Affect Microtubule Organization Research Articles

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle.

The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.

Quantitative Analysis of Microtubule Organization in Leaf Epidermis Pavement Cells.

Leaf epidermis pavement cells form highly complex shapes with interlocking lobes and necks at their anticlinal walls. The microtubule cytoskeleton plays essential roles in pavement cell morphogenesis, in particular at necks. Vice versa, shape generates stress patterns that regulate microtubule organization. Genetic or pharmacological perturbations that affect pavement cell shape often affect microtubule organization. Pavement cell shape and microtubule organization are therefore closely interconnected. Here, we present commonly used approaches for the quantitative analysis of pavement cell shape characteristics and of microtubule organization. In combination with ablation experiments, these methods can be applied to investigate how different genotypes (or treatments) affect the organization and stress responsiveness of the microtubule cytoskeleton.

Scale-type-specific requirement for the mosquito Aedes aegypti Spindle-F homologue by regulating microtubule organization.

Insect epithelial cells contain unique cellular extensions such as bristles, hairs, and scales. In contrast to bristle and hair, which are not divergent in their shape, scale morphology shows high diversity. In our attempt to characterize the role of the insect-specific gene, Spindle-F (spn-F), in mosquito development, we revealed a scale-type specific requirement for the mosquito Aedes aegypti spn-F homologue. Using CRISPR-Cas9, we generated Ae-spn-F mutants and found that Ae-spn-F is an essential gene, but we were able to recover a few adult escapers. These escapers could not fly nor move, and died after 3 to 4 days. We found that in Ae-spn-F mutants, only the tip part of the bristle was affected with bulbous with misoriented ribs. We also show that in Ae-spn-F mutants, only in falcate scales, which are curved with a sharp or narrowly rounded apex, and not in other scale types, the tip region is strongly affected. Our analysis also revealed that in contrast to Drosophila spn-F, which show strong defects in both the actin and microtubule (MT) network in the bristle, the Ae-spn-F gene is required only for MT organization in scales and bristles. In summary, our results reveal that Ae-spn-F is required for shaping tapered epithelial cellular extension structures, namely, the bristle and falcate scales by affecting MT organization.

Crowder and surface effects on self-organization of microtubules

Microtubules are an essential physical building block of cellular systems. They are organized using specific crosslinkers, motors, and influencers of nucleation and growth. With the addition of antiparallel crosslinkers, microtubule self-organization patterns go through a transition from fanlike structures to homogeneous tactoid condensates in vitro. Tactoids are reminiscent of biological mitotic spindles, the cell division machinery. To create these organizations, we previously used polymer crowding agents. Here we study how altering the properties of the crowders, such as type, size, and molecular weight, affects microtubule organization. Comparing simulations with experiments, we observe a scaling law associated with the fanlike patterns in the absence of crosslinkers. Tactoids formed in the presence of crosslinkers show variable length, depending on the crowders. We correlate the subtle differences to filament contour length changes, affected by nucleation and growth rate changes induced by the polymers in solution. Using quantitative image analysis, we deduce that the tactoids differ from traditional liquid crystal organization, as they are limited in width irrespective of crowders and surfaces, and behave as solidlike condensates.

Regulation of microtubule plus end dynamics by septin 9.

Septins are GTP-binding proteins that associate with the microtubule (MT) and actin cytoskeleton. Septins affect MT organization and posttranslational modifications, but their role in MT dynamics is less understood. Here, we reconstituted MT dynamics in the presence of the MT-binding septin (SEPT9) using an in vitro cell-free assay, which images the polymerization of tubulin from guanosine-5'-[(α,β)-methyleno]triphosphate (GMPCPP)-stabilized MT seeds. We found that submicromolar concentrations of SEPT9 suppress MT catastrophe and enhance the growth of MT plus ends to great lengths, while low micromolar concentrations of SEPT9 stabilize MTs by inhibiting dynamic instability. We show that SEPT9 associates preferentially with the lattice of GMPCPP-stabilized MT seeds and surprisingly recruits soluble tubulin to the MT lattice. Notably, the effects of SEPT9 on MT dynamics are dependent on its G-G dimerization interface, which is formed by the pockets of the GTP-binding domains. A mutation (H530D) that disrupts G-G dimerization abrogates the effects of SEPT9 on MT dynamics and diminishes its ability to recruit tubulin to the MT lattice. Taken together, these results suggest that SEPT9 promotes the formation and maintenance of long stable MTs through a mechanism that may involve recruitment of unpolymerized tubulin to the MT lattice.

Katanin: A Sword Cutting Microtubules for Cellular, Developmental, and Physiological Purposes.

KATANIN is a well-studied microtubule severing protein affecting microtubule organization and dynamic properties in higher plants. By regulating mitotic and cytokinetic and cortical microtubule arrays it is involved in the progression of cell division and cell division plane orientation. KATANIN is also involved in cell elongation and morphogenesis during plant growth. In this way KATANIN plays critical roles in diverse plant developmental processes including the development of pollen, embryo, seed, meristem, root, hypocotyl, cotyledon, leaf, shoot, and silique. KATANIN-dependent microtubule regulation seems to be under the control of plant hormones. This minireview provides an overview on available KATANIN mutants and discusses advances in our understanding of KATANIN biological roles in plants.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Formin' bridges between microtubules and actin filaments in axonal growth cones.

Cadmium affects microtubule organization and post-translational modifications of tubulin in seedlings of soybean (Glycine max L.).

Cadmium (Cd) is a non-essential heavy metal, toxic to all living organisms. The microtubule (MT) cytoskeleton appears to be one of the main targets of Cd action. In this study we present, with the use of various immunological approaches, the effect of Cd at moderate (85 μM) and high (170 μM) concentrations on the structure and functioning of the MT cytoskeleton in the root cells of soybean seedlings. As the result of heavy metal action, root growth was significantly diminished and was accompanied by a reduction in mitotic activity and disturbance in the structure of the MT arrays, including randomization of the cortical MT arrangement, distorted mitotic arrays and complete depolymerization of the MTs. Biochemical analysis revealed decreased levels of various α- and β-tubulin isoforms with a parallel down-regulation of most examined α-tubulin genes. Simultaneously, Cd treatment led to differentiated changes in the level of tubulin post-translational modifications, including tyrosination, detyrosination, acetylation, and polyglutamylation. Decreased tyrosination and polyglutamylation of particular tubulin isoforms accompanied by increase in the level of specific detyrosinated and acetylated isoforms implies augmented stability and reduced turnover of the MTs during stress conditions. Taken together, the obtained results indicate the significant impact of Cd on gene expression levels and subsequent post-translational processing of tubulin, which may be related to the impairment of MT cytoskeleton functioning in root cells.

Katanin Activity is Regulated by Common Cellular Components

Proper organization of the microtubule cytoskeletal network is required to perform necessary cellular functions. Network organization is achieved through remodeling by microtubule associated proteins (MAPs) that control microtubule dynamics by selectively stabilizing and destabilizing microtubules. Stabilizing MAPs are relatively well understood, while less is known about destabilizing MAPs, such as severing enzymes. Katanin, the first-discovered microtubule severing enzyme, is a AAA+ enzyme that oligomerizes into hexamers and uses ATP hydrolysis to sever microtubules. Using single molecule biophysics techniques in vitro we investigate how katanin and its severing activity can be regulated by the type of microtubule, free tubulin, ATP concentration, and the neuronal MAP tau isoform 4RL. This work provides evidence that katanin activity is regulated by common cellular contents that will ultimately affect microtubule organization in cells.

Transcription factor NF-κB associates with microtubules and stimulates apoptosis in response to suppression of microtubule dynamics in MCF-7 cells

NF-κB, a master regulator of several signaling cascades, is known to be actively transported in the nucleus in response to various stimuli. Here, we found that NF-κB is associated with polymeric tubulin and co-localized with microtubules in MCF-7 cells. Using TN16, a known microtubule targeting agent, we found that microtubule dynamics plays a critical role in NF-κB-microtubule interaction. Treatment of cells with low concentrations of TN16 (25 and 50nM) that suppressed microtubule dynamics without visibly affecting microtubule organization enhanced the association of NF-κB with microtubules and facilitated nuclear translocation of NF-κB. Colchicine and vinblastine also produced similar nuclear translocation of NF-κB. Further, nuclear import of NF-κB activated apoptotic pathway in the cells that were blocked in mitosis by TN16 treatment suggesting that NF-κB acts as a pro-apoptotic protein in response to the suppression of microtubule dynamics. Interestingly, in the presence of high concentrations of TN16 that extensively disrupted the microtubule network, though there was an increase in the apoptotic cell death, the interaction of NF-κB with microtubules and its nuclear import were significantly reduced. Under these conditions, we detected an increase in the level of phosphorylation and nuclear accumulation of ERK, a MAP kinase, suggesting that the induction of apoptosis was caused by ERK signaling. The results indicate that the interaction of NF-κB with microtubules, its nuclear accumulation and subsequent gene transcription are critically dependent on microtubule dynamics. The data suggest a correlation between the functional status of microtubules and different apoptotic mechanisms invoked in response to microtubule inhibitors.

Dynamic Regulation of Cortical Microtubule Organization through Prefoldin-DELLA Interaction

Plant morphogenesis relies on specific patterns of cell division and expansion. It is well established that cortical microtubules influence the direction of cell expansion, but less is known about the molecular mechanisms that regulate microtubule arrangement. Here we show that the phytohormones gibberellins (GAs) regulate microtubule orientation through physical interaction between the nuclear-localized DELLA proteins and the prefoldin complex, a cochaperone required for tubulin folding. In the presence of GA, DELLA proteins are degraded, and the prefoldin complex stays in the cytoplasm and is functional. In the absence of GA, the prefoldin complex is localized to the nucleus, which severely compromises α/β-tubulin heterodimer availability, affecting microtubule organization. The physiological relevance of this molecular mechanism was confirmed by the observation that the daily rhythm of plant growth was accompanied by coordinated oscillation of DELLA accumulation, prefoldin subcellular localization, and cortical microtubule reorientation.

A Pericentrin-Related Protein Homolog in Aspergillus nidulans Plays Important Roles in Nucleus Positioning and Cell Polarity by Affecting Microtubule Organization

Pericentrin is a large coiled-coil protein in mammalian centrosomes that serves as a multifunctional scaffold for anchoring numerous proteins. Recent studies have linked numerous human disorders with mutated or elevated levels of pericentrin, suggesting unrecognized contributions of pericentrin-related proteins to the development of these disorders. In this study, we characterized AnPcpA, a putative homolog of pericentrin-related protein in the model filamentous fungus Aspergillus nidulans, and found that it is essential for conidial germination and hyphal development. Compared to the hyphal apex localization pattern of calmodulin (CaM), which has been identified as an interactive partner of the pericentrin homolog, GFP-AnPcpA fluorescence dots are associated mainly with nuclei, while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA. In addition, the depletion of AnPcpA by an inducible alcA promoter repression results in severe growth defects and abnormal nuclear segregation. Most interestingly, in mature hyphal cells, knockdown of pericentrin was able to significantly induce changes in cell shape and cytoskeletal remodeling; it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well. Moreover, defects in AnPcpA significantly disrupted the microtubule organization and nucleation, suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.

Stabilization of actin filaments prevents germinal vesicle breakdown and affects microtubule organization in Xenopus oocytes

In Xenopus oocytes, extremely giant nuclei, termed germinal vesicles, contain a large amount of actin filaments most likely for mechanical integrity. Here, we show that microinjection of phalloidin, an F-actin-stabilizing drug, prevents the germinal vesicle breakdown (GVBD) in oocytes treated with progesterone. These nuclei remained for more 12 h after control oocytes underwent GVBD. Immunostaining showed significant elevation of actin in the remaining nuclei and many actin filament bundles in the cytoplasm. Furthermore, microtubules formed unusual structures in both nuclei and cytoplasm of phalloidin-injected oocytes stimulated by progesterone. Cytoplasmic microtubule arrays and intranuclear microtubules initially formed in phalloidin-injected oocytes as control oocytes exhibited white maturation spots; these structures gradually disappeared and finally converged upon intranuclear short bundles when control oocytes completed maturation. In contrast, treatment of oocytes with jasplakinolide, a cell membrane-permeable actin filament-stabilizing drug, did not affect GVBD. This drug preferentially induced accumulation of actin filaments at the cortex without any increase in cytoplasmic actin staining. Based on these results, intranuclear and cytoplasmic actin filament dynamics appear to be required for the completion of GVBD and critically involved in the regulation of microtubule assembly during oocyte maturation in Xenopus laevis.

Kinetic stabilization of microtubule dynamics by indanocine perturbs EB1 localization, induces defects in cell polarity and inhibits migration of MDA-MB-231 cells

Cell motility is an essential aspect of metastatic spread of cancer. Microtubule-targeted agents exhibit anti-metastatic properties, the underlying mechanism of which remains understudied. In this study, we have investigated the role of microtubule dynamics in migration of cancer cells using indanocine, a synthetic small molecule inhibitor of tubulin. We found that indanocine, at concentrations that did not visibly affect microtubule organization, suppressed dynamic instability of microtubules and reduced the rate of migration of highly metastatic MDA-MB-231 cells. Indanocine-treated cells were defective in lamellipodium formation and could not develop polarized morphology. The kinetic stabilization of microtubules was associated with a marked increase in their acetylation level and a perturbation in the localization of EB1, a microtubule plus end binding protein. Using standard scratch wound healing assay and immunofluorescence analysis; we found that microtubule acetylation occurred in the direction of migration in vehicle-treated cells, whereas indanocine treatment led to a global acetylation of microtubules. The results together suggested that selective stabilization of microtubules was perturbed in the presence of indanocine that possibly resulted in lack of cell polarization and a concurrent reduction in migration of cells. Moreover, microtubule stabilization by indanocine affected adhesion turnover and impaired the polarized pattern of adhesion sites in cells. Together the results indicated that the regulation of microtubule dynamics is required to coordinate cell polarization as well as adhesion asymmetry and support the hypothesis that the perturbation of microtubule dynamics by tubulin-targeted agents can be exploited to restrict the migration of tumor cells.

The novel functions of kinesin motor proteins in plants

Kinesin superfamily proteins are important microtubule-based motor proteins with a kinesin motor domain that is conserved among all eukaryotic organisms. They are responsible for unidirectionally transporting various cargos, including membranous organelles, protein complexes, and mRNAs. They also play critical roles in mitosis, morphogenesis, and signal transduction. Most kinesins in plants are evolutionarily divergent from their counterparts in animals and fungi. The mitotic kinesins in the plant kinesin-5 and kinesin-14 subfamilies appear to be similar to those in fungi and animals. However, others with nonmotor sequences are unique to plants. The kinesins affect microtubule organization, organelle distribution, vesicle transport, and cellulose microfibril order. Ultimately, plant kinesins contribute directly or indirectly to cell division and cell growth in various tissues. Here, we review a novel function of kinesins with transcription activation activity in regulating gibberellin biosynthesis and cell growth. These findings will open exciting new areas of kinesin research.

A New Hypothesis about Neuronal Degeneration Appeared after a Rat Model of Menopause

Objective: Estrogen is suggested to be one of the most important regulators of neuronal function, including neuronal proliferation, survival and plasticity. There is a broad consensus that the loss of ovarian hormones is associated with neurodegeneration in the hippocampus that leads to cognitive impairment. Methods: A total of 8 female rats which were subjected to bilateral ovariectomy were included in this study. After ovariectomy, the rats were housed for 123 days in a standard laboratory. At the end of the 123 days, the rats were euthanized and the brain sections were investigated by conventional light microscopic and electron microscopic techniques. Results: The regular structure of almost all axon extensions was lost. The majority of these extensions had a sawtooth-like appearance in longitudinal section profiles. Especially in transfer section profiles of myelinated axons, some morphological changes were shown which may be matched up with light microscopic findings. Conclusions: Deficiency of estrogen will initially affect microtubule organization. When this organization breaks down, it will physically cause the distribution of the normal structure of axonal plasmalemma. This in turn will lead to the distribution of physical organizations of estrogen and other different types of receptors which are placed in both the membrane and microtubules in the axon.

The effect of okadaic acid on Arabidopsis thaliana root morphology and microtubule organization in its cells

To investigate the role of protein hyperphosphorylation in plant cells, the effect of okadaic acid, a specific inhibitor of protein phosphatases PPI and PP2A, on the general morphology of Arabidopsis thaliana primary roots and the structural-functional characteristics of cortical microtubules in different cell types in all primary root growth zones was studied. It was found that okadaic acid affects microtubule organization in a different manner depending on the type of cells and functional zones of the primary root. Cortical microtubules in the epidermis and cortex cells of the elongation zone proved to be most sensitive to 0.1, 1, and 10 nM okadaic acid which completely depolymerized after inhibitor treatment. In trichoblasts, atrichoblasts of differentiation zone treatment with okadaic acid caused the microtubules stabilization. The treatment with okadaic acid significantly affected the morphology of root hairs, causing their swelling and branching as a result of abnormal microtubule orientation. The results of this study suggest that induction of protein hyperphosphorylation as a result of protein phosphatase inhibition plays a crucial key in microtubule organization in plant cells.

Rapid effects of acute anoxia on spindle kinetochore interactions activate the mitotic spindle checkpoint

The dramatic chromosome instability in certain tumors might reflect a synergy of spindle checkpoint defects with hypoxic conditions. In Caenorhabditis elegans and Drosophila melanogaster, spindle checkpoint activation has been implicated in the response to acute anoxia. The activation mechanism is unknown. Our analyses in D. melanogaster demonstrate that oxygen deprivation affects microtubule organization within minutes. The rapid effects of anoxia are identical in wild-type and spindle checkpoint-deficient Mps1 mutant embryos. Therefore, the anoxia effects on the mitotic spindle are not a secondary consequence of spindle checkpoint activation. Some motor, centrosome and kinetochore proteins (dynein, Kin-8, Cnn, TACC, Cenp-C, Nuf2) are rapidly relocalized after oxygen deprivation. Kinetochores congress inefficiently into the metaphase plate and do not experience normal pulling forces. Spindle checkpoint proteins accumulate mainly within the spindle midzone and inhibit anaphase onset. In checkpoint-deficient embryos, mitosis is still completed after oxygen deprivation, although accompanied by massive chromosome missegregation. Inhibitors of oxidative phosphorylation mimic anoxia effects. We conclude that oxygen deprivation impairs the chromosome segregation machinery more rapidly than spindle checkpoint function. Although involving adenosine triphosphate (ATP)-consuming kinases, the spindle checkpoint can therefore be activated by spindle damage in response to acute anoxia and protect against aneuploidies.

Totarol Inhibits Bacterial Cytokinesis by Perturbing the Assembly Dynamics of FtsZ

Totarol, a diterpenoid phenol, has been shown to inhibit the proliferation of several pathogenic Gram-positive bacteria including Mycobacterium tuberculosis. In this study, totarol was found to inhibit the proliferation of Bacillus subtilis cells with a minimum inhibitory concentration of 2 microM. It did not detectably perturb the membrane structure of B. subtilis; it strongly induced the filamentation in B. subtilis cells, suggesting that it inhibits bacterial cytokinesis. Totarol (1.5 microM) reduced the frequency of the Z-ring occurrence per micrometer of the bacterial cell length but did not affect the nucleoid frequency, suggesting that it blocks cytokinesis by inhibiting the formation of the Z-ring. The assembly dynamics of FtsZ is thought to play an important role in the formation and functioning of the Z-ring, a machine that engineers cytokinesis in bacteria. Since totarol was shown to inhibit the proliferation of M. tuberculosis, we examined the effects of totarol on the assembly dynamics of M. tuberculosis FtsZ (MtbFtsZ) in vitro. Totarol decreased the assembly of MtbFtsZ protofilaments and potently suppressed the GTPase activity of MtbFtsZ. It bound to MtbFtsZ with a dissociation constant of 11 +/- 2.3 microM. It increased the fluorescence intensity of the MtbFtsZ-1-anilinonaphthalene-8-sulfonic acid complex and inhibited the fluorescence intensity of N-(1-pyrene)maleimide-labeled MtbFtsZ, suggesting that totarol induces conformational changes in MtbFtsZ. The results indicated that totarol can perturb the assembly dynamics of FtsZ protofilaments in the Z-ring. Totarol exhibited extremely weak inhibitory effects on HeLa cell proliferation. It did not affect microtubule organization in HeLa cells. The results suggest that totarol inhibits bacterial proliferation by targeting FtsZ and it may be useful as a lead compound to develop an effective antitubercular drug.

Constitutive activation of neuronal Src causes aberrant dendritic morphogenesis in mouse cerebellar Purkinje cells

Src family tyrosine kinases are essential for neural development, but their in vivo functions remain elusive because of functional compensation among family members. To elucidate the roles of individual Src family members in vivo, we generated transgenic mice expressing the neuronal form of c-Src (n-Src), Fyn, and their constitutively active forms in cerebellar Purkinje cells using the L7 promoter. The expression of the constitutively active n-Src retarded the postnatal development of Purkinje cells and disrupted dendritic morphogenesis, whereas the wild-type n-Src had only moderate effects. Neither wild-type nor constitutively active Fyn over-expression significantly affected Purkinje-cell morphology. The aberrant Purkinje cells in n-Src transgenic mice retained multiple dendritic shafts extending in non-polarized directions and were located heterotopically in the molecular layer. Ultrastructural observation of the dendritic shafts revealed that the microtubules of n-Src transgenic mice were more densely and irregularly arranged, and had structural deformities. In primary culture, Purkinje cells from n-Src transgenic mice developed abnormally thick dendritic shafts and large growth-cone-like structures with poorly extended dendrites, which could be rescued by treatment with a selective inhibitor of Src family kinases, PP2. These results suggest that n-Src activity regulates the dendritic morphogenesis of Purkinje cells through affecting microtubule organization.