Centromere-dependent binding of yeast minichromosomes to microtubules in vitro

We present an in vitro assay for yeast centromere function; isolated yeast minichromosomes require a functional centromere to bind to bovine microtubules and sediment with them. Centromere-bovine microtubule complexes form at physiological microtubule concentrations. Two of the three centromere DNA elements, which are necessary for centromere function in vivo, are also necessary for centromeres to bind microtubules in vitro. However, purified centromere DNA alone does not bind to microtubules. These results suggest that microtubule binding must be mediated by the two centromere DNA elements and factors that associate with one or both of them. The percent of centromeres with microtubule-binding activity is 7- to 10-fold higher in lysates made from nocodazole-arrested G2-M cells than from alpha factor G1 cells, suggesting that this centromere activity is regulated during the cell cycle. The potential of this assay for dissecting centromere assembly, function, and regulation is discussed.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Function and Assembly of DNA Looping,Clustering, and Microtubule Attachment Complexes within a Eukaryotic Kinetochore

The kinetochore is a complex protein–DNA assembly that provides the mechanical linkage between microtubules and the centromere DNA of each chromosome. Centromere DNA in all eukaryotes is wrapped around a unique nucleosome that contains the histone H3 variant CENP-A (Cse4p in Saccharomyces cerevisiae). Here, we report that the inner kinetochore complex (CBF3) is required for pericentric DNA looping at the Cse4p-containing nucleosome. DNA within the pericentric loop occupies a spatially confined area that is radially displaced from the interpolar central spindle. Microtubule-binding kinetochore complexes are not involved in pericentric DNA looping but are required for the geometric organization of DNA loops around the spindle microtubules in metaphase. Thus, the mitotic segregation apparatus is a composite structure composed of kinetochore and interpolar microtubules, the kinetochore, and organized pericentric DNA loops. The linkage of microtubule-binding to centromere DNA-looping complexes positions the pericentric chromatin loops and stabilizes the dynamic properties of individual kinetochore complexes in mitosis.     

Transport of African swine fever virus from assembly sites to the plasma membrane is dependent on microtubules and conventional kinesin

African swine fever virus (ASFV) is a large DNA virus that assembles in perinuclear viral factories located close to the microtubule organizing center. In this study, we have investigated the mechanism by which ASFV reaches the cell surface from the site of assembly. Immunofluorescence microscopy revealed that at 16 h postinfection, mature virions were aligned along microtubules. Furthermore, virus movement to the cell periphery was inhibited when microtubules were depolymerized by nocodazole. In addition, ASFV infection resulted in the increased acetylation of microtubules as well as their protection against depolymerization by nocodazole. Immunofluorescence microscopy showed that conventional kinesin was recruited to virus factories and to a large fraction of virus particles in the cytoplasm. Consistent with a role for conventional kinesin during ASFV egress to the cell periphery, overexpression of the cargo-binding domain of the kinesin light chain severely inhibited the movement of particles to the plasma membrane. Based on our observations, we propose that ASFV is recognized as cargo by conventional kinesin and uses this plus-end microtubule motor to move from perinuclear assembly sites to the plasma membrane.     

Molecular architecture of the kinetochore-microtubule interface

Segregation of the replicated genome during cell division in eukaryotes requires the kinetochore to link centromeric DNA to spindle microtubules. The kinetochore is composed of a number of conserved protein complexes that direct its specification and assembly, bind to spindle microtubules and regulate chromosome segregation. Recent studies have identified more than 80 kinetochore components, and are revealing how these proteins are organized into the higher order kinetochore structure, as well as how they function to achieve proper chromosome segregation.     

Effects of proteolysis of the extending parts of the high-molecular-weight microtubule-associated proteins on interactions between microtubules

Digestion of assembled microtubules with agarose-bound trypsin was performed to obtain microtubules which lack the extending projections, the non-tubulin-binding part of the high-molecular-weight microtubule-associated proteins. The assembly kinetics and the minimum protein concentration for assembly were the same for these trypsinated microtubules as for normal, untreated microtubules. Furthermore, the digested microtubules gave rise to the same change in turbidity per polymer mass as that found for normal microtubules. However, electron microscopy of pelleted microtubules revealed a closer packing after trypsin treatment. A substantially lower increase in specific viscosity was found upon assembly. At concentrations of above approx. 1.5 mg/ml, the viscosity of trypsin-treated microtubules was almost independent of the protein concentration, in contrast to the turbidity, which still increased. Both microtubules and the trypsin-digested microtubules were easily oriented by shear, although the flow linear dichroism signal for the microtubules after trypsin treatment was only half of that found for perfectly oriented normal microtubules. At higher shear force gradients, digested microtubules aggregated side by side as shown by electron microscopy. This was not found for normal microtubules. Even although the extending parts of the high-molecular-weight proteins are not needed for assembly, they were found to play an important role in microtubule orientation and interactions between microtubules, probably by acting as spacers between microtubules.     

The initiation of neurite outgrowth by sympathetic neurons grown in vitro does not depend on assembly of microtubules [published erratum appears in J Cell Biol 1995 Feb;128(3):443]

Neurite formation by dissociated chick sympathetic neurons in vitro begins when one of the many filopodia that emanate from the cell body of a neuron is invaded by cytoplasm containing microtubules and other components of axoplasm (Smith, 1994). This study was undertaken to determine whether this process depends on assembly of microtubules. To inhibit microtubule assembly, neurons were grown in medium containing nocodazole or colchicine. In one series of experiments, neurons first were exposed to the microtubule-stabilizing drug, taxol, so that existing microtubules would remain intact while assembly of new microtubules was inhibited. The ability of neurons to form neurites was assessed by time-lapse video microscopy. Neurons subsequently were stained with antibodies against the tyrosinated and acetylated forms of alpha-tubulin and examined by laser confocal microscopy to visualize microtubules. Neurons were able to form short processes despite inhibition of microtubule assembly and they did so in a way that closely resembled process formation in control medium. Processes formed by neurons that had not been pretreated with taxol were devoid of microtubules. However, microtubules were present in processes of taxol- pretreated neurons. These microtubules contained acetylated alpha- tubulin, as is typical of stable microtubules, but not tyrosinated alpha-tubulin, the form present in recently assembled microtubules. These findings show that the initial steps in neurite formation do not depend on microtubule assembly and suggest that microtubules assembled in the cell body can be translocated into developing neurites as they emerge. The results are compatible with models of neurite formation which postulate that cytoplasm from the cell body is transported into filopodia by actomyosin-based motility mechanisms.     

Cdk activity couples epigenetic centromere inheritance to cell cycle progression

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.     

The Cik1/Kar3 motor complex is required for the proper kinetochore-microtubule interaction after stressful DNA replication

In budding yeast Saccharomyces cerevisiae, kinetochores are attached by microtubules during most of the cell cycle, but the duplication of centromeric DNA disassembles kinetochores, which results in a brief dissociation of chromosomes from microtubules. Kinetochore assembly is delayed in the presence of hydroxyurea, a DNA synthesis inhibitor, presumably due to the longer time required for centromeric DNA duplication. Some kinetochore mutants are sensitive to stressful DNA replication as these kinetochore proteins become essential for the establishment of the kinetochore-microtubule interaction after treatment with hydroxyurea. To identify more genes required for the efficient kinetochore-microtubule interaction under stressful DNA replication conditions, we carried out a genome-wide screen for yeast mutants sensitive to hydroxyurea. From this screen, cik1 and kar3 mutants were isolated. Kar3 is the minus-end-directed motor protein; Cik1 binds to Kar3 and is required for its motor function. After exposure to hydroxyurea, cik1 and kar3 mutant cells exhibit normal DNA synthesis kinetics, but they display a significant anaphase entry delay. Our results indicate that cik1 cells exhibit a defect in the establishment of chromosome bipolar attachment in the presence of hydroxyurea. Since Kar3 has been shown to drive the poleward chromosome movement along microtubules, our data support the possibility that this chromosome movement promotes chromosome bipolar attachment after stressful DNA replication.     

Microtubule dynamics in the chromosomal spindle fiber: analysis by fluorescence and high-resolution polarization microscopy

We describe preliminary results from two studies exploring the dynamics of microtubule assembly and organization within chromosomal spindle fibers. In the first study, we microinjected fluorescently labeled tubulin into mitotic PtK1 cells and measured fluorescence redistribution after photobleaching (FRAP) to determine the assembly dynamics of the microtubules within the chromosomal fibers in metaphase cells depleted of nonkinetochore microtubules by cooling to 23-24 degrees C. FRAP measurements showed that the tubulin throughout at least 72% of the microtubules within the chromosomal fibers exchanges with the cellular tubulin pool with a half-time of 77 sec. There was no observable poleward flux of subunits. If the assembly of the kinetochore microtubules is governed by dynamic instability, our results indicate that the half-life of microtubule attachment to the kinetochore is less than several min at 23-24 degrees C. In the second study, we used high-resolution polarization microscopy to observe microtubule dynamics during mitosis in newt lung epithelial cells. We obtained evidence from 150-nm-thick optical sections that microtubules throughout the spindle laterally associate for several sec into "rods" composed of a few microtubules. These transient lateral associations between microtubules appeared to produce the clustering of nonkinetochore and kinetochore microtubules into the chromosomal fibers. Our results indicate that the chromosomal fiber is a dynamic structure, because microtubule assembly is transient, lateral interactions between microtubules are transient, and the attachment of the kinetochores to microtubules may also be transient.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

A kinase-independent role for Aurora A in the assembly of mitotic spindle microtubules in Caenorhabditis elegans embryos

The assembly of a functional mitotic spindle is crucial for achieving successful mitosis. Aurora A kinase is one of the key regulators of mitotic events, including mitotic entry, centrosome maturation and spindle bipolarity. Caenorhabditis elegans Aurora A (AIR-1) is responsible for the assembly of γ-tubulin-independent microtubules in early embryos; however, the mechanism by which AIR-1 contributes to microtubule assembly during mitosis has been unclear. Here we show by live-cell imaging and RNA-mediated interference (RNAi)-based modulation of gene activity that AIR-1 has a crucial role in the assembly of chromatin-stimulated microtubules that is independent of the γ-tubulin complex. Surprisingly, the kinase activity of AIR-1 is dispensable for this process. Although the kinase-inactive form of AIR-1 was detected along the microtubules as well as on centrosomes, the kinase-active form of AIR-1 was restricted to centrosomes. Thus, we propose that AIR-1 has a kinase-dependent role at centrosomes and a kinase-independent role for stabilizing spindle microtubules and that coordination of these two roles is crucial for the assembly of mitotic spindles.     

Nucleation and transport organize microtubules in metaphase spindles

Spindles are arrays of microtubules that segregate chromosomes during cell division. It has been difficult to validate models of spindle assembly due to a lack of information on the organization of microtubules in these structures. Here we present a method, based on femtosecond laser ablation, capable of measuring the detailed architecture of spindles. We used this method to study the metaphase spindle in Xenopus laevis egg extracts and found that microtubules are shortest near poles and become progressively longer toward the center of the spindle. These data, in combination with mathematical modeling, imaging, and biochemical perturbations, are sufficient to reject previously proposed mechanisms of spindle assembly. Our results support a model of spindle assembly in which microtubule polymerization dynamics are not spatially regulated, and the proper organization of microtubules in the spindle is determined by nonuniform microtubule nucleation and the local sorting of microtubules by transport.     

Keratan sulfate and related murine glycosylation can suppress murine cartilage damage in vitro and in vivo

Nucleolar spindle associated protein (NuSAP) is a microtubule-stabilizing protein that localizes to chromosome arms and chromosome-proximal microtubules during mitosis and to the nucleus, with enrichment in the nucleoli, during interphase. The critical function of NuSAP is underscored by the finding that its depletion in HeLa cells results in various mitotic defects. Moreover, NuSAP is found overexpressed in multiple cancers and its expression levels often correlate with the aggressiveness of cancer. Due to its localization on chromosome arms and combination of microtubule-stabilizing and DNA-binding properties, NuSAP takes a special place within the extensive group of spindle assembly factors. In this study, we identify a SAP-like domain that shows DNA binding in vitro with a preference for dsDNA. Deletion of the SAP-like domain abolishes chromosome arm binding of NuSAP during mitosis, but is not sufficient to abrogate its chromosome-proximal localization after anaphase onset. Fluorescence recovery after photobleaching experiments revealed the highly dynamic nature of this NuSAP-chromatin interaction during mitosis. In interphase cells, NuSAP also interacts with chromatin through its SAP-like domain, as evident from its enrichment on dense chromatin regions and intranuclear mobility, measured by fluorescence correlation spectroscopy.The obtained results are in agreement with a model where NuSAP dynamically stabilizes newly formed microtubules on mitotic chromosomes to enhance chromosome positioning without immobilizing these microtubules. Interphase NuSAP-chromatin interaction suggests additional functions for NuSAP, as recently identified for other nuclear spindle assembly factors with a role in gene expression or DNA damage response.     

The murine DNA glycosylase NEIL2 (mNEIL2) and human DNA polymerase beta bind microtubules in situ and in vitro

8-oxoguanine DNA glycosylase (OGG1), a major DNA repair enzyme in mammalian cells and a component of the base excision repair (BER) pathway, was recently shown to be associated with the microtubule network and the centriole at interphase and the spindle assembly at mitosis. In this study, we determined whether other participants in the BER pathway also bind microtubules in situ and in vitro. Purified recombinant human DNA polymerase beta (DNA Pol beta) and purified recombinant mNEIL2 were chemically conjugated to fluorochromes and photosensitive dyes and used in in situ localization and binding experiments. Results from in situ localization, microtubule co-precipitation and site-directed photochemical experiments showed that recombinant human DNA Pol beta and recombinant mNEIL2 associated with microtubules in situ and in vitro in a manner similar to that shown earlier for another BER pathway component, OGG1. Observations reported in this study suggest that these BER pathway components are microtubule-associated proteins (MAPs) themselves or utilize yet to be identified MAPs to bind microtubules in order to regulate their intracellular trafficking and activities during the cell cycle.     

The association of heterotrimeric GTP-binding protein (Go) with microtubules

The heterotrimeric GTP-binding regulatory proteins (G proteins) play an important role in the regulation of membrane signal transduction. Recently, we identified the association of Go protein with mitotic spindles. Here we have investigated the relationship between Go protein and microtubules. We used temperature-dependent reversible assembly and taxol methods to purify microtubules from bovine brains. Goalpha and Gbeta proteins were identified in the microtubular fraction by both methods. The Goalpha subunit in the microtubular fraction could be ADP ribosylated by pertussis toxin. Co-immunoprecipitation data also revealed that Go protein can interact with microtubules. Exogenous Go protein could be incorporated into the assembled microtubular fraction, and 5 microg/ml (60 nM) of Go protein inhibited 40% of microtubule assembly. Western blot analysis of Goalpha-1 and Goalpha-2 in microtubular fractions showed that only Goalpha-1 is associated with microtubules. We conclude that the Goalpha-1betagamma proteins are associated with microtubules and may play some role in regulating the assembly and disassembly of microtubules.     

Microtubule assembly in rat brain extracts. Further characterization of the effects of zinc on assembly and cold stability

Microtubule assembly has been studied turbidometrically in supernatant fluids prepared from rat brain by high-speed centrifugation. It was confirmed that assembly occurred in the absence of added GTP. Zinc ions (500 microM, but in the presence of 1 mM EGTA) stimulated assembly under these conditions. Zinc-stimulated assembly produced microtubules with normal characteristics, as judged by electron microscopy, SDS-polyacrylamide gel electrophoresis and inhibition of assembly by fructose-6-phosphate or colchicine. However, microtubules formed in the presence of such zinc concentrations were more stable to cold than controls, although the rate constant for the disassembly reaction was unchanged. Neither the stimulation of assembly by zinc nor the effect on cold stability was affected by trifluoperazine suggesting that a calmodulin-related mechanism is not involved. Microtubule "seeds" had little effect in the presence of zinc, suggesting that it may be acting on the nucleation phase of the assembly reaction. This was supported by the findings that zinc reduced the critical concentration of brain supernatant necessary for assembly and that zinc did not affect the rate constant for assembly. The results suggest zinc can in some way stabilize microtubules; possible mechanisms are discussed.     

Incorporation of GDP-tubulin during elongation of microtubules in vitro

Removal of GDP from tubulin E-site is not obligatory for the in vitro assembly of microtubule protein in 0.5 mM GMPPCP. This assembly, which is significantly enhanced by glycerol, produces microtubules of normal morphology and with normal composition of microtubule-associated proteins (MAPs). [3H]-GDP initially present at the E-site is shown to be incorporated directly into microtubules during assembly; this incorporation, maximally 60% of the assembled polymer, is dependent upon MAPs. These results are consistent with oligomeric species composed principally of GDP-tubulin plus MAPs, being incorporated directly into microtubules. The finding that stoichiometric GTP-tubulin formation is not an essential prerequisite for microtubule assembly may have important implications for the energetics of microtubule formation.     

Different assembly properties of cod, bovine, and rat brain microtubules.

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.     

Enhanced microtubule binding and tubulin assembly properties of conformationally constrained paclitaxel derivatives

Microtubule binding and tubulin assembly promotion by a series of conformationally restricted paclitaxel (PTX) derivatives was investigated. In these derivatives, the C-4 acetate of the taxane is tethered to the C-3' phenyl at ortho and meta positions with different length linkers. The apparent affinity of these derivatives for GMPCPP-stabilized microtubules was assessed by a competition assay, and their influence on microtubule polymerization was evaluated by measuring the critical concentration of GDP-tubulin in the presence of the respective molecule. In general, taxane derivatives with higher apparent affinity for microtubules induced tubulin assembly more efficiently. Among the derivatives, molecules with the shortest tether display the strongest affinity for microtubules. These derivatives exhibited enhanced microtubule stabilization properties and efficiently induced GDP-tubulin assembly into microtubules at low temperature of 12 degrees C and in the absence of Mg2+ ions in 0.1 M PIPES. Based on molecular dynamics simulations, we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.