Affect Microtubule Dynamics Research Articles

Direct targeting of host microtubule and actin cytoskeletons by a chlamydial pathogenic effector protein.

To propagate within a eukaryotic cell, pathogenic bacteria hijack and remodulate host cell functions. The Gram-negative obligate intracellular Chlamydiaceae, which pose a serious threat to human and animal health, attach to host cells and inject effector proteins that reprogram host cell machineries. Members of the conserved chlamydial TarP family have been characterized as major early effectors that bind to and remodel the host actin cytoskeleton. We now describe a new function for the Chlamydia pneumoniae TarP member CPn0572, namely the ability to bind and alter the microtubule cytoskeleton. Thus, CPn0572 is unique in being the only prokaryotic protein that directly modulates both dynamic cytoskeletons of a eukaryotic cell. Ectopically expressed GFP-CPn0572 associates in a dose-independent manner with either cytoskeleton singly or simultaneously. In vitro, CPn0572 binds directly to microtubules. Expression of a microtubule-only CPn0572 variant resulted in the formation of an aberrantly thick, stabilized microtubule network. Intriguingly, during infection, secreted CPn0572 also colocalized with altered microtubules, suggesting that this protein also affects microtubule dynamics during infection. Our analysis points to a crosstalk between actin and microtubule cytoskeletons via chlamydial CPn0572.

Damage-repair events increase the instability of cortical microtubules in Arabidopsis.

Microtubules rely on dynamic assembly and disassembly for their functions. Increasing evidences support that the damage-repair of microtubule lattices can affect microtubule dynamics in vitro and in animal cells. Here we successfully established a way for visualizing damage-repair sites on microtubule lattices in plant cells, via labeling the tubulin proteins with the photoconvertible fluorescent protein mEOS3.2. We observed that the crossovers of the microtubule lattice were more prone to be damaged and repaired, with the frequency of damage-repair events positively correlated with the crossing angle between microtubules. The microtubules with damage-repair events displayed shorter lifespans and significantly increased severing frequency compared with the undamaged microtubules. These observations suggested that the damage-repair events promoted instability of cortical microtubules in plant cells.

Efficient axonal transport of endolysosomes relies on the balanced ratio of microtubule tyrosination and detyrosination.

In neurons, the microtubule (MT) cytoskeleton forms the basis for long-distance protein transport from the cell body into and out of dendrites and axons. To maintain neuronal polarity, the axon initial segment (AIS) serves as a physical barrier, separating the axon from the somatodendritic compartment and acting as a filter for axonal cargo. Selective trafficking is further instructed by axonal enrichment of MT post-translational modifications, which affect MT dynamics and the activity of motor proteins. Here, we compared two knockout mouse lines lacking the respective enzymes for MT tyrosination and detyrosination, and found that both knockouts led to a shortening of the AIS. Neurons from both lines also showed an increased immobile fraction of endolysosomes present in the axon, whereas mobile organelles displayed shortened run distances in the retrograde direction. Overall, our results highlight the importance of maintaining the balance of tyrosinated and detyrosinated MTs for proper AIS length and axonal transport processes.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

Quantifying Yeast Microtubules and Spindles Using the Toolkit for Automated Microtubule Tracking (TAMiT).

Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

Glutamylation is a negative regulator of microtubule growth.

Microtubules are noncovalent polymers built from αβ-tubulin dimers. The disordered C-terminal tubulin tails are functionalized with multiple glutamate chains of variable lengths added and removed by tubulin tyrosine ligases (TTLLs) and carboxypeptidases (CCPs). Glutamylation is abundant on stable microtubule arrays such as in axonemes and axons, and its dysregulation leads to human pathologies. Despite this, the effects of glutamylation on intrinsic microtubule dynamics are unclear. Here we generate tubulin with short and long glutamate chains and show that glutamylation slows the rate of microtubule growth and increases catastrophes as a function of glutamylation levels. This implies that the higher stability of glutamylated microtubules in cells is due to effectors. Interestingly, EB1 is minimally affected by glutamylation and thus can report on the growth rates of both unmodified and glutamylated microtubules. Finally, we show that glutamate removal by CCP1 and 5 is synergistic and occurs preferentially on soluble tubulin, unlike TTLL enzymes that prefer microtubules. This substrate preference establishes an asymmetry whereby once the microtubule depolymerizes, the released tubulin is reset to a less-modified state, while polymerized tubulin accumulates the glutamylation mark. Our work shows that a modification on the disordered tubulin tails can directly affect microtubule dynamics and furthers our understanding of the mechanistic underpinnings of the tubulin code.

Structural Perspective of Benzophenones Targeting Tubulin as Anticancer Agents.

Cancer is the leading cause of death and the most significant determinant of life expectancy in almost every country in this twenty-first century. According to the World Health Organization (WHO), cancer is responsible for the leading cause of death globally. Benzophenone derivatives are found in a variety of naturally occurring compounds which are known to be pharmacologically efficacious against a variety of diseases, including cancer. Microtubules are thought to be a good target for cancer chemotherapies. Microtubule polymerization and depolymerization are induced by a variety of natural, synthetic, and semisynthetic chemicals having a benzophenone nucleus, affecting tubulin dynamics. Several medications that affect microtubule dynamics are in various stages of clinical trials, including Combretastatins (phase II), Vincristine (clinically approved), Paclitaxel (in clinical usage), and epothilone (phase III), and only a few have been patented. Benzophenone derivatives target the colchicine binding site of microtubules, damage them and cause cell cycle arrest in the G2-M phase. Belonging to this class of molecules, phenstatin, a potent inhibitor of tubulin polymerization, has shown strongly inhibit cancer cell growth and arrest the G2/M phase of the cell cycle by targeting the colchicine binding site of microtubules. In the present manuscript, we described the benzophenone as tubulin polymerization inhibitors, their Structure-Activity Relationships (SARs) and molecular docking studies that reveal its binding affinity with the colchicine binding site.

α-Tubulin detyrosination links the suppression of MCAK activity with taxol cytotoxicity.

α/β-Tubulin posttranslational modifications (PTMs) generate microtubule diversity, but whether they account for cancer cell resistance to microtubule-targeting drugs remains unknown. Here, we performed a pilot dissection of the "cancer tubulin code" using the NCI-60 cancer cell panel. We found that acetylated, detyrosinated, and ∆2-α-tubulin that typically accumulate on stable microtubules were uncoupled in many cancer cells. Acetylated α-tubulin did not affect microtubule dynamics, whereas its levels correlated with, but were not required for, taxol-induced cytotoxicity. In contrast, experimental increase of α-tubulin detyrosination, and/or depletion of the detyrosination-sensitive microtubule-depolymerizing enzyme MCAK, enhanced taxol-induced cytotoxicity by promoting cell death in mitosis and the subsequent interphase, without causing a cumulative effect. Interestingly, only increased detyrosinated α-tubulin aggravated taxol-induced spindle multipolarity. Overall, we identified high α-tubulin acetylation as a potential biomarker for cancer cell response to taxol and uncovered a mechanistic link between α-tubulin detyrosination and the suppression of MCAK activity in taxol-induced cytotoxicity, likely by promoting chromosome missegregation, regardless of spindle defects.

Microtubule-associated ROP interactors affect microtubule dynamics and modulate cell wall patterning and root hair growth.

Rho of plant (ROP) proteins and the interactor of constitutively active ROP (ICR) family member ICR5/MIDD1 have been implicated to function as signaling modules that regulate metaxylem secondary cell wall patterning. Yet, loss-of-function mutants of ICR5 and its closest homologs have not been studied and, hence, the functions of these ICR family members are not fully established. Here, we studied the functions of ICR2 and its homolog ICR5. We show that ICR2 is a microtubule-associated protein that affects microtubule dynamics. Secondary cell wall pits in the metaxylem of Arabidopsis icr2 and icr5 single mutants and icr2 icr5 double mutants are smaller than those in wild-type Col-0 seedlings; however, they are remarkably denser, implying a complex function of ICRs in secondary cell wall patterning. ICR5 has a unique function in protoxylem secondary cell wall patterning, whereas icr2, but not icr5, mutants develop split root hairs, demonstrating functional diversification. Taken together, our results show that ICR2 and ICR5 have unique and cooperative functions as microtubule-associated proteins and as ROP effectors.

Theory of tip structure–dependent microtubule catastrophes and damage-induced microtubule rescues

Microtubules are essential cytoskeletal polymers that exhibit stochastic switches between tubulin assembly and disassembly. Here, we examine possible mechanisms for these switches, called catastrophes and rescues. We formulate a four-state Monte Carlo model, explicitly considering two biochemical and two conformational states of tubulin, based on a recently conceived view of microtubule assembly with flared ends. The model predicts that high activation energy barriers for lateral tubulin interactions can cause lagging of curled protofilaments, leading to a ragged appearance of the growing tip. Changes in the extent of tip raggedness explain some important but poorly understood features of microtubule catastrophe: weak dependence on tubulin concentration and an increase in its probability over time, known as aging. The model predicts a vanishingly rare frequency of spontaneous rescue unless patches of guanosine triphosphate tubulin are artificially embedded into microtubule lattice. To test our model, we used invitro reconstitution, designed to minimize artifacts induced by microtubule interaction with nearby surfaces. Microtubules were assembled from seeds overhanging from microfabricated pedestals and thus well separated from the coverslip. This geometry reduced the rescue frequency and the incorporation of tubulins into the microtubule shaft compared with the conventional assay, producing data consistent with the model. Moreover, the rescue positions of microtubules nucleated from coverslip-immobilized seeds displayed a nonexponential distribution, confirming that coverslips can affect microtubule dynamics. Overall, our study establishes a unified theory accounting for microtubule assembly with flared ends, a tip structure-dependent catastrophe frequency, and a microtubule rescue frequency dependent on lattice damage and repair.

SETDB1 regulates microtubule dynamics.

SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we looked for cytoplasmic functions of SETDB1. SETDB1 association with microtubules was detected by immunofluorescence and co-sedimentation. Microtubule dynamics were analysed during recovery from nocodazole treatment and by tracking microtubule plus-ends in live cells. Live cell imaging was used to study mitotic kinetics and protein-protein interaction was identified by co-immunoprecipitation. SETDB1 co-sedimented with microtubules and partially colocalized with microtubules. SETDB1 partial silencing led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 may affect microtubule dynamics by a methylation-independent mechanism. Moreover, SETDB1 co-immunoprecipitated with HDAC6 and tubulin acetylation levels were increased upon silencing of SETDB1. Taken together, our study suggests a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.

MDP25 mediates the fine-tuning of microtubule organization in response to salt stress.

Microtubules are dynamic cytoskeleton structures playing fundamental roles in plant responses to salt stress. The precise mechanisms by which microtubule organization is regulated under salt stress are largely unknown. Here, we report that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN 25 (MDP25; also known as PLASMA MEMBRANE-ASSOCIATED CATION-BINDING PROTEIN 1 (PCaP1)) helps regulate microtubule organization. Under salt treatment, elevated cytosolic Ca2+ concentration caused MDP25 to partially dissociate from the plasma membrane, promoting microtubule depolymerization. When Ca2+ signaling was blocked by BAPTA-AM or LaCl3 , microtubule depolymerization in wild-type and MDP25-overexpressing cells was slower, while there was no obvious change in mdp25 cells. Knockout of MDP25 improved microtubule reassembly and was conducive to microtubule integrity under long-term salt treatment and microtubule recovery after salt stress. Moreover, mdp25 seedlings exhibited a higher survival rate under salt stress. The presence microtubule-disrupting reagent oryzalin or microtubule-stabilizing reagent paclitaxel differentially affected the survival rates of different genotypes under salt stress. MDP25 promoted microtubule instability by affecting the catastrophe and rescue frequencies, shrinkage rate and time in pause phase at the microtubule plus-end and the depolymerization rate at the microtubule minus-end. These findings reveal a role for MDP25 in regulating microtubule organization under salt treatment by affecting microtubule dynamics.

Stathmins and Motor Neuron Diseases: Pathophysiology and Therapeutic Targets.

Motor neuron diseases (MNDs) are a group of fatal, neurodegenerative disorders with different etiology, clinical course and presentation, caused by the loss of upper and lower motor neurons (MNs). MNs are highly specialized cells equipped with long, axonal processes; axonal defects are some of the main players underlying the pathogenesis of these disorders. Microtubules are key components of the neuronal cytoskeleton characterized by dynamic instability, switching between rapid polymerization and shrinkage. Proteins of the stathmin family affect microtubule dynamics regulating the assembly and the dismantling of tubulin. Stathmin-2 (STMN2) is one of the most abundantly expressed genes in MNs. Following axonal injury, STMN2 expression is upregulated, and the protein is transported toward the growth cones of regenerating axons. STMN2 has a critical role in axonal maintenance, and its dysregulation plays an important role in neurodegenerative processes. Stathmin-1 (STMN1) is a ubiquitous protein that is highly expressed during the development of the nervous system, and its phosphorylation controls microtubule dynamics. In the present review, we summarize what is currently known about the involvement of stathmin alterations in MNDs and the potential therapeutic effect of their modulation, with a specific focus on the most common forms of MND, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA).

A new view of how cytoplasmic viscosity affects microtubule dynamics.

How the physical nature of the cytoplasm shapes cellular processes has been increasingly studied in recent years. Work in this issue demonstrates that cytoplasmic viscosity affects the dynamic instability of microtubules. Surprisingly, both polymerization and depolymerization slow with increasing viscosity. This raises interesting questions for future work.

Microtubule affinity regulating kinase 4 promoted activation of the NLRP3 inflammasome-mediated pyroptosis in periodontitis

ABSTRACT Background Microtubule dynamics plays a crucial role in the spatial arrangement of cell organelles and activation of the NLRP3 inflammasome. Purpose This study aimed to explore whether microtubule affinity regulating kinase 4 (MARK4) can be a therapeutic target of periodontitis by affecting microtubule dynamics and NLRP3 inflammasome-mediated pyroptosis in macrophages. Materials and Methods The NLRP3 inflammasome-related genes and MARK4 were measured in the healthy and inflamedhuman gingival tissues. Bone marrow-derived macrophages (BMDMs) were infected with Porphyromonas gingivalis,while the MARK4 inhibitors (OTSSP167 and Compound 50) and small interference RNAwere utilized to restrain MARK4. Apoptosis-associated speck-like protein (ASC)speck was detected by confocal, and levels of interleukin-1β (IL-1β), as wellas IL-18, were assessed by ELISA. Results Increased staining and transcription of MARK4, NLRP3, ASC, and Caspase-1 were observed in the inflamed gingiva. P. gingivalis infection promoted MARK4 expression and the NLRP3 inflammasome in BMDMs. Inhibition of MARK4 decreased LDH release, IL-1β and IL-18 production, ASC speck formation, and the pyroptosis-related genes transcription. Furthermore, MARK4 inhibition reduced microtubule polymerization and acetylation in P. gingivalis-infected BMDMs. Conclusions MARK4 promoted NLRP3 inflammasome activation and pyroptosis in P. gingivalis-infected BMDMs by affecting microtubule dynamics. MARK4 inhibition might be a potential target in regulating the NLRP3 inflammasome during periodontitis progress.

Plectin-Mediated Intermediate Filament Functions: Why Isoforms Matter.

This essay focuses on the role of plectin and its various isoforms in mediating intermediate filament (IF) network functions. It is based on previous studies that provided comprehensive evidence for a concept where plectin acts as an IF recruiter, and plectin-mediated IF networking and anchoring are key elements in IF function execution. Here, plectin’s global role as modulator of IF functionality is viewed from different perspectives, including the mechanical stabilization of IF networks and their docking platforms, contribution to cellular viscoelasticity and mechanotransduction, compartmentalization and control of the actomyosin machinery, connections to the microtubule system, and mechanisms and specificity of isoform targeting. Arguments for IF networks and plectin acting as mutually dependent partners are also given. Lastly, a working model is presented that describes a unifying mechanism underlying how plectin–IF networks mechanically control and propagate actomyosin-generated forces, affect microtubule dynamics, and contribute to mechanotransduction.

Vimentin intermediate filaments stabilize dynamic microtubules by direct interactions

The cytoskeleton determines cell mechanics and lies at the heart of important cellular functions. Growing evidence suggests that the manifold tasks of the cytoskeleton rely on the interactions between its filamentous components—actin filaments, intermediate filaments, and microtubules. However, the nature of these interactions and their impact on cytoskeletal dynamics are largely unknown. Here, we show in a reconstituted in vitro system that vimentin intermediate filaments stabilize microtubules against depolymerization and support microtubule rescue. To understand these stabilizing effects, we directly measure the interaction forces between individual microtubules and vimentin filaments. Combined with numerical simulations, our observations provide detailed insight into the physical nature of the interactions and how they affect microtubule dynamics. Thus, we describe an additional, direct mechanism by which cells establish the fundamental cross talk of cytoskeletal components alongside linker proteins. Moreover, we suggest a strategy to estimate the binding energy of tubulin dimers within the microtubule lattice.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αβ-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential invivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

New Insights Into Functions of IQ67-Domain Proteins.

IQ67-domain (IQD) proteins, first identified in Arabidopsis and rice, are plant-specific calmodulin-binding proteins containing highly conserved motifs. They play a critical role in plant defenses, organ development and shape, and drought tolerance. Driven by comprehensive genome identification and analysis efforts, IQDs have now been characterized in several species and have been shown to act as microtubule-associated proteins, participating in microtubule-related signaling pathways. However, the precise molecular mechanisms underpinning their biological functions remain incompletely understood. Here we review current knowledge on how IQD family members are thought to regulate plant growth and development by affecting microtubule dynamics or participating in microtubule-related signaling pathways in different plant species and propose some new insights.

Neuronal Delamination and Outer Radial Glia Generation in Neocortical Development.

During neocortical development, many neuronally differentiating cells (neurons and intermediate progenitor cells) are generated at the apical/ventricular surface by the division of neural progenitor cells (apical radial glial cells, aRGs). Neurogenic cell delamination, in which these neuronally differentiating cells retract their apical processes and depart from the apical surface, is the first step of their migration. Since the microenvironment established by the apical endfeet is crucial for maintaining neuroepithelial (NE)/aRGs, proper timing of the detachment of the apical endfeet is critical for the quantitative control of neurogenesis in cerebral development. During delamination, the microtubule–actin–AJ (adherens junction) configuration at the apical endfeet shows dynamic changes, concurrent with the constriction of the AJ ring at the apical endfeet and downregulation of cadherin expression. This process is mediated by transcriptional suppression of AJ-related molecules and multiple cascades to regulate cell adhesion and cytoskeletal architecture in a posttranscriptional manner. Recent advances have added molecules to the latter category: the interphase centrosome protein AKNA affects microtubule dynamics to destabilize the microtubule–actin–AJ complex, and the microtubule-associated protein Lzts1 inhibits microtubule assembly and activates actomyosin systems at the apical endfeet of differentiating cells. Moreover, Lzts1 induces the oblique division of aRGs, and loss of Lzts1 reduces the generation of outer radial glia (oRGs, also called basal radial glia, bRGs), another type of neural progenitor cell in the subventricular zone. These findings suggest that neurogenic cell delamination, and in some cases oRG generation, could be caused by a spectrum of interlinked mechanisms.