regular and elongated in the direction of blood flow) and loaded by biaxial deformation, blood pressure, and shear load from blood flow – natural physiological conditions of the arterial endothelium – aiming to investigate the “in situ” mechanical response of the cell. ResultsThe impact of individual components of loads on the nucleus deformation (more specifically on the first principal strain) potentially influencing mechanotransduction was investigated and the role of the cytoskeleton and its constituents in the mechanical response of the endothelial cell was assessed. The results show (i) the impact of pulsating blood pressure on cyclic deformations of the nucleus, which increase substantially with decreasing axial pre-stretch of the cell, (ii) the importance of relatively low shear stresses in the cell response and nucleus deformation. ConclusionNot only the pulsatile blood pressure but also the wall shear stress may induce significant deformation of the nucleus and thus trigger remodelation processes in endothelial cells.

Simulation to clarify the role of microtubules in cell growth

Plant materials sometimes have high mechanical strength in its outer structure which is derived from the cell wall at the microscopic level. Since the deposition of the cell wall is thought to be induced by the microtubule alignment inside cells, it is important to know the microtubule dynamics from the viewpoints of both plant and materials sciences. To investigate the microtubule dynamics in silico, we constructed an agent-based model of microtubule that simulates the growth and shrinkage of microtubules with microtubule-microtubule interactions. In addition to this initial setup, we further considered the condition of the edge-catastrophe that microtubule is to shrink when it hits the edge of the geometry, or the condition of the tension-catastrophe that cell surface tension induces the shrinkage. By introducing these conditions to our model, we aim to elucidate how the ring-like microtubules are formed during plant zygote development. Our formulation suggests a control parameter of the orientation order from the collective behavior of microtubules. It would greatly help to test some hypotheses on the microtubule ring formation that have been experimentally difficult to investigate.

TEM Imaging of Membrane Choreography During Mitosis of Drosophila Tissue Culture Cells.

Schneider 2 (S2) cells are one of the most widely used Drosophila cell lines, and are specifically suitable for genetic dissection of biological processes by RNA interference. We have recently developed a method that allows an easy preparation of samples for transmission electron microscopy (TEM) analysis of S2 cells. This method is based on the collection and pelleting of the cells in test tubes, followed by fixation and staining of pellets in the same tubes. Pellets are then embedded in resin and used to prepare ultrathin sections for TEM observation. Our Method allows clear visualization of the complex membrane transformations that characterize mitosis in S2 cells. It also allows precise analysis of microtubule behavior during the different mitotic phases. Although the method was specifically developed for S2 cells, our preliminary results indicate that it can be successfully applied to other types of Drosophila tissue culture cells.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons.

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

Dynamic behaviour of microtubules around the critical temperature and effect of the electric field produced by these vibrations on its environment

Dynamic behaviour of microtubules around the critical temperature and effect of the electric field produced by these vibrations on its environment

The drebrin/EB3 pathway regulates cytoskeletal dynamics to drive neuritogenesis in embryonic cortical neurons.

Co-ordinating the dynamic behaviour of actin filaments (F-actin) and microtubules in filopodia is an important underlying process in neuritogenesis, but the molecular pathways involved are ill-defined. The drebrin/end-binding protein 3 (EB3) pathway is a candidate pathway for linking F-actin to microtubules in filopodia. Drebrin binds F-actin and, simultaneously, the microtubule-binding protein EB3 when bound to microtubule plus-ends. We assessed the effect on neuritogenesis of gain- or loss-of-function of proteins in the drebrin/EB3 pathway in rat embryonic cortical neurons in culture. Loss-of-function of drebrin by gene editing or pharmacological inhibition of drebrin binding to F-actin reduced the number of dynamic microtubules in the cell periphery and simultaneously delayed the initiation of neuritogenesis, whereas over-expression of drebrin induced supernumerary neurites. Similarly, loss of EB3 inhibited neuritogenesis, whereas loss of end-binding protein 1 (EB1), a related protein that does not bind to drebrin, did not affect neuritogenesis. Over-expression of EB3, but not EB1, induced supernumerary neurites. We discovered that EB3 is more proximally located at dynamic microtubule plus-ends than EB1 in growth cone filopodia allowing for continuous microtubule elongation as the drebrin/EB3 pathway zippers microtubules to F-actin in filopodia. Finally, we showed that preventing the entry of dynamic microtubules into filopodia using a pharmacological inhibitor of microtubule dynamics is associated with a loss of EB3, but not EB1, from microtubule plus-ends and a concurrent attenuation of neuritogenesis. Collectively, these findings support the idea that neuritogenesis depends on microtubule/F-actin zippering in filopodia orchestrated by the drebrin/EB3 pathway.

Investigation of wave propagation in architected uniform triangle mass center fractal nano-bio-filters based on microtubules

Microtubules, polymer tubes stretched from the cell nucleus to the cell membrane, are the major parts of the cytoskeleton that provide the mechanical rigidity, organization, and shape retention for the cytoplasm of eukaryotic cells. These structures play a key role in some cellular processes such as cell division, intracellular transport, and the internal organization of cells. In all the above applications, the network structure of microtubules is the main reason for the importance of in-depth studies of their mechanical properties. In this paper, the propagation of elastic waves in periodic networks based on two-dimensional fractal microtubules of fixed mass-center triangles is analyzed. This study begins with the selection of a suitable beam model for a microtubule and examines the dynamic behavior of microtubules by creating periodic structures. To obtain dispersion curves, finite element models of microtubules and their networks are developed, and the bandgap equations are calculated based on Bloch's theory. The results show that depending on the topology of the selected unit cells as well as the considered periods, it is possible to design a frequency gap in specific ranges for the application of low and high-frequency bio-filters. This study helps researchers control or absorb some unwanted vibrations using periodic structures, and thanks to their better biocompatibility, these networks can be used in next-generation nanomechanical devices such as implantable biosensors.

Modeling the Mechanical Response of Microtubule Lattices to Pressure.

Microtubules, the largest and stiffest filaments of the cytoskeleton, have to be well adapted to the high levels of crowdedness in cells to perform their multitude of functions. Furthermore, fundamental processes that involve microtubules, such as the maintenance of the cellular shape and cellular motion, are known to be highly dependent on external pressure. In light of the importance of pressure for the functioning of microtubules, numerous studies interrogated the response of these cytoskeletal filaments to osmotic pressure, resulting from crowding by osmolytes, such as poly(ethylene glycol)/poly(ethylene oxide) (PEG/PEO) molecules, or to direct applied pressure. The interpretation of experiments is usually based on the assumptions that PEG molecules have unfavorable interactions with the microtubule lattices and that the behavior of microtubules under pressure can be described by using continuous models. We probed directly these two assumptions. First, we characterized the interaction between the main interfaces in a microtubule filament and PEG molecules of various sizes using a combination of docking and molecular dynamics simulations. Second, we studied the response of a microtubule filament to compression using a coarse-grained model that allows for the breaking of lattice interfaces. Our results show that medium length PEG molecules do not alter the energetics of the lateral interfaces in microtubules but rather target and can penetrate into the voids between tubulin monomers at these interfaces, which can lead to a rapid loss of lateral interfaces under pressure. Compression of a microtubule under conditions corresponding to high osmotic pressure results in the formation of the deformed phase found in experiments. Our simulations show that the breaking of lateral interfaces, rather than the buckling of the filament inferred from the continuous models, accounts for the deformation.

Dual Impact of a Benzimidazole Resistant β-Tubulin on Microtubule Behavior in Fission Yeast.

The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target β-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on β-tubulin remains controversial. Here, we characterize the S. pombe β-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral β-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant β-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant β-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant β-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to β-tubulin.

Tell the Difference Between Mitosis and Meiosis: Interplay Between Chromosomes, Cytoskeleton, and Cell Cycle Regulation.

Meiosis is a specialized style of cell division conserved in eukaryotes, particularly designed for the production of gametes. A huge number of studies to date have demonstrated how chromosomes behave and how meiotic events are controlled. Yeast substantially contributed to the understanding of the molecular mechanisms of meiosis in the past decades. Recently, evidence began to accumulate to draw a perspective landscape showing that chromosomes and microtubules are mutually influenced: microtubules regulate chromosomes, whereas chromosomes also regulate microtubule behaviors. Here we focus on lessons from recent advancement in genetical and cytological studies of the fission yeast Schizosaccharomyces pombe, revealing how chromosomes, cytoskeleton, and cell cycle progression are organized and particularly how these are differentiated in mitosis and meiosis. These studies illuminate that meiosis is strategically designed to fulfill two missions: faithful segregation of genetic materials and production of genetic diversity in descendants through elaboration by meiosis-specific factors in collaboration with general factors.

Finite Element Simulations of Mechanical Behaviour of Endothelial Cells.

Biomechanical models based on the finite element method have already shown their potential in the simulation of the mechanical behaviour of cells. For instance, development of atherosclerosis is accelerated by damage of the endothelium, a monolayer of endothelial cells on the inner surface of arteries. Finite element models enable us to investigate mechanical factors not only at the level of the arterial wall but also at the level of individual cells. To achieve this, several finite element models of endothelial cells with different shapes are presented in this paper. Implementing the recently proposed bendotensegrity concept, these models consider the flexural behaviour of microtubules and incorporate also waviness of intermediate filaments. The suspended and adherent cell models are validated by comparison of their simulated force-deformation curves with experiments from the literature. The flat and dome cell models, mimicking natural cell shapes inside the endothelial layer, are then used to simulate their response in compression and shear which represent typical loads in a vascular wall. The models enable us to analyse the role of individual cytoskeletal components in the mechanical responses, as well as to quantify the nucleus deformation which is hypothesized to be the quantity decisive for mechanotransduction.

Collective Effects of Xmap215, EB1, CLASP2, and MCAK Lead to Robust Microtubule Treadmilling

Microtubule network remodeling is essential for fundamental cellular processes including cell division, differentiation, and motility. Microtubules are active biological polymers whose ends stochastically and independently switch between phases of growth and shrinkage. Microtubule treadmilling, in which the microtubule plus end grows while the minus end shrinks, is observed in cells; however, the underlying mechanisms are not known. Here, we use a combination of computational and in vitro reconstitution approaches to determine the conditions leading to robust microtubule treadmilling. We find that microtubules polymerized from tubulin alone can treadmill, albeit with opposite directionality and order-of-magnitude slower rates than observed in cells. We then employ computational simulations to predict that the combinatory effects of four microtubule-associated proteins (MAPs), namely EB1, XMAP215, CLASP2, and MCAK, can promote fast and sustained plus-end-leading treadmilling. Finally, we experimentally confirm the predictions of our computational model using a multi-MAP, in vitro microtubule dynamics assay to reconstitute robust plus-end-leading treadmilling, consistent with observations in cells. Our results demonstrate how microtubule dynamics can be modulated to achieve a dynamic balance between assembly and disassembly at opposite polymer ends, resulting in treadmilling over long periods of time. Overall, we show how the collective effects of multiple components give rise to complex microtubule behavior that may be used for global network remodeling in cells.

Persistent growth of microtubules at low density.

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability—transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density–dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.

Revealing and Attenuating the Electrostatic Properties of Tubulin and Its Polymers.

Tubulin is an electrostatically negative protein that forms cylindrical polymers termed microtubules, which are crucial for a variety of intracellular roles. Exploiting the electrostatic behavior of tubulin and microtubules within functional microfluidic and optoelectronic devices is limited due to the lack of understanding of tubulin behavior as a function of solvent composition. This work displays the tunability of tubulin surface charge using dimethyl sulfoxide (DMSO) for the first time. Increasing the DMSO volume fractions leads to the lowering of tubulin's negative surface charge, eventually causing it to become positive in solutions >80% DMSO. As determined by electrophoretic mobility measurements, this change in surface charge is directionally reversible, i.e., permitting control between -1.5 and + 0.2cm2 (Vs)-1 . When usually negative microtubules are exposed to these conditions, the positively charged tubulin forms tubulin sheets and aggregates, as revealed by an electrophoretic transport assay. Fluorescence-based experiments also indicate that tubulin sheets and aggregates colocalize with negatively charged g-C3 N4 sheets while microtubules do not, further verifying the presence of a positive surface charge. This study illustrates that tubulin and its polymers, in addition to being mechanically robust, are also electrically tunable.

The p53/p73 - p21CIP1 tumor suppressor axis guards against chromosomal instability by restraining CDK1 in human cancer cells

Whole chromosome instability (W-CIN) is a hallmark of human cancer and contributes to the evolvement of aneuploidy. W-CIN can be induced by abnormally increased microtubule plus end assembly rates during mitosis leading to the generation of lagging chromosomes during anaphase as a major form of mitotic errors in human cancer cells. Here, we show that loss of the tumor suppressor genes TP53 and TP73 can trigger increased mitotic microtubule assembly rates, lagging chromosomes, and W-CIN. CDKN1A, encoding for the CDK inhibitor p21CIP1, represents a critical target gene of p53/p73. Loss of p21CIP1 unleashes CDK1 activity which causes W-CIN in otherwise chromosomally stable cancer cells. Consequently, induction of CDK1 is sufficient to induce abnormal microtubule assembly rates and W-CIN. Vice versa, partial inhibition of CDK1 activity in chromosomally unstable cancer cells corrects abnormal microtubule behavior and suppresses W-CIN. Thus, our study shows that the p53/p73 - p21CIP1 tumor suppressor axis, whose loss is associated with W-CIN in human cancer, safeguards against chromosome missegregation and aneuploidy by preventing abnormally increased CDK1 activity.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

C-Terminal Tail Polyglycylation and Polyglutamylation Alter Microtubule Mechanical Properties

Microtubules are biopolymers that perform diverse cellular functions. Microtubule behavior regulation occurs in part through post-translational modification of both the α- and β-subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and/or glutamate residues to the disordered C-terminal tails (CTTs) of tubulin. Because of their prevalence in stable, high-stress cellular structures such as cilia, we sought to determine if these modifications alter microtubules’ intrinsic stiffness. Here, we describe the purification and characterization of differentially modified pools of tubulin from Tetrahymena thermophila. We found that post-translational modifications do affect microtubule stiffness but do not affect the number of protofilaments incorporated into microtubules. We measured the spin dynamics of nuclei in the CTT backbone by NMR spectroscopy to explore the mechanism of this change. Our results show that the α-tubulin CTT does not protrude out from the microtubule surface, as is commonly depicted in models, but instead interacts with the dimer’s surface. This suggests that the interactions of the α-tubulin CTT with the tubulin body contributes to the stiffness of the assembled microtubule, thus providing insight into the mechanism by which polyglycylation and polyglutamylation can alter microtubule mechanical properties.

Modified heptapeptide from tau binds both tubulin and microtubules.

BackgroundMicrotubules are the major cytoskeletal component in eukaryotes which are essential for a large spectrum of cellular activities. Monitoring the behavior of microtubules is helpful for a better understanding of the regulatory mechanism governing microtubule architecture and microtubule‐based activities. Here, we characterized the binding capability of a modified heptapeptide from tau to both tubulin and microtubules and sought to develop it as a fluorescent peptide for monitoring microtubules.MethodsTo deliver the fluorescent peptide into the cells, a cell‐penetrating peptide was conjugated to the modified heptapeptide from tau and synthesized. The affinity of the modified heptapeptide was determined by microscale thermophoresis. The microtubule labeling ability was determined by adding the peptide into the polymerized microtubule solutions or cultured HeLa cells.;ResultsAffinity determination revealed that the tau‐derived peptide specifically bound to tubulin. In addition, the peptide was able to label polymerized microtubules in solution, although no obvious microtubule filaments were observed clearly in living cells, probably due to the inadequate affinity.ConclusionsThese results suggest that using a peptide‐based strategy for imaging microtubules might be plausible and attempts to improve its affinity is warranted in the future.

Kv10.1 Regulates Microtubule Dynamics during Mitosis.

Kv10.1 (potassium voltage-gated channel subfamily H member 1, known as EAG1 or Ether-à-go-go 1), is a voltage-gated potassium channel, prevailingly expressed in the central nervous system. The aberrant expression of Kv10.1 is detected in over 70% of all human tumor tissues and correlates with poorer prognosis. In peripheral tissues, Kv10.1 is expressed almost exclusively during the G2/M phase of the cell cycle and regulates its progression—downregulation of Kv10.1 extends the duration of the G2/M phase both in cancer and healthy cells. Here, using biochemical and imaging techniques, such as live-cell measurements of microtubule growth and of cytosolic calcium, we elucidate the mechanisms of Kv10.1-mediated regulation at the G2/M phase. We show that Kv10.1 has a dual effect on mitotic microtubule dynamics. Through the functional interaction with ORAI1 (calcium release-activated calcium channel protein 1), it modulates cytosolic calcium oscillations, thereby changing microtubule behavior. The inhibition of either Kv10.1 or ORAI1 stabilizes the microtubules. In contrast, the knockdown of Kv10.1 increases the dynamicity of mitotic microtubules, resulting in a stronger spindle assembly checkpoint, greater mitotic spindle angle, and a decrease in lagging chromosomes. Understanding of Kv10.1-mediated modulation of the microtubule architecture will help to comprehend how cancer tissue benefits from the presence of Kv10.1, and thereby increase the efficacy and safety of Kv10.1-directed therapeutic strategies.

Kinetochore proteins suppress neuronal microtubule dynamics and promote dendrite regeneration.

Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.