Microtubule Assembly Research Articles

TACC3-ch-TOG interaction regulates spindle microtubule assembly by controlling centrosomal recruitment of γ-TuRC.

γ-Tubulin ring complex (γ-TuRC), composed of γ-tubulin and multiple γ-tubulin complex proteins (GCPs), serves as the major microtubule nucleating complex in animal cells. However, several γ-TuRC-associated proteins have been shown to control its function. Centrosomal adaptor protein, TACC3, is one such γ-TuRC-interacting factor that is essential for proper mitotic spindle assembly across organisms. ch-TOG is another microtubule assembly promoting protein, which interacts with TACC3 and cooperates in mitotic spindle assembly. However, the mechanism how TACC3-ch-TOG interaction regulates microtubule assembly and the γ-TuRC functions at the centrosomes remain unclear. Here, we show that deletion of the ch-TOG-binding region in TACC3 enhances recruitment of the γ-TuRC proteins to centrosomes and aggravates spindle microtubule assembly in human cells. Loss of TACC3-ch-TOG binding imparts stabilization on TACC3 interaction with the γ-TuRC proteins and it does so by stimulating TACC3 phosphorylation and thereby enhancing phospho-TACC3 recruitment to the centrosomes. We also show that localization of ch-TOG at the centrosomes is substantially reduced and the same on the spindle microtubules is increased in its TACC3-unbound condition. Additional results reveal that ch-TOG depletion stimulates γ-tubulin localization on the spindles without significantly affecting the centrosomal γ-tubulin level. The results indicate that ch-TOG binding to TACC3 controls TACC3 phosphorylation and TACC3-mediated stabilization of the γ-TuRCs at the centrosomes. They also implicate that the spatio-temporal control of TACC3 phosphorylation via ch-TOG-binding ensures mitotic spindle assembly to the optimal level.

Tau protein plays a role in the mechanism of cognitive disorders induced by anesthetic drugs.

Cognitive disorders are mental health disorders that can affect cognitive ability. Surgery and anesthesia have been proposed to increase the incidence of cognitive dysfunction, including declines in memory, learning, attention and executive function. Tau protein is a microtubule-associated protein located in the axons of neurons and is important for microtubule assembly and stability; its biological function is mainly regulated by phosphorylation. Phosphorylated tau protein has been associated with cognitive dysfunction mediated by disrupting the stability of the microtubule structure. There is an increasing consensus that anesthetic drugs can cause cognitive impairment. Herein, we reviewed the latest literature and compared the relationship between tau protein and cognitive impairment caused by different anesthetics. Our results substantiated that tau protein phosphorylation is essential in cognitive dysfunction caused by anesthetic drugs, and the possible mechanism can be summarized as "anesthetic drugs-kinase/phosphatase-p-Tau-cognitive impairment".

Thiouronium Salt Derivatives Based on Vicinal Diamines as Potential Neuroprotectors

Most of the medicinal products that are currently approved and used in clinical practice for neurodegenerative diseases, in particular Alzheimer’s disease, have a compensatory mechanism of action that enhances neurotransmitter signalling. It is an urgent need to develop new medicinal products combining cognitive-enhancing, neuroprotective, and disease-specific effects resulting from a multi-target mechanism of action including, in particular, prevention of glutamate-induced neuronal calcium uptake and stabilisation of microtubules.The aim of this study was to search for potentially neuroprotective and tauopathy-alleviating medicines amongst new thiouronium salt derivatives based on vicinal diamines.Materials and methods. The study investigated the ability of thiouronium salts to block glutamate-induced 45Ca2+ uptake by synaptosomes prepared from the brain of Wistar rats. The authors evaluated effects of these new compounds on polymerisation of a preparation of C57bl mouse brain tubulin and microtubule-associated proteins. The evaluation was carried out in the presence of guanosine triphosphate (GTP) and based on specific absorbance changes at 355 nm due to formation of microtubules. The authors analysed the structure of these microtubules, using negative staining followed by transmission electron microscopy. The IC50 determination and the statistical analysis were performed using standard software (Excel and PRISM 6.02).Results. The authors developed a screening algorithm for a number of new thiouronium salt derivatives based on vicinal diamines and studied biological activity of these derivatives by the effects on glutamate-induced calcium uptake by synaptosomes and on microtubule assembly processes. The authors identified compounds suppressing glutamate-induced calcium uptake by synaptosomes, i.e. compounds with neuroprotective potential. In addition, a number of new compounds were able to stimulate GTP-dependent microtubule assembly processes. The authors observed formation of microtubules with a normal structure in the presence of isopropyl-N’-[2-(benzoylamino)-1,2-diphenylethyl]-N-ethylimidothiocarbamate hydrobromide and considered the compound a promising scaffold for further optimisation.Conclusions. Chemical modification of thiouronium salts is a promising direction for developing effective neuroprotectors and microtubule stabilisers.

Vacuole Proteins with Optimized Microtubule Assembly Is Required for Fum1 Protein Localization and Fumonisin Biosynthesis in Mycotoxigenic Fungus Fusarium verticillioides.

Fumonisin contamination of corn caused by Fusarium verticillioides is a major concern worldwide. While key genes involved in fumonisin biosynthesis are known, the location within the fungal cell where this process occurs has yet to be fully characterized. In this study, three key enzymes, i.e., Fum1, Fum8, and Fum6, associated with early steps of fumonisin biosynthesis pathway, were tagged with GFP, and we examined their cellular localization. Results showed that these three proteins co-localized with the vacuole. To further understand the role of the vacuole in fumonisin B1 (FB1) biosynthesis, we disrupted two predicted vacuole associated proteins, FvRab7 and FvVam7, resulting in a significant reduction of FB1 biosynthesis and a lack of Fum1-GFP fluorescence signal. Furthermore, we used the microtubule-targeting drug carbendazim to show that proper microtubule assembly is critical for proper Fum1 protein localization and FB1 biosynthesis. Additionally, we found that α1 tubulin is a negative regulator in FB1 biosynthesis. We concluded that vacuole proteins with optimized microtubule assembly play a crucial role in proper Fum1 protein localization and fumonisin production in F. verticillioides.

Herbicide‐resistant barnyardgrass (Echinochloa crus‐galli) in global rice production

AbstractBarnyardgrass (Echinochloa crus‐galli (L.) Beauv.), an annual species of the family Poaceae, is a major weed problem in rice‐producing countries throughout the globe. Synthetic herbicides can effectively control this grass in rice paddies, but the development of resistant biotypes after the continuous use of the same active ingredients has led to low herbicide efficacy and yield losses. In this review, a summary of resistant‐barnyardgrass cases in global rice production is reported based on data from the International Herbicide‐Resistant Weed Database. The first case of resistant barnyardgrass in rice paddies was to the photosystem‐II inhibitor propanil in the late 1980s. Eighty‐five (85) out of 116 cases in the period from 1986 to 2022 refer to resistant barnyardgrass (E. crus‐galli var. crus‐galli, E. crus‐galli var. formosensis and E. crus‐galli var. zelayensis) in 16 countries. Barnyardgrass has been found resistant to acetolactate synthase (ALS) inhibitors (34 cases), acetyl‐CoA carboxylase (ACCase) inhibitors (23 cases), photosystem‐II inhibitors (11 cases), auxin mimics/cellulose biosynthesis inhibitors (9 cases), very long chain fatty acid inhibitors (6 cases), and microtubule assembly inhibitors (1 case). The majority of all resistance cases reported to the active ingredients penoxsulam, bispyribac‐sodium, and imazamox (ALS inhibitors), cyhalofop‐butyl and fenoxaprop‐ethyl (ACCase inhibitors), propanil (photosystem‐II inhibitors), and quinclorac (auxin mimics/cellulose biosynthesis inhibitors). Although target‐site resistance with specific mutations has been identified, non‐target site resistance mainly through herbicide detoxification is also of great concern increasing the chance of multiple herbicide resistance evolution. Rotation of herbicides should be adopted concerning the modes of action used as well as the application methods to mitigate resistance evolution of this weed in rice paddies.

A new electrochemical method that mimics phosphorylation of the core tau peptide K18 enables kinetic and structural analysis of intermediates and assembly

Tau protein's reversible assembly and binding of microtubules in brain neurons are regulated by charge-neutralizing phosphorylation, while its hyperphosphorylation drives the irreversible formation of cytotoxic filaments associated with neurodegenerative diseases. However, the structural changes that facilitate these diverse functions are unclear. Here, we analyzed K18, a core peptide of tau, using newly developed spectroelectrochemical instrumentation that enables electroreduction as a surrogate for charge neutralization by phosphorylation, with simultaneous, real-time quantitative analyses of the resulting conformational transitions and assembly. We observed a tipping point between behaviors that paralleled the transition between tau's physiologically required, reversible folding and assembly and the irreversibility of assemblies. The resulting rapidly electroassembled structures represent the first fibrillar tangles of K18 that have been formed invitro at room temperature without using heparin or other charge-complementary anionic partners. These methods make it possible to (i) trigger and analyze in real time the early stages of conformational transitions and assembly without the need for preformed seeds, heterogenous coacervation, or crowding; (ii) kinetically resolve and potentially isolate never-before-seen early intermediates in these processes; and (iii) develop assays for additional factors and mechanisms that can direct the trajectory of assembly from physiologically benign and reversible to potentially pathological and irreversible structures. We anticipate wide applicability of these methods to other amyloidogenic systems and beyond.

Targeted Dephosphorylation of Tau by Phosphorylation Targeting Chimeras (PhosTACs) as a Therapeutic Modality.

Microtubule-associated protein tau is essential for microtubule assembly and stabilization. Hyperphosphorylation of the microtubule-associated protein tau plays an important pathological role in the development of Alzheimer's disease and other tauopathies. In vivo studies using kinase inhibitors suggest that reducing tau phosphorylation levels has therapeutic potential; however, such approaches showed limited benefits. We sought to further develop our phosphorylation targeting chimera (PhosTAC) technology to specifically induce tau dephosphorylation. Herein, we use small molecule-based PhosTACs to recruit tau to PP2A, a native tau phosphatase. PhosTACs induced the formation of a stable ternary complex, leading to rapid, efficient, and sustained tau dephosphorylation, which also correlated with the enhanced downregulation of tau protein. Mass spectrometry data validated that PhosTACs downregulated multiple phosphorylation sites of tau. We believe that PhosTAC possesses several advantages over current strategies to modulate tau phosphorylation and represents a new avenue for disease-modifying therapies for tauopathies.

Sepsis and Acute Pancreatitis in Tumor-Stage Mycosis Fungoides and Treatment with Brentuximab-Vedotin

Dear Editor, we report a case of a 67-year-old male patient who was first diagnosed with tumor-stage Mycosis Fungoides (MF) (T3N0M0B0a, clinical stage IIB – current ISCL/EORTC classification) in 2017. During the course of the disease of MF the patient was treated with bexarotene, gemcitabine and chlorambucil. All these treatments helped only short-term, quickly followed by relapses of the skin lesions. In September 2018, the patient developed a rapidly progressive disease with erythematous patches, plaques and ulcerated tumors (mSWAT 20) (Fig. 1). CD30 expression was determined in several skin biopsies, each with an expression rate of 2-5% (Fig. 1). Despite this relatively low amount, the tumor board decision was made favoring the monoclonal CD30-antibody Brentuximab-Vedotin (BV) application. In October 2018, BV was first administered with the maximum dose of 180 mg (1.8mg/kg of body weight). Five days after the infusion, the patient developed abdominal pain, diarrhea, chills and fever. Blood cultures were positive for Methicillin-Resistant Staphylococcus Aureus (MRSA) and a MRSA-sepsis was diagnosed. Intensive medical care was requiredand under symptomatic treatment, the patient recovered completely. Two months later – in December 2018 – the patient presented again in our department to evaluate the skin lesions, displaying a partial response (mSWAT 12) (Fig. 1). At this time, the sepsis was not considered in direct context with the medication. The patient had several ulcerations and had been a known MRSA-patient already before the initiation of the treatment. Therefore, the patient received BV for a second time in the total dose of 180 mg. Nine days after the infusion, abdominal pain with vomiting, diarrhea and unconsciousness occurred. The patient was readmitted to the intensive care unit and an acute pancreatitis was diagnosed (lipase 1502 U/l). Under symptomatic therapy and close monitoring, pancreatitis resolved within two weeks. BV is a CD30 antibody-drug conjugate. The cytotoxic component is monomethylauristatin E, a potent inhibitor of microtubule assembly [1]. It is approved for the treatment of patients with relapsed or refractory Hodgkin’s Lymphoma (HL), systemic Anaplastic Large-T-cell Lymphoma (sALCL) and in addition, for the treatment of CD30-positive Cutaneous T-cell Lymphoma (CTCL) [2]. This case report describes two serious complications after the administration of BV in the same patient. Both belong in the spectrum of rare to infrequent side effects. Awareness of the possibility of pancreatitis as a side effect of BV is essential, especially because it seems to occur not immediately but within days or weeks after the treatment application. The literature describes pancreatitis in patients with HL, ALCL and cutaneous Gamma Delta T-cell Lymphoma (GD-TCL) [3]. The onset of pancreatitis in most cases seems to be delayed with a range of 7-33 days [1,4]. In our patient the first symptoms presented nine days after the BV-administration. Acute pancreatitis can lead to fatal outcomes. BV reinduction should be carefully discussed. However, in patients without any other treatment options, it might be possible to reinduce BV without the risk of side effect recurrence [1,3].

Tau-RNA complexes inhibit microtubule polymerization and drive disease-relevant conformation change.

Alzheimer's disease and related disorders feature neurofibrillary tangles and other neuropathological lesions composed of detergent-insoluble tau protein. In recent structural biology studies of tau proteinopathy, aggregated tau forms a distinct set of conformational variants specific to the different types of tauopathy disorders. However, the constituents driving the formation of distinct pathological tau conformations on pathway to tau-mediated neurodegeneration remain unknown. Previous work demonstrated RNA can serve as a driver of tau aggregation, and RNA associates with tau containing lesions, but tools for evaluating tau/RNA interactions remain limited. Here, we employed molecular interaction studies to measure the impact of tau/RNA binding on tau microtubule binding and aggregation. To investigate the importance of tau/RNA complexes (TRCs) in neurodegenerative disease, we raised a monoclonal antibody (TRC35) against aggregated tau/RNA complexes. We showed that native tau binds RNA with high affinity but low specificity, and tau binding to RNA competes with tau-mediated microtubule assembly functions. Tau/RNA interaction in vitro promotes the formation of higher molecular weight tau/RNA complexes, which represent an oligomeric tau species. Coexpression of tau and poly(A)45 RNA transgenes in Caenorhabditis elegans exacerbates tau-related phenotypes including neuronal dysfunction and pathological tau accumulation. TRC35 exhibits specificity for Alzheimer's disease-derived detergent-insoluble tau relative to soluble recombinant tau. Immunostaining with TRC35 labels a wide variety of pathological tau lesions in animal models of tauopathy, which are reduced in mice lacking the RNA binding protein MSUT2. TRC-positive lesions are evident in many human tauopathies including Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease. We also identified ocular pharyngeal muscular dystrophy as a novel tauopathy disorder, where loss of function in the poly(A) RNA binding protein (PABPN1) causes accumulation of pathological tau in tissue from post-mortem human brain. Tau/RNA binding drives tau conformational change and aggregation inhibiting tau-mediated microtubule assembly. Our findings implicate cellular tau/RNA interactions as modulators of both normal tau function and pathological tau toxicity in tauopathy disorders and suggest feasibility for novel therapeutic approaches targeting TRCs.

Regulation of actin and microtubules by TDP-43 and profilin.

TDP-43 is a classic DNA/RNA binding protein important for the normal function of numerous cellular activities that directly and indirectly impinge on the actin and microtubule cytoskeletons. When TDP-43 levels are reduced, neurons display phenotypes consistent with the loss of actin-microtubule crosstalk functions including aberrant morphology, fewer dendritic branches, and diminished area of growth cones. TDP-43 is direct binding partner of profilin (kD = 115.2 nM), which also regulates actin and microtubule dynamics, and variants of either protein are considered causative agents in several neurodegenerative diseases. First, we explored whether TDP-43 directly interacts with the actin or microtubules cytoskeletons and whether TDP-43 could mediate cytoskeletal crosstalk by forming phase-separated biomolecular condensates. We used TIRF microscopy assays to measure the impact of purified TDP-43 on actin and microtubule assembly. TDP-43 bound and sequestered both actin and tubulin (kD = 22.7 nM). inhibiting the assembly of either polymer. We also explored how interactions between profilin and TDP-43 modulated the dynamics of either cytoskeleton or phase separation. The addition of profilin increased TDP-43 condensate formation (number and size), further inhibited actin assembly, but stabilized microtubule assembly. To investigate this in a more physiological context, we imaged the cytoskeleton and TDP-43 in Neuroblastoma-2a cells. Cells lacking profilin displayed less robust actin and microtubule arrays compared to endogenous or rescue controls. In addition, TDP-43 is mislocalized to the cytoplasm, mirroring the disease state of amyotrophic lateral sclerosis (ALS). Rescue experiments expressing wild type profilin on plasmids restored actin and microtubule arrays and the nuclear localization of TDP-43, whereas profilin plasmids harboring ALS-related mutations did not. Thus, profilin and TDP-43 are important regulators of actin and microtubule dynamics in cells and in the context of ALS.

Abl2 mediates microtubule nucleation and repair via tubulin co-condensation.

Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation both in vitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics were unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We discovered that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo phase separation and form co-condensates with tubulin. A naturally occurring tubulin binding-deficient Abl2 splice isoform fails to promote nucleation. Abl2 binds to regions adjacent to MT damage and facilitates their repair via fresh tubulin recruitment and increases MT rescue frequency and lifetime. MT recovery after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared to wild type cells. Our results lead us to propose a model in which Abl2 locally concentrates tubulin and recruits it to MT tips or to defects in the MT lattice to promote MT repair, rescue, and nucleation.

Characterization of pseudophosphorylated tau protein.

Tau protein facilitates assembly, organization, and stability of microtubules found in neuronal axons. Hyperphosphorylation results in a loss of tau function from decreased microtubule binding affinity which is prominent in Alzheimer's Disease. The current study employed 7-Phos—a tau mutant with seven specific site pseudophosphorylation mutations—to mimic the characteristic changes found in hyperphosphorylation. In vivo, tau has six naturally occurring isoforms that vary in the length of the N-terminal projection domains, three of which were investigated in this study. Biophysical characterization of these isoforms was accomplished with several techniques. SDS-PAGE was used to identify an upward band shift, a feature of 7-Phos. A thioflavin fluorescence assay was performed to compare the arachidonic acid induced polymerization of the 7-Phos variants with their wild-type counterparts. Atomic force microscopy was used to measure the length of 7-Phos tau polymers over several days and to analyze the intermolecular interactions between molecules of 7-Phos. We determined that the total amount of polymerization measured by thioflavin fluorescence of the two longer 7-Phos isoforms, 1N4R and 2N4R, were similar to their corresponding wild-type proteins. The total amount of polymerization of the shortest isoform, 0N4R, was greater than its wild-type. Atomic force microscopy (AFM) imaging revealed that 7-Phos isoforms form longer polymers than their wild-type. Atomic force microscopy of immobilized tau layers allowed comparison of projection domain lengths between 7-Phos and wild-type. These results demonstrate some novel AFM-based measurements to characterize tau polymerization, projection domain extension/conformation and intermolecular interactions.

A structural mechano-chemical model of microtubule nucleation.

Microtubules (MTs) are the backbone of cytoskeleton and crucial for many essential processes, such as mitosis, morphogenesis and migration. A critical step in the assembly and regulation of cellular MT network is nucleation. There are two types of templates for MT nucleation in cells: existing MT and γ-tubulin ring complex. Both types of nucleations have been reconstituted in vitro, but their mechanisms remain poorly understood. Here, we developed a physically rigorous structure-based mechano-chemical model that provides an unified mechanism for both types of nucleations. For nucleation from seed MTs, the key observations are: 1) the critical tubulin concentration for nucleation is much higher than that for growth, 2) the concentration-dependent time lags. Our model shows that the bottleneck for nucleation is frequent catastrophes (i.e., the switching from MT growth to shortening) that keep removing nascent MTs from the seed, which is reduced as tubulin concentration increases. The observed time lag is due to repeated assembly and removal of nascent MTs, which delay nucleation. For nucleation from γ-tubulin ring, the most intriguing question is the effects of ring closure, a complex-wide conformational change required for the ring complex to match the tube-like geometry of MT. Our model shows that ring closure helps tubulins to first form a sheet, which is then closed into tube with the help of the ring closure process. Because catastrophe cannot develop while sheet is open, MTs more likely grow longer, increasing the chance of nucleation. Thus, ring closure allows more regulated and efficient MT nucleation.

Differentiating R5l and wildtype tau using atomic force microscopy.

Tau is a microtubule associated protein found in neuronal axons which regulates microtubule assembly and organization. Aberrant tau polymerization into neurofibrillary tangles is highly correlated with neurodegenerative diseases such as Alzheimer's disease. As a natively unstructured protein, it has eluded traditional protein structural characterization methods such as crystallography, but analysis with atomic force microscopy (AFM) offers a novel method to interrogate tau structure. When tau is adsorbed on the AFM tip and onto a flat mica surface, the N-terminal projection domains of the protein extend outwards to form polyelectrolyte brushes on both the tip and surface, allowing measurement of forces between the projection domains of the opposing layers of tau as the tip approaches the surface.

Exploring generic principles of compartmentalization in a developmental in vitro model.

Self-organization of cells into higher-order structures is key for multicellular organisms, for example via repetitive replication of template-like founder cells or syncytial energids. Yet, very similar spatial arrangements of cell-like compartments ('protocells') are also seen in a minimal model system of Xenopus egg extracts in the absence of template structures and chromatin, with dynamic microtubule assemblies driving the self-organization process. Quantifying geometrical features over time, we show here that protocell patterns are highly organized with a spatial arrangement and coarsening dynamics similar to that of two-dimensional foams but without the long-range ordering expected for hexagonal patterns. These features remain invariant when enforcing smaller protocells by adding taxol, i.e. patterns are dominated by a single, microtubule-derived length scale. Comparing our data to generic models, we conclude that protocell patterns emerge by simultaneous formation of randomly assembling protocells that grow at a uniform rate towards a frustrated arrangement before fusion of adjacent protocells eventually drives coarsening. The similarity of protocell patterns to arrays of energids and cells in developing organisms, but also to epithelial monolayers, suggests generic mechanical cues to drive self-organized space compartmentalization.

Low-Dose Taxol Promotes Neuronal Axons Extension and Functional Recovery after Spinal Cord Injury.

Axonal regeneration has been the research focus in the field of clinical treatment for spinal cord injury (SCI). The growth and extension of neuronal axons is a dynamic biological process mediated by the cytoskeleton, and microtubule plays an important role in axonal growth. Moderate stabilization of microtubule promotes axonal growth and eliminates various intra- and extracellular mechanisms that impede axonal regeneration. After SCI, the damaged axons rapidly form a growth cone, wherein the stability of tubulin decreases, impairing axonal regeneration. Taxol with proven clinical safety is commonly used as a broad-spectrum antitumor drug. Importantly, Taxol can promote axonal extension by enhancing and stabilizing the microtubule assembly. In our study, we systematically investigated the differentiation of neural stem cells (NSCs) in vitro and functional recovery in injured rats in vivo following Taxol treatment. Low-dose Taxol promoted differentiation of NSCs to neurons and significantly extended the axons in vitro. In vivo, Taxol promoted the expression of βIII-tubulin in the injured areas and motor function recovery after SCI. Low-dose Taxol is a promising clinical agent to promote axonal regeneration after SCI.

Microtubule cytoskeleton inhibition induced by taxol and vinorelbine affects encystation, proliferation, morphology, and gene expression of Cryptocaryon irritans protomont

Cryptocaryon irritans protomont is important for encystation and division to continue the reinfection events. Microtubules play a determinant role in multiple cell processes such as: motility, intracellular transport, division, encystation and excystation. To evaluate the significance of microtubule cytoskeleton in Cryptocaryon irritans encystation and cyst proliferation, taxol (a tubulin polymerizing agent) and vinorelbine (a tubulin depolymerizing agent) were used. Using a structure-activity approach, their effects on the regulation of encystment, cyst proliferation, division, morphology, and encystment-related gene expression were assessed. Following collection, protomonts were exposed to serial concentrations of taxol (25, 50, 100, 250, and 500 μM) and vinorelbine (100, 250, 500, 750, and 1000 μM) for 1, 2, 3, and 12 h. Survival, encystation rate, excystation number of theronts, and theront body size decreased with varying degrees of time and concentration. Furthermore, the effects of anti-tubulins on the micro- and ultrastructure of protomonts were examined using the most feasible concentrations of 250 μM taxol and 500 μM vinorelbine. Microstructure observations for one cell cycle revealed delayed/incomplete encystation, change in cell symmetry, and delayed excystation process. Ultrastructure observations showed aggregation of microtubule bundles and disruption of microtubule assembly in the cytoplasm and around the cell wall of taxol- and vinorelbine-treated protomonts respectively. Inhibited microtubules altered ultrastructure resulting in fragile/fragmentary cyst wall formation. mRNA expression of α- and β-tubulin in the taxol-treated cells was significantly up-regulated when compared with the control (P˂0.05). In contrast, vinorelbine treatment markedly down-regulated mRNA expression levels of α- and β-tubulin at one and four hours (P˂0.05). mRNA level of EF-1α was enhanced in taxol-treated protomonts, but reduced in vinorelbine-treated cells when compared with the control (P˂0.05). While mRNA level of actin-cytoplasmic1 remained almost stable after treatment with both anti-tubulin drugs, mRNA expressions of other targeted genes- Rab-7, 40s ribosomal protein, and cathepsin were insignificantly down-regulated in both treatments when compared with control (P > 0.05). In general, target-specific taxol and vinorelbine were found to disturb the rate of encystation, cyst growth, and morphology. In addition, these drugs caused irregular mRNA expression of α- and β-tubulins. This study elucidates the constructive role of tubulins in protomont encystation and cyst proliferation.

Alternative Approaches to Understand Microtubule Cap Morphology and Function.

Microtubules (MTs) are essential cellular machines built from concatenated αβ-tubulin heterodimers. They are responsible for two central and opposite functions from the dynamic point of view: scaffolding (static filaments) and force generation (dynamic MTs). These roles engage multiple physiological processes, including cell shape, polarization, division and movement, and intracellular long-distance transport. At the most basic level, the MT regulation is chemical because GTP binding and hydrolysis have the ability to promote assembly and disassembly in the absence of any other constraint. Due to the stochastic GTP hydrolysis, a chemical gradient from GTP-bound to GDP-bound tubulin is created at the MT growing end (GTP cap), which is translated into a cascade of structural regulatory changes known as MT maturation. This is an area of intense research, and several models have been proposed based on information mostly gathered from macromolecular crystallography and cryo-electron microscopy studies. However, these classical structural biology methods lack temporal resolution and can be complemented, as shown in this mini-review, by other approaches such as time-resolved fiber diffraction and computational modeling. Together with studies on structurally similar tubulins from the prokaryotic world, these inputs can provide novel insights on MT assembly, dynamics, and the GTP cap.

Morphological Evidence for Novel Roles of Microtubules in Macrophage Phagocytosis

Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

The ring saga: looking back at the discovery of γ-tubulin and γ-tubulin ring complexes

For many years, two central, unanswered questions in cytoskeleton research were how microtubule assembly is nucleated and microtubule polarity established. The discoveries of γ-tubulin and γ-tubulin ring complexes were key advances that allowed these questions to be substantially answered. The discovery of γ-tubulin was the product of a genetic screen in Aspergillus nidulans for genes important for microtubule function. γ-Tubulin is a member of the tubulin superfamily of proteins, closely related to α- and β-tubulin but distinct from both. It is ubiquitous in eukaryotes, and in many organisms there are small families of γ-tubulin genes. γ-Tubulin and associated proteins form ring-like complexes that localize to microtubule-organizing centers (MTOCs) and play an important role in the nucleation of microtubule assembly from MTOCs and the establishment of microtubule polarity.