Continuous Microtubules Research Articles

NuSAP regulates microtubule flux and Kif2A localization to ensure accurate chromosome congression.

Precise chromosome congression and segregation requires the proper assembly of a steady-state metaphase spindle, which is dynamic and maintained by continuous microtubule flux. NuSAP is a microtubule-stabilizing and -bundling protein that promotes chromosome-dependent spindle assembly. However, its function in spindle dynamics remains unclear. Here, we demonstrate that NuSAP regulates the metaphase spindle length control. Mechanistically, NuSAP facilitates kinetochore capture and spindle assembly by promoting Eg5 binding to microtubules. It also prevents excessive microtubule depolymerization through interaction with Kif2A, which reduces Kif2A spindle-pole localization. NuSAP is phosphorylated by Aurora A at Ser-240 during mitosis, and this phosphorylation promotes its interaction with Kif2A on the spindle body and reduces its localization with the spindle poles, thus maintaining proper spindle microtubule flux. NuSAP knockout resulted in the formation of shorter spindles with faster microtubule flux and chromosome misalignment. Taken together, we uncover that NuSAP participates in spindle assembly, dynamics, and metaphase spindle length control through the regulation of microtubule flux and Kif2A localization.

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

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

Fast super-resolution imaging with ultra-high labeling density achieved by joint tagging super-resolution optical fluctuation imaging.

Previous stochastic localization-based super-resolution techniques are largely limited by the labeling density and the fidelity to the morphology of specimen. We report on an optical super-resolution imaging scheme implementing joint tagging using multiple fluorescent blinking dyes associated with super-resolution optical fluctuation imaging (JT-SOFI), achieving ultra-high labeling density super-resolution imaging. To demonstrate the feasibility of JT-SOFI, quantum dots with different emission spectra were jointly labeled to the tubulin in COS7 cells, creating ultra-high density labeling. After analyzing and combining the fluorescence intermittency images emanating from spectrally resolved quantum dots, the microtubule networks are capable of being investigated with high fidelity and remarkably enhanced contrast at sub-diffraction resolution. The spectral separation also significantly decreased the frame number required for SOFI, enabling fast super-resolution microscopy through simultaneous data acquisition. As the joint-tagging scheme can decrease the labeling density in each spectral channel, thereby bring it closer to single-molecule state, we can faithfully reconstruct the continuous microtubule structure with high resolution through collection of only 100 frames per channel. The improved continuity of the microtubule structure is quantitatively validated with image skeletonization, thus demonstrating the advantage of JT-SOFI over other localization-based super-resolution methods.

Transfer of mitochondria via tunneling nanotubes rescues apoptotic PC12 cells.

Tunneling nanotubes (TNTs) are F-actin-based membrane tubes that form between cells in culture and in tissues. They mediate intercellular communication ranging from electrical signalling to the transfer of organelles. Here, we studied the role of TNTs in the interaction between apoptotic and healthy cells. We found that pheochromocytoma (PC) 12 cells treated with ultraviolet light (UV) were rescued when cocultured with untreated PC12 cells. UV-treated cells formed a different type of TNT with untreated PC12 cells, which was characterized by continuous microtubule localized inside these TNTs. The dynamic behaviour of mCherry-tagged end-binding protein 3 and the accumulation of detyrosinated tubulin in these TNTs indicate that they are regulated structures. In addition, these TNTs show different biophysical properties, for example, increased diameter allowing dye entry, prolonged lifetime and decreased membrane fluidity. Further studies demonstrated that microtubule-containing TNTs were formed by stressed cells, which had lost cytochrome c but did not enter into the execution phase of apoptosis characterized by caspase-3 activation. Moreover, mitochondria colocalized with microtubules in TNTs and transited along these structures from healthy to stressed cells. Importantly, impaired formation of TNTs and untreated cells carrying defective mitochondria were unable to rescue UV-treated cells in the coculture. We conclude that TNT-mediated transfer of functional mitochondria reverse stressed cells in the early stages of apoptosis. This provides new insights into the survival mechanisms of damaged cells in a multicellular context.

Microtubule organization in the final stages of cytokinesis as revealed by cryo-electron tomography

The completion of cytokinesis is dominated by the midbody, a tightly-packed microtubule (MT)-based bridge that transiently connects the two daughter cells. Assembled from condensed, spindle-MTs and numerous associated proteins, the midbody gradually narrows down until daughter cell partitioning occurs at this site. Although described many years ago, detailed understanding of the abscission process remains lacking. Applying cryo-electron tomography to purified midbodies, in combination with fluorescence microscopy, we present here new insight into MT organization within the midbody. We find that the midbody is spatially divided into a core bundle of MTs that traverses the electron-dense overlap region (continuous MTs), surrounded by MTs that terminate within the overlap region (polar MTs). Residual continuous MTs remained intact up to the verge of abscission, whereas the residual polar MTs lost their organization and retreated from the overlap region at late cytokinesis stages. A detailed localization of the microtubule-bundling protein PRC1 supports the above notion. Our study thus provides a detailed account of the abscission process and suggests that the midbody, having acquired a distinct MT architecture as compared to the preceding central spindle, actively facilitates the final stage of cytokinesis.

Antimitotic action of glycosides of Nerium oleander L.

The effects of mixtures of glycosides from Nerium oleander L. on root tips of Allium cepa L. and endosperm of Haemanthus katherinae Bak. were studied. The root tips were exposed to the solutions for different time intervals (6 to 48 hours) and their recovery in tap water was also observed. Disturbances in Allium were studied exclusively by means of the squash preparation technique. Endosperm of Haemanthus was studied both in vitro and in the electron microscope. The following disturbances were found: (1) Disturbances in the formation of a normal bi-polar mitotic spindle. Distinct continuous fibers are not present although kinetochore-fibers are well developed. It is suggested that continuous microtubules arranged as distinct bundles are necessary for a normal bipolar anaphase. Irregularities at anaphase resulted in: a) Formation of restitution nuclei from whole chromosomes (kinetochore regions unsplit) or chromatids. b) Separation of chromosomes or chromatids into a few irregular groups which form restitution nuclei of different sizes. (2) Disturbances in phragmoplast development, often leading to the formation of multipolar phragmoplasts and a branched cell plate. During recovery in tap water, aberrant mitoses are gradually replaced by normal ones both in polyploid and hypoploid nuclei (even those containing only two chromosomes).

A Novel Microtubule-Depolymerizing Kinesin Involved in Length Control of a Eukaryotic Flagellum

Cilia and flagella are complex, microtubule (MT)-filled cell organelles of which the structure is evolutionarily conserved from protistan cells to mammalian sperm and the size is regulated. The best-established model for flagellar length (FL) control is set by the balance of continuous MT assembly and disassembly occurring at the flagellar tip. Because steady-state assembly of tubulin onto the distal end of the flagellum requires intraflagellar transport (IFT)--a bidirectional movement of large protein complexes that occurs within the flagellum--FL control must rely upon the regulation of IFT. This does not preclude that other pathways might "directly" affect MT assembly and disassembly. Now, among the superfamily of kinesins, family-13 (MCAK/KIF2) members exhibit a MT-depolymerizing activity responsible for their essential functions in mitosis. Here we present a novel family-13 kinesin from the flagellated protozoan parasite Leishmania major, that localizes essentially to the flagellum, and whose overexpression produces flagellar shortening and knockdown yields long flagella. Using negative mutants, we demonstrate that this phenotype is linked with the MT-binding and -depolymerizing activity of this kinesin. This is the first report of an effector protein involved in FL control through a direct action in MT dynamics, thus this finding complements the assembly-disassembly model.

Head-head coordination is required for the processive motion of cytoplasmic dynein, an AAA + molecular motor

Cytoplasmic dynein is an AAA +-type molecular motor whose major components are two identical heavy chains containing six AAA + modules in tandem. It moves along a single microtubule in multiple steps which are accompanied with multiple ATP hydrolysis. This processive sliding is crucial for cargo transports in vivo. To examine how cytoplasmic dynein exhibits this processivity, we performed in vitro motility assays of two-headed full-length or truncated single-headed heavy chains. The results indicated that four to five molecules of the single-headed heavy chain were required for continuous microtubule sliding, while approximately one molecule of the two-headed full-length heavy chain was enough for the continuous sliding. The ratio of the stroking time to the total ATPase cycle time, which is a quantitative indicator of the processivity, was ∼0.2 for the single-headed heavy chain, while it was ∼0.6 for the full-length molecule. When two single-headed heavy chains were artificially linked by a coiled-coil of myosin, the processivity was restored. These results suggest that the two heads of a single cytoplasmic dynein communicate with each other to take processive steps along a microtubule.

Crm1 is a mitotic effector of Ran-GTP in somatic cells

The Ran GTPase controls multiple cellular processes, including nuclear transport, mitotic checkpoints, spindle assembly and post-mitotic nuclear envelope reassembly. Here we examine the mitotic function of Crm1, the Ran-GTP-binding nuclear export receptor for leucine-rich cargo (bearing nuclear export sequence) and Snurportin-1 (ref. 3). We find that Crm1 localizes to kinetochores, and that Crm1 ternary complex assembly is essential for Ran-GTP-dependent recruitment of Ran GTPase-activating protein 1 (Ran-GAP1) and Ran-binding protein 2 (Ran-BP2) to kinetochores. We further show that Crm1 inhibition by leptomycin B disrupts mitotic progression and chromosome segregation. Analysis of spindles within leptomycin B-treated cells shows that their centromeres were under increased tension. In leptomycin B-treated cells, centromeres frequently associated with continuous microtubule bundles that spanned the centromeres, indicating that their kinetochores do not maintain discrete end-on attachments to single kinetochore fibres. Similar spindle defects were observed in temperature-sensitive Ran pathway mutants (tsBN2 cells). Taken together, our findings demonstrate that Crm1 and Ran-GTP are essential for Ran-BP2/Ran-GAP1 recruitment to kinetochores, for definition of kinetochore fibres and for chromosome segregation at anaphase. Thus, Crm1 is a critical Ran-GTP effector for mitotic spindle assembly and function in somatic cells.

Mitosis and mitotic wave propagation in the coenocytic alga, Vaucheria terrestris sensu Goetz.

Mitosis and cytoplasmic microtubule (MT) dynamics were observed for the first time in Vaucheria terrestris sensu Goetz. Mitosis could occasionally be seen in part of the cylindrical coenocytic cell. The frequency of encountering cells with dividing nuclei was highest (ca 12%) 4 h after the onset of light in 12 h light/12 h dark regimes; it decreased thereafter and approached zero during the dark period. From the anterior end of every interphase nucleus a unique, long MT bundle extended. Differential-interference optics reveals that there is a filamentous structure in front of the moving nucleus. In prophase, the interphase bundle disappeared and shorter MT bundles emanated from both ends of the nucleus. In metaphase, the cytoplasmic MTs completely disappeared, probably being recycled to spindles. Continuous MTs elongated in anaphase and developed into an interzonal spindle in telophase; this elongated up to as much as 10 microm. The daughter nuclei were pushed away from each other by the interzonal spindle. Mitosis started synchronously in a relatively narrow region, and the mitotic stage propagated as a mitotic wave to adjacent regions, most frequently from tip to base. The role of the mitotic wave in tip growth and morphogenesis of a coenocytic cell is discussed.

Mutations in the PILZ group genes disrupt the microtubule cytoskeleton and uncouple cell cycle progression from cell division in Arabidopsis embryo and endosperm

Organised cell division and expansion play important roles in plant embryogenesis. To address their cellular basis, we have analysed Arabidopsis abnormal-embryo mutants which were isolated for their characteristic phenotype: mutant embryos are small, mushroom-shaped ("pilz") and consist of only one or few large cells each containing one or more variably enlarged nuclei and often cell wall stubs. These 23 mutants represent four genes, PFIFFERLING, HALLIMASCH, CHAMPIGNON, and PORCINO, which map to different chromosomes. All four genes have very similar mutant phenotypes although porcino embryos often consisted of only one large cell. The endosperm did not cellularise and contained a variably reduced number of highly enlarged nuclei. By contrast, genetic evidence suggests that these genes are not required for gametophyte development. Expression of cell cycle genes, Cdc2a, CyclinA2 and CyclinB1, and the cytokinesis-specific KNOLLE gene was not altered in mutant embryos. However, KNOLLE syntaxin accumulated in patches but no KNOLLE-positive structure resembling a forming cell plate occurred in mitotic cells. A general defect in microtubule assembly was observed in all mutants. Interphase cells lacked cortical microtubules, and spindles were absent from mitotic nuclei although in rare cases, short stubs of microtubules were attached to partially condensed chromosomes. Our results suggest that the cellular components affected by the pilz group mutations are necessary for continuous microtubule organisation, mitotic division and cytokinesis but do not mediate cell cycle progression.

Maintenance of membrane sheets by cultured oligodendrocytes requires continuous microtubule turnover and Golgi transport

Oligodendrocytes in murine shakeoff cultures elaborate extensive membrane sheets containing networks of microtubules. Several membrane components, including proteolipid protein (PLP) and sulfatide, are transported through the Golgi en route to the plasma membrane or myelin (1,2). This transport is essential for membrane assembly, but its role in continuing maintenance of the sheets is not known. We examined the stability of the membrane sheets following microtubule stabilization with taxol or block of transport into the Golgi with brefeldin A. Within one to three hours, both agents had marked effects on the membrane sheets. While some oligodendrocytes maintained regions of normal membrane sheets, many showed retraction of the sheets, with the majority now exhibiting multiple processes rather than sheets. The distribution of sulfatide, PLP and tubulin in cell bodies, processes and sheets was altered in treated cells, as analyzed by immunocytochemical staining with antibodies to these components. The Golgi apparatus also showed reorganization in the presence of taxol, as visualized by binding of wheat germ agglutinin, a lectin with high affinity for distal Golgi vesicles. All of these effects were reversible when the agents were removed after 3 hours. Thus, maintenance of membrane sheets by oligodendrocytes in culture is a dynamic process, requiring ongoing microtubule turnover and transport of molecules through the Golgi.

Three-dimensional reconstruction and analysis of mitotic spindles from the yeast, Schizosaccharomyces pombe.

Mitotic spindles of Schizosaccharomyces pombe have been studied by EM, using serial cross sections to reconstruct 12 spindles from cells that were ultrarapidly frozen and fixed by freeze substitution. The resulting distributions of microtubules (MTs) have been analyzed by computer. Short spindles contain two kinds of MTs: continuous ones that run from pole to pole and MTs that originate at one pole and end in the body of the spindle. Among the latter there are three pairs of MT bundles that end on fibrous, darkly staining structures that we interpret as kinetochores. The number of MTs ending at each putative kinetochore ranges from two to four; all kinetochore-associated MTs disappear as the spindle elongates from 3-6 microns. At this and greater spindle lengths, there are no continuous MTs, only polar MTs that interdigitate at the spindle midzone, but the spindle continues to elongate. An analysis of the density of neighboring MTs at the midzone of long spindles shows that their most common spacing is approximately 40 nm, center to center, and that there is a preferred angular separation of 90 degrees. Only hints of such square-packing are found at the midzone of short spindles, and near the poles there is no apparent order at any mitotic stage. Our data suggest that the kinetochore MTs (KMTs) do not interact directly with nonkinetochore MTs, but that interdigitating MTs from the two spindle poles do interact to form a mechanically stable bundle that connects the poles. As the spindle elongates, the number of MTs decreases while the mean length of the MTs that remain increases. We conclude that the chromosomes of S. pombe become attached to the spindle by kinetochore MTs, that these MTs disappear as the chromosomes segregate, that increased separation of daughter nuclei is accompanied by a sliding apart of anti-parallel MTs, and that the mitotic processes of S. pombe are much like those in other eukaryotic cells.

Microtubule disruption and cognitive defects: Effect of colchicine on learning behavior in rats

Neuropathological findings in Alzheimer's disease (AD) suggest a possible involvement of microtubule dysfunction in neurodegenerative process pathogenesis. Because microtubules have a major role in neuronal plasticity, microtubule disruption could be also directly responsible for cognitive defects in AD. We report that in rats, continuous microtubule disruption induced by chronic colchicine administration results in a dose-dependent learning deficit. In addition, retention is also impaired. These cognitive defects are specific, as chronic colchicine induces no other behavioral toxicity within the study dose range. Colchicine-induced cognitive defects resemble those of AD, which are characterised by amnesia of recent learning and loss of formerly established memories. This new procedure of pharmacologically induced cognitive impairment may prove useful, both towards understanding AD pathogenesis and towards drug screening.

Cortical microtubule organization in Vitis protoplasts as affected by concentration of enzyme isolation medium and duration of incubation

Protoplasts of Vitis rotundifolia Michx. cv. Summit were isolated from mesophyll of axenic shoot cultures under different enzyme concentrations and digestion times. Viability and plating efficiency were assessed and related to the cortical microtubule network, visualized using immunofluorescence. Higher concentrations of enzyme isolation medium significantly decreased protoplast viability and plating efficiency. However, the cortical microtubule network appeared stable, at all concentrations with dense, continuous microtubule strands in both random and parallel arrays. In contrast, longer vs shorter enzyme incubation duration resulted in significantly lower plating efficiency, which was correlated with changes in cortical microtubule organization. With longer incubation, the frequency of parallel microtubule strands decreased; microtubule organization showed increasing disruption, microtubule strands were shortened, fragmented and exhibited only a weak fluorescence labeling. Both high enzyme concentration and prolonged incubation periods negatively affected protoplast regenerability, but in different ways. Microtubule organization was sensitive to duration of incubation, but not to enzyme concentration. It is concluded that the presence of a well‐developed cortical microtubule network does not gurantee regeneration. Other factors related to isolation appear to be involved.

THE ELECTRON MICROSCOPIC STUDY OF MICROTUBULES AND THE EFFECT OF COLCHICINE DURING MITOSIS IN Glycine max (L.) Merr. (Soybean)

This study was carried to investigate the formation and inhibition of microtubules in ultrastructural level by analysing ali the stages of celi division in the control and colchicine treated celIs of root tip meristematic ccIis of Glycine max (L.) Merr. (Soybean). In control cells, circular sections of microtubules were observed near the celi walls in interphase. At the end of the metaphase, it was observed that the nuclear envelope at the poles ruptured and microtubules entered the karyoplasm from there. The number of microtubules reached its marimum level during metaphase. Three different types of microtubules were distinguis- hed namely kinetochoral, free and continuous microtubules. The number of microrubules were observed to decrease starting from the anaphase. There observed some phragmoplast microtubules in the telophase. The maximum effect of colchicine was observed at metaphase and there was no microtubules found in those root tip cells treated with colchicine. It was also determined that the chromosomes could not seperate from each other and stayed cons- tantly at metaphase.

Direct isolation of neuronal microtubule skeletons.

The microtubule skeletons of cultured neuronal cells have been isolated in their original form utilizing taxol and rigorous extraction conditions. Continuous microtubules are visible throughout the cell body and into the processes. These preparations include the complex set of microtubule-associated proteins of 69,000 and 80,000 daltons previously identified in these cells.

Direct isolation of neuronal microtubule skeletons.

The microtubule skeletons of cultured neuronal cells have been isolated in their original form utilizing taxol and rigorous extraction conditions. Continuous microtubules are visible throughout the cell body and into the processes. These preparations include the complex set of microtubule-associated proteins of 69,000 and 80,000 daltons previously identified in these cells.

A regulatory model for spindle function during mitosis

Structural information on the mitotic spindle of Saccharomyces cerevisiae obtained from isolated whole mount preparations has shown that the spindle undergoes a two-fold increase in length whilst comprising only a single microtubule continuous between the two spindle pole bodies. Further data from immunofluorescence microscopy on the timing of anaphase B has suggested that microtubules do not directly produce the required force, but instead have a more passive role. Here a regulatory function for spindle microtubules during mitosis is explored and the existence of a non-microtubule force-generating system is postulated. Thus it is suggested that the continuous microtubules govern the velocity of anaphase B by providing a resistive force that is itself regulated by the number of microtubules and their rate of polymerization. On this basis a model for the forces acting on a spindle pole body during anaphase is proposed.

Microtubule structure in Caenorhabditis elegans neurons.

Microtubules (MTs) are ubiquitous components of neuronal processes, and although they have been implicated in neurite outgrowth, shape maintenance, axonal transport, and sensory transduction, their function remains unclear (for review, see Porter 1966; Wuerker and Kirkpatrick 1972; Dustin 1978; Bray and Gilbert 1981; Lasek and Shelanski 1981). The MTs in the neurons of the nematode Caenorhabditis elegans have unusual structures that permit a comparative approach to the relationship of microtubule structure and function. A set of six touch-receptor neurons (the microtubule cells) (Chalfie and Thomson 1979) contain prominent arrays of large MTs. These MTs have more protofilaments than do MTs in other neurons (15 as opposed to 11), and they respond differently to antimicrotubule drugs, fixation protocols, temperature, and mutation. Studies of C. elegans neurotubules (Chalfie and Thomson 1979 and in prep.) suggest that most MT functions do not require long, continuous MTs or MTs with a specific number of...