Microtubule Dynamic Instability Research Articles

Abstract LB261: Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line

Abstract Triple-negative breast cancer (TNBC) is a lethal female cancer, of which paclitaxel resistance is a major factor limiting treatment outcomes, and continued unmet needs for more effective therapeutic strategies are arduous. The aim of this study is to establish a paclitaxel-resistant TNBC cell line and to characterize the underlying mechanism for potential strategies to overcome the resistance. The human TNBC cell line MDA-MB-231 was exposed to stepwise escalating paclitaxel concentration from 0.5 to 50 nM and a resistant cell line T50R was selected. The IC50 of paclitaxel was 2 and >100 nM in MDA-MB-231 and T50R cells respectively. However, T50R cells required 50 nM Taxol for proliferation. When cultured in the absence of paclitaxel, T50R cells arrested at mitotic stage with high levels of spindle abnormalities. Since induction of microtubule (MT) stabilization plays critical roles in paclitaxel’s potency, we thus measure cell MT polymerization and kinetochore-MT stability. The results showed that, compared to parental MDA-MB-231 cells, the interphase MTs were less polymerized and the kinetochore-MTs were less stable in T50R cells, indicating that the MT dynamic instability was significantly increased in T50R cells. Thus, the increased dynamic unstable MTs in T50R cells might contribute to drug requirement for cell growth and counteract with paclitaxel effects, and consequently lead to drug-resistant phenotype. Identification and interference of endogenous regulators involved in the increased MT instability in T50R cells might provide leads to overcome paclitaxel resistance. Citation Format: Ling-Huei Yih, Hsiao-Hui Kuo, Chieh-Ting Fang. Establishment and characterization of a paclitaxel-resistant human triple-negative breast cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB261.

Emergence of Dynamic Instability by Hybridizing Synthetic Self-Assembled Dipeptide Fibers with Surfactant Micelles.

Supramolecular chemistry currently faces the challenge of controlling nonequilibrium dynamics such as the dynamic instability of microtubules. In this study, we explored the emergence of dynamic instability through the hybridization of peptide-type supramolecular nanofibers with surfactant micelles. Using real-time confocal imaging, we discovered that the addition of micelles to nanofibers induced the simultaneous but asynchronous growth and shrinkage of nanofibers during which the total number of fibers decreased monotonically. This dynamic phenomenon unexpectedly persisted for 6 days and was driven not by chemical reactions but by noncovalent supramolecular interactions between peptide-type nanofibers and surfactant micelles. This study demonstrates a strategy for inducing autonomous supramolecular dynamics, which will open up possibilities for developing soft materials applicable to biomedicine and soft robotics.

CSPP1 stabilizes microtubules by capping both plus and minus ends.

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

Optimization of a machine-learning-based approach to quantify and characterize microtubule dynamic instability

Optimization of a machine-learning-based approach to quantify and characterize microtubule dynamic instability

A force balance model for a cell size-dependent meiotic nuclear oscillation in fission yeast.

Fission yeast undergoes premeiotic nuclear oscillation, which is dependent on microtubules and is driven by cytoplasmic dynein. Although the molecular mechanisms have been analyzed, how a robust oscillation is generated despite the dynamic behaviors of microtubules has yet to be elucidated. Here, we show that the oscillation exhibits cell length-dependent frequency and requires a balance between microtubule and viscous drag forces, as well as proper microtubule dynamics. Comparison of the oscillations observed in living cells with a simulation model based on microtubule dynamic instability reveals that the period of oscillation correlates with cell length. Genetic alterations that reduce cargo size suggest that the nuclear movement depends on viscous drag forces. Deletion of a gene encoding Kinesin-8 inhibits microtubule catastrophe at the cell cortex and results in perturbation of oscillation, indicating that nuclear movement also depends on microtubule dynamic instability. Our findings link numerical parameters from the simulation model with cellular functions required for generating the oscillation and provide a basis for understanding the physical properties of microtubule-dependent nuclear movements.

Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities

Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer79) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. In this study, we found that modulating pSer79-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. We then showed that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. We also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, our study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

Enhancing image resolution of confocal fluorescence microscopy with deep learning

Super-resolution optical imaging is crucial to the study of cellular processes. Current super-resolution fluorescence microscopy is restricted by the need of special fluorophores or sophisticated optical systems, or long acquisition and computational times. In this work, we present a deep-learning-based super-resolution technique of confocal microscopy. We devise a two-channel attention network (TCAN), which takes advantage of both spatial representations and frequency contents to learn a more precise mapping from low-resolution images to high-resolution ones. This scheme is robust against changes in the pixel size and the imaging setup, enabling the optimal model to generalize to different fluorescence microscopy modalities unseen in the training set. Our algorithm is validated on diverse biological structures and dual-color confocal images of actin-microtubules, improving the resolution from ~ 230 nm to ~ 110 nm. Last but not least, we demonstrate live-cell super-resolution imaging by revealing the detailed structures and dynamic instability of microtubules.

Myotoxin-3 from the Pacific Rattlesnake Crotalus oreganus oreganus Venom Is a New Microtubule-Targeting Agent

Microtubule targeting agents (MTA) are anti-cancer molecules that bind tubulin and interfere with the microtubule functions, eventually leading to cell death. In the present study, we used an in vitro microtubule polymerization assay to screen several venom families for the presence of anti-microtubule activity. We isolated myotoxin-3, a peptide of the crotamine family, and three isoforms from the venom of the Northern Pacific rattlesnake Crotalus oreganus oreganus, which was able to increase tubulin polymerization. Myotoxin-3 turned out to be a cell-penetrating peptide that slightly diminished the viability of U87 glioblastoma and MCF7 breast carcinoma cells. Myotoxin 3 also induced remodeling of the U87 microtubule network and decreased MCF-7 microtubule dynamic instability. These effects are likely due to direct interaction with tubulin. Indeed, we showed that myotoxin-3 binds to tubulin heterodimer with a Kd of 5.3 µM and stoichiometry of two molecules of peptide per tubulin dimer. Our results demonstrate that exogenous peptides are good candidates for developing new MTA and highlight the richness of venoms as a source of pharmacologically active molecules.

Beyond the GTP-cap: Elucidating the molecular mechanisms of microtubule catastrophe.

Almost 40years since the discovery of microtubule dynamic instability, the molecular mechanisms underlying microtubule dynamics remain an area of intense research interest. The "standard model" of microtubule dynamics implicates a "cap" of GTP-bound tubulin dimers at the growing microtubule end as the main determinant of microtubule stability. Loss of the GTP-cap leads to microtubule "catastrophe," a switch-like transition from microtubule growth to shrinkage. However, recent studies, using biochemical in vitro reconstitution, cryo-EM, and computational modeling approaches, challenge the simple GTP-cap model. Instead, a new perspective on the mechanisms of microtubule dynamics is emerging. In this view, highly dynamic transitions between different structural conformations of the growing microtubule end - which may or may not be directly linked to the nucleotide content at the microtubule end - ultimately drive microtubule catastrophe.

Modeling microtubule dynamic instability: Microtubule growth, shortening and pause

Microtubules (MTs) are protein polymers found in all eukaryotic cells. They are crucial for normal cell development, providing structural support for the cell and aiding in the transportation of proteins and organelles. In order to perform these functions, MTs go through periods of relatively slow polymerization (growth) and very fast depolymerization (shortening), where the switch from growth to shortening is called a catastrophe and the switch from shortening to growth is called a rescue. Although MT dynamic instability has traditionally been described solely in terms of growth and shortening, MTs have been shown to pause for extended periods of time, however the reason for pausing is not well understood. Here, we present a new mathematical model to describe MT dynamics in terms of growth, shortening, and pausing. Typically, MT dynamics are defined by four key parameters which include the MT growth rate, shortening rate, frequency of catastrophe, and the frequency of rescue. We derive a mathematical expression for the catastrophe frequency in the presence of pausing, as well as expressions to describe the total time that MTs spend in a state of growth and pause. In addition to exploring MT dynamics in a control-like setting, we explore the implicit effect of stabilizing MT associated proteins (MAPs) and stabilizing and destabilizing chemotherapeutic drugs that target MTs on MT dynamics through variations in model parameters.

Radiosynthesis and evaluation of [11C]AG-488, a dual anti-angiogenetic and anti-tubulin PET ligand

Combinations of antiangiogenic and cytotoxic agents show promising results in the treatment of cancer. However, there is a lack of single agent with both antiangiogenic and cytotoxic activities for clinical application. AG-488 aka FLAG-003 is a novel ligand with established antiangiogenetic properties via activation of receptor thymidine kinase (RTK) and anti-tubulin properties in tumor cells. AG-488 is also reported to reduce tumor volume and prolong survival in preclinical animal models of glioblastoma multiforme, breast cancer and is in clinical stage. Higher expression of RTKs and tubulins is reported in various cancers. This study reveals the development of [11C]AG-488, a high affinity dual target inhibitor binding to RTK and anti-tubulin activities. We rationale that antiangiogenic RTK and anti-tubulin activity of [11C]AG-488 may enhance the tumor to tissue ratio, assisting in cancer drug development. [11C]AG-488 was synthesized in 35 ± 5 % radiochemical yield by radiomethylating the corresponding phenolate using [11C]CH3I. MicroPET studies in mice indicated blood-brain barrier penetration of [11C]AG-488 and retention in the brain. However, blocking studies with antitubulin and RTK agent HD-800 and microtubule depolymerizing agent MPC-6827 show increased binding of [11C]AG-488 in brain. The pattern of tracer binding in blocking conditions is similar to the baseline conditions. The higher binding may be due to the increased plasma uptake of radiotracer or the formation of more free tubulins due to microtubule dynamic instability during the blocking conditions.

Cell Fate following Irradiation of MDA-MB-231 and MCF-7 Breast Cancer Cells Pre-Exposed to the Tetrahydroisoquinoline Sulfamate Microtubule Disruptor STX3451

A tetrahydroisoquinoline (THIQ) core is able to mimic the A and B rings of 2-methoxyestradiol (2ME2), an endogenous estrogen metabolite that demonstrates promising anticancer properties primarily by disrupting microtubule dynamic instability parameters, but has very poor pharmaceutical properties that can be improved by sulfamoylation. The non-steroidal THIQ-based microtubule disruptor 2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (STX3451), with enhanced pharmacokinetic and pharmacodynamic profiles, was explored for the first time in radiation biology. We investigated whether 24 h pre-treatment with STX3451 could pre-sensitize MCF-7 and MDA-MB-231 breast cancer cells to radiation. This regimen showed a clear increase in cytotoxicity compared to the individual modalities, results that were contiguous in spectrophotometric analysis, flow cytometric quantification of apoptosis induction, clonogenic studies and microscopy techniques. Drug pre-treatment increased radiation-induced DNA damage, with statistically more double-strand (ds) DNA breaks demonstrated. The latter could be due to the induction of a radiation-sensitive metaphase block or the increased levels of reactive oxygen species, both evident after compound exposure. STX3451 pre-exposure may also delay DNA repair mechanisms, as the DNA damage response element ataxia telangiectasia mutated (ATM) was depressed. These in vitro findings may translate into in vivo models, with the ultimate aim of reducing both radiation and drug doses for maximal clinical effect with minimal adverse effects.

Divalent cations induce non-equilibrium phase transitions in tau-mediated microtubule bundles undergoing dynamic instability

The microtubule-associated protein tau is known for its ability to alter microtubule (MT) dynamic instability in developing and mature neurons and for its role in the pathology of many neurodegenerative diseases. Bundling of Mts into small linear arrays (or fascicles) is a cardinal feature of the axon initial segment. While tau plays an important role in the structural stability of MT fascicles, the exact mechanism by which tau imparts MT bundle stabilization remains unclear. To help elucidate the nature of tau's role in physiological bundling of Mts, we have expanded on a recent platform for studying paclitaxel-free in vitro reaction mixtures of tubulin, GTP, and wild-type tau under dissipative, out-of-equilibrium conditions at 37 °C.

SSNA1 stabilizes dynamic microtubules and detects microtubule damage

Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. We find that SSNA1 modulates all parameters of microtubule dynamic instability—slowing down the rates of growth, shrinkage and catastrophe, and promoting rescue.

Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules

Microtubules (MTs) are polymers of αβ-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.

Visualization and Quantification of Microtubule Self-Repair.

Since its discovery, several decades ago, microtubule dynamic instability has been the subject of countless studies that demonstrate its impact on cellular behavior in health and disease. Recent studies reveal a new dimension of microtubule dynamics. Microtubules are not only dynamic at their tips but also exhibit loss and incorporation of tubulin subunits along their lattice far from the tips. Although this phenomenon has been observed to occur under various conditions in vitro as well as in cells, many questions remain regarding the regulation of lattice dynamics and their contribution to overall microtubule network organization and function. Compared to microtubule tip dynamics, the dynamics of tubulin incorporation along the lattice are more challenging to investigate as they are hidden in classical experimental setups, which is likely the reason they were overlooked for a long time. In this chapter, we present a strategy to visualize and quantify the incorporation of tubulin subunits into the microtubule lattice in vitro. The proposed method does not require specialized equipment and can thus be carried out readily in most research laboratories.

Microtubule assembly and disassembly dynamics model: Exploring dynamic instability and identifying features of Microtubules’ Growth, Catastrophe, Shortening, and Rescue

Microtubules (MTs), a cellular structure element, exhibit dynamic instability and can switch stochastically from growth to shortening; but the factors that trigger these processes at the molecular level are not understood. We developed a 3D Microtubule Assembly and Disassembly DYnamics (MADDY) model, based upon a bead-per-monomer representation of the αβ-tubulin dimers forming an MT lattice, stabilized by the lateral and longitudinal interactions between tubulin subunits. The model was parameterized against the experimental rates of MT growth and shortening, and pushing forces on the Dam1 protein complex due to protofilaments splaying out. Using the MADDY model, we carried out GPU-accelerated Langevin simulations to access dynamic instability behavior. By applying Machine Learning techniques, we identified the MT characteristics that distinguish simultaneously all four kinetic states: growth, catastrophe, shortening, and rescue. At the cellular 25 μM tubulin concentration, the most important quantities are the MT length L, average longitudinal curvature κlong, MT tip width w, total energy of longitudinal interactions in MT lattice Ulong, and the energies of longitudinal and lateral interactions required to complete MT to full cylinder Ulongadd and Ulatadd. At high 250 μM tubulin concentration, the most important characteristics are L, κlong, number of hydrolyzed αβ-tubulin dimers nhyd and number of lateral interactions per helical pitch nlat in MT lattice, energy of lateral interactions in MT lattice Ulat, and energy of longitudinal interactions in MT tip ulong. These results allow greater insights into what brings about kinetic state stability and the transitions between states involved in MT dynamic instability behavior.

SSNA1 stabilizes dynamic microtubules and detects microtubule damage.

Sjögren's syndrome nuclear autoantigen-1 (SSNA1/NA14) is a microtubule-associated protein with important functions in cilia, dividing cells, and developing neurons. However, the direct effects of SSNA1 on microtubules are not known. We employed in vitro reconstitution with purified proteins and TIRF microscopy to investigate the activity of human SSNA1 on dynamic microtubule ends and lattices. Our results show that SSNA1 modulates all parameters of microtubule dynamic instability-slowing down the rates of growth, shrinkage, and catastrophe, and promoting rescue. We find that SSNA1 forms stretches along growing microtubule ends and binds cooperatively to the microtubule lattice. Furthermore, SSNA1 is enriched on microtubule damage sites, occurring both naturally, as well as induced by the microtubule severing enzyme spastin. Finally, SSNA1 binding protects microtubules against spastin's severing activity. Taken together, our results demonstrate that SSNA1 is both a potent microtubule-stabilizing protein and a novel sensor of microtubule damage; activities that likely underlie SSNA1's functions on microtubule structures in cells.

The Tubulin Code and Tubulin-Modifying Enzymes in Autophagy and Cancer

Microtubules are key components of the cytoskeleton of eukaryotic cells. Microtubule dynamic instability together with the "tubulin code" generated by the choice of different α- and β- tubulin isoforms and tubulin post-translational modifications have essential roles in the control of a variety of cellular processes, such as cell shape, cell motility, and intracellular trafficking, that are deregulated in cancer. In this review, we will discuss available evidence that highlights the crucial role of the tubulin code in determining different cancer phenotypes, including metastatic cell migration, drug resistance, and tumor vascularization, and the influence of modulating tubulin-modifying enzymes on cancer cell survival and aggressiveness. We will also discuss the role of post-translationally modified microtubules in autophagy-the lysosomal-mediated cellular degradation pathway-that exerts a dual role in many cancer types, either promoting or suppressing cancer growth. We will give particular emphasis to the role of tubulin post-translational modifications and their regulating enzymes in controlling the different stages of the autophagic process in cancer cells, and consider how the experimental modulation of tubulin-modifying enzymes influences the autophagic process in cancer cells and impacts on cancer cell survival and thereby represents a new and fruitful avenue in cancer therapy.

Regulation of microtubule dynamics, mechanics and function through the growing tip.

Microtubule dynamics and their control are essential for the normal function and division of all eukaryotic cells. This plethora of functions is, in large part, supported by dynamic microtubule tips, which can bind to various intracellular targets, generate mechanical forces and couple with actin microfilaments. Here, we review progress in the understanding of microtubule assembly and dynamics, focusing on new information about the structure of microtubule tips. First, we discuss evidence for the widely accepted GTP cap model of microtubule dynamics. Next, we address microtubule dynamic instability in the context of structural information about assembly intermediates at microtubule tips. Three currently discussed models of microtubule assembly and dynamics are reviewed. These are considered in the context of established facts and recent data, which suggest that some long-held views must be re-evaluated. Finally, we review structural observations about the tips of microtubules in cells and describe their implications for understanding the mechanisms of microtubule regulation by associated proteins, by mechanical forces and by microtubule-targeting drugs, prominently including cancer chemotherapeutics.