Docking study of ligands into the colchicine binding site of tubulin

Cancer is a major cause of mortality in developed countries, following only cardiovascular diseases. Death of cancerous cells can be achieved by stopping mitosis and the antimitotic class of drugs formed by the spindle poisons can be used for this purpose. Their role is to disorganize the mitotic spindle by targeting its main constituent, the microtubules, themselves made of heterodimers of alpha and beta-tubulin. They disrupt the dynamics of the microtubules either by stabilizing them, as do paclitaxel or epothilones, or destabilizing them, as do colchicine. The binding site of colchicine seems to lie between the two units of the tubulin dimer. Here, we report on the characterization of this site by the docking of a series of reference compounds, and the subsequent docking of ligands prepared in our laboratory.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of spindle microtubules in the timing of the cell cycle in echinoderm eggs

Spindle microtubules play an important role in the mechanisms that control the timing of cell cycle events in the eggs of the sea urchins L. variegatus and L. pictus. However, recent work which used colchicine to block microtubule assembly in the eggs of two other echinoderms, S. purpuratus and D. excentricus, has raised serious questions about the generality of this role for spindle microtubules. Thus, we have systematically examined the role of spindle microtubules in the timing of the cell cycle in the fertilized eggs of these latter species. We treated eggs of both species with 5-10 microM Colcemid for several minutes starting 30 min after fertilization to completely prevent spindle microtubule assembly for several h. We used Colcemid, instead of colchicine, because it is effective at lower doses and, at these doses, shows no detectable toxic side effects. We compared for control and treated eggs the time course of nuclear envelope breakdown/reformation and DNA synthesis. We found for both species that the eggs continue to cycle without spindle microtubules; mitosis is up to twice the normal duration while interphase remains essentially unaffected. To test for the possible toxic side effects of the 1-2 mM colchicine used earlier on S. purpuratus and D. excentricus, we treated eggs of these two species, and also those of L. variegatus, with 1 mM lumi-colchicine. This photo-inactivated form of colchicine, which does not bind to tubulin, substantially prolongs mitosis and, to a lesser extent, interphase. Thus, the results of the earlier work are most easily explained by the combination of specific and nonspecific effects of the 1-2 mM colchicine used. Our present results indicate that the importance of spindle microtubules in the mechanisms that control the timing of the mitosis portion of the cell cycle is a general phenomenon.     

The effects of cytochalasin B and colchicine on the morphogenesis of mitochondria in Drosophila hydei during meiosis and early spermiogenesis

Summary Morphogenesis of mitochondria in male germ cells in cultivated cytocysts begins in early prophase I at which time mitochondria thicken and become ordered along the spindle apparatus during meiosis. At the end of the second meiotic division they aggregate to form the Nebenkern.In the presence of colchicine or cytochalasin B mitochondria are able to begin differentiation, although the correct course of meiosis is not guaranteed. In medium supplemented with colchicine they undergo normal thickening but do not aggregate, in a pattern known from untreated cultures. This may indicate that microtubules are involved in the aggregation process of mitochondria as colchicine is known to inhibit microtubule formation. Moreover, in cell cultures treated with cytochalasin B mitochondrial aggregation does occur; it is concluded that microfilaments, which are sensitive to cytochalasin B, do not play a detectable role in the aggregation of mitochondria.     

Effect of Colchicine and Vinblastine on the Agglutination of Polymorphonuclear Leucocytes by Concanavalin A

IT is generally accepted that microtubules serve as supportive elements within cell structures such as the mitotic spindle and a role for microtubules in the mechanical stabilization of cell surfaces was suggested by the microcinematographic experiments of Vasiliev, Gelfand et al.¹. On contact, surfaces lost their ruffled movement and this immobilization could be abolished by colcemid which, like colchicine and vinblastine, binds to and dissolves microtubules. We tested the possibility that microtubular proteins might also determine or maintain the topographical organization of surface elements in several functional systems in which this is believed important.     

Rapid rate of tubulin dissociation from microtubules in the mitotic spindle in vivo measured by blocking polymerization with colchicine

At metaphase, the amount of tubulin assembled into spindle microtubules is relatively constant; the rate of tubulin association equals the rate of dissociation. To measure the intrinsic rate of dissociation, we microinjected high concentrations of colchicine, or its derivative colcemid, into sea urchin embryos at metaphase to bind the free tubulin, thereby rapidly blocking polymerization. The rate of microtubule disassembly was measured from a calibrated video signal by the change in birefringent retardation (BR). After an initial delay after injection of colchicine or colcemid at final intracellular concentrations of 0.1-3.0 mM, BR decreased rapidly and simultaneously throughout the central spindle and aster. Measured BR in the central half-spindle decreased exponentially to 10% of its initial value within a characteristic period of approximately 20 s; the rate constant, k = 0.11 +/- 0.023 s-1, and the corresponding half-time, t 1/2, of BR decay was approximately 6.5 +/- 1.1 s in this concentration range. Below 0.1 mM colchicine or colcemid, the rate at which BR decreased was concentration dependent. Electron micrographs showed that the rapid decrease in BR corresponded to the disappearance of nonkinetochore microtubules; kinetochore fiber microtubules were differentially stable. As a control, lumicolchicine, which does not bind to tubulin with high affinity, was shown to have no effect on spindle BR at intracellular concentrations of 0.5 mM. If colchicine and colcemid block only polymerization, then the initial rate of tubulin dissociation from nonkinetochore spindle microtubules is in the range of 180-992 dimers per second. This range of rates is based on k = 11% of the initial polymer per second and an estimate from electron micrographs that the average length of a half-spindle microtubule is 1- 5.5 micron. Much slower rates of tubulin association are predicted from the characteristics of end-dependent microtubule assembly measured previously in vitro when the association rate constant is corrected for the lower rate of tubulin diffusion in the embryo cytoplasm. Various possibilities for this discrepancy are discussed.     

Cellular Effects of Curcumin on Plasmodium falciparum Include Disruption of Microtubules

Curcumin has been widely investigated for its myriad cellular effects resulting in reduced proliferation of various eukaryotic cells including cancer cells and the human malaria parasite Plasmodium falciparum. Studies with human cancer cell lines HT-29, Caco-2, and MCF-7 suggest that curcumin can bind to tubulin and induce alterations in microtubule structure. Based on this finding, we investigated whether curcumin has any effect on P. falciparum microtubules, considering that mammalian and parasite tubulin are 83% identical. IC₅₀ of curcumin was found to be 5 µM as compared to 20 µM reported before. Immunofluorescence images of parasites treated with 5 or 20 µM curcumin showed a concentration-dependent effect on parasite microtubules resulting in diffuse staining contrasting with the discrete hemispindles and subpellicular microtubules observed in untreated parasites. The effect on P. falciparum microtubules was evident only in the second cycle for both concentrations tested. This diffuse pattern of tubulin fluorescence in curcumin treated parasites was similar to the effect of a microtubule destabilizing drug vinblastine on P. falciparum. Molecular docking predicted the binding site of curcumin at the interface of alpha and beta tubulin, similar to another destabilizing drug colchicine. Data from predicted drug binding is supported by results from drug combination assays showing antagonistic interactions between curcumin and colchicine, sharing a similar binding site, and additive/synergistic interactions of curcumin with paclitaxel and vinblastine, having different binding sites. This evidence suggests that cellular effects of curcumin are at least, in part, due to its perturbing effect on P. falciparum microtubules. The action of curcumin, both direct and indirect, on P. falciparum microtubules is discussed.     

A nuclear-derived proteinaceous matrix embeds the microtubule spindle apparatus during mitosis

The concept of a spindle matrix has long been proposed. Whether such a structure exists, however, and what its molecular and structural composition are have remained controversial. In this study, using a live-imaging approach in Drosophila syncytial embryos, we demonstrate that nuclear proteins reorganize during mitosis to form a highly dynamic, viscous spindle matrix that embeds the microtubule spindle apparatus, stretching from pole to pole. We show that this "internal" matrix is a distinct structure from the microtubule spindle and from a lamin B-containing spindle envelope. By injection of 2000-kDa dextran, we show that the disassembling nuclear envelope does not present a diffusion barrier. Furthermore, when microtubules are depolymerized with colchicine just before metaphase the spindle matrix contracts and coalesces around the chromosomes, suggesting that microtubules act as "struts" stretching the spindle matrix. In addition, we demonstrate that the spindle matrix protein Megator requires its coiled-coil amino-terminal domain for spindle matrix localization, suggesting that specific interactions between spindle matrix molecules are necessary for them to form a complex confined to the spindle region. The demonstration of an embedding spindle matrix lays the groundwork for a more complete understanding of microtubule dynamics and of the viscoelastic properties of the spindle during cell division.     

The Effect of Colchicine on Regenerating Membranellar Cilia in Stentor coeruleus

SYNOPSIS. Stentors treated with toxic substances can be induced to shed their oral bands (19, Fig. 1), complex structures composed of many cilia organized into membranelles. Regenerating membranellar bands were observed in control stentors removed from toxic (urea-containing) medium at about 3.5 hours. At 8 hours regenerated control organisms were indistinguishable from normal unshed stentors. Experimental animals replaced into colchicine medium were inhibited from regeneration at low, nontoxic concentrations of this mitotic spindle inhibitor. Upon removal of the colchicine and replacement of the shed animals into normal medium or normal medium to which GTP had been added, complete and normal regeneration of the membranellar band ensued. Our observations are consistent with many suggesting that colchicine acts by reversibly binding with a protein during processes involving microtubule formation. Colchicine inhibition of membranellar band formation further indicates that oral membranelles are specialized evolutionary homologs to other centriole (= basal body, = kinetosome) derivatives such as mitotic spindle fibers, cilia and flagella, axopods, etc. (structures containing the ubiquitous microtubules of eukaryotic cells).     

Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length

Changes in birefringence retardation (BR) and length of Chaetopterus meiotic metaphase-arrested spindles produced by increased hydrostatic pressure were observed with polarized-light microscopy using a newly developed optical pressure chamber. Increased pressure produced rapid, reversible decreases in spindle BR and length. Pressures of 3,500 psi or higher at 22 degrees C caused complete disappearance of spindle BR within 3 min. Up to 6,000 psi, the rates of both BR decay and spindle shortening increased progressively with increasing pressure. At 6,000 psi or above, the BR decreased rapidly but there was no evidence of spindle shortening. The general observations are consistent with results of earlier classical experiments on effects of pressure on mitosis, and with experiments that used colchicine or low temperature as microtubule-depolymerizing agents. The kinetics of spindle depolymerization and repolymerization showed two phases: an initial phase of rapid decreases or increase in half-spindle microtubule BR; and a second phase of nearly constant BR during which most of the spindle shortening or growth occurs. BR is assumed to be directly related to the number of microtubules in a spindle cross section. It is hypothesized that microtubules in the spindle have different stabilities depending on the attachment of nonattachment of their ends. This hypothesis is used to explain the two phases of spindle depolymerization and repolymerization as well as several other observations.     

Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation

The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.     

Structure-based virtual screening of novel tubulin inhibitors and their characterization as anti-mitotic agents

Microtubule cytoskeletons are involved in many essential functions throughout the life cycle of cells, including transport of materials into cells, cell movement, and proper progression of cell division. Small compounds that can bind at the colchicine site of tubulin have drawn great attention because these agents can suppress or inhibit microtubule dynamics and tubulin polymerization. To find novel tubulin polymerization inhibitors as anti-mitotic agents, we performed a virtual screening study of the colchicine binding site on tubulin. Novel tubulin inhibitors were identified and characterized by their inhibitory activities on tubulin polymerization in vitro. The structural basis for the interaction of novel inhibitors with tubulin was investigated by molecular modeling, and we have proposed binding models for these hit compounds with tubulin. The proposed docking models were very similar to the binding pattern of colchicine or podophyllotoxin with tubulin. These new hit compound derivatives exerted growth inhibitory effects on the HL60 cell lines tested and exhibited strong cell cycle arrest at G2/M phase. Furthermore, these compounds induced apoptosis after cell cycle arrest. In this study, we show that the validated derivatives of compound 11 could serve as potent lead compounds for designing novel anti-cancer agents that target microtubules.     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

The interaction of the B-ring of colchicine with alpha-tubulin: a novel footprinting approach

Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on alpha and some supporting a binding site on beta. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the alpha-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on alpha-tubulin; these residues are located well away from the alpha/beta interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the alpha/beta interface of tubulin with the B-ring on the alpha-subunit and the A and the C-rings on the beta-subunit.     

Microtubule functions.

Microtubules, with intermediate filaments and microfilaments, are the components of the cell skeleton which determinates the shape of a cell. Microtubules are involved in different functions including the assembly of mitotic spindle, in dividing cells, or axon extension, in neurons. In the first case, microtubules are highly dynamic, while in the second case microtubules are quite stable, suggesting that microtubule with different physical properties (stability) are involved in different functions. Thus, to understand the mechanisms of microtubule functions it is very important to understand microtubule dynamics. Historically, tubulin, the main component of microtubules, was first characterized as the major component of the mitotic spindle that binds to colchicine. Afterwards, it was found that tubulin is particularly more abundant in brain than in other tissues. Therefore, the roles of microtubules in mitosis, and in neurons, have been more extensively analyzed and, in this review, these roles will be discussed.     

Structures of potent anticancer compounds bound to tubulin

Small molecules that bind to tubulin exert powerful effects on cell division and apoptosis (programmed cell death). Cell‐based high‐throughput screening combined with chemo/bioinformatic and biochemical analyses recently revealed a novel compound MI‐181 as a potent mitotic inhibitor with heightened activity towards melanomas. MI‐181 causes tubulin depolymerization, activates the spindle assembly checkpoint arresting cells in mitosis, and induces apoptotic cell death. C2 is an unrelated compound previously shown to have lethal effects on microtubules in tumorigenic cell lines. We report 2.60 Å and 3.75 Å resolution structures of MI‐181 and C2, respectively, bound to a ternary complex of αβ‐tubulin, the tubulin‐binding protein stathmin, and tubulin tyrosine ligase. In the first of these structures, our crystallographic results reveal a unique binding mode for MI‐181 extending unusually deep into the well‐studied colchicine‐binding site on β‐tubulin. In the second structure the C2 compound occupies the colchicine‐binding site on β‐tubulin with two chemical moieties recapitulating contacts made by colchicine, in combination with another system of atomic contacts. These insights reveal the source of the observed effects of MI‐181 and C2 on microtubules, mitosis, and cultured cancer cell lines. The structural details of the interaction between tubulin and the described compounds may guide the development of improved derivative compounds as therapeutic candidates or molecular probes to study cancer cell division.     

The effect of colchicine on the sleeve element of microtubules

The microtubules in the nutritive tubes of telotrophic insect ovaries, like those in many other situations, are surrounded by an electron clear zone or ‘sleeve’ after conventional preparative procedures for electron microscopy. Ribosomes, which also pack the nutritive tubes, do not encroach into this region, and although microtubules are often closely opposed, they are rarely seen to touch. The composition of the microtubule sleeve zone is unknown. This study shows that colchicine not only destroys the microtubules in the nutritive tubes, but also the sleeve zone which surrounds them, suggesting that the integrity of the microtubules is essential for the existence of the sleeves.     

Swinging a sword: how microtubules search for their targets

The cell interior is in constant movement, which is to a large extent determined by microtubules, thin and long filaments that permeate the cytoplasm. To move large objects, microtubules need to connect them to the site of their destination. For example, during cell division, microtubules connect chromosomes with the spindle poles via kinetochores, protein complexes on the chromosomes. A general question is how microtubules, while being bound to one structure, find the target that needs to be connected to this structure. Here we review the mechanisms of how microtubules search for kinetochores, with emphasis on the recently discovered microtubule feature to explore space by pivoting around the spindle pole. In addition to accelerating the search for kinetochores, pivoting helps the microtubules to search for cortical anchors, as well as to self-organize into parallel arrays and asters to target specific regions of the cell. Thus, microtubule pivoting constitutes a mechanism by which they locate targets in different cellular contexts.     

Keratin filaments restrict organelle migration into the forming spindle of newt pneumocytes.

When viewed by light microscopy the mitotic spindle of newt pneumocytes appears to assemble in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which is often much larger than the spindle. This clear area is also frequently larger than the region previously occupied by the nucleus. It forms even in prometaphase cells depleted of microtubules prior to nuclear envelope breakdown by colchicine treatment. Time-lapse video microscopy reveals that as prometaphase proceeds this clear area slowly and progressively collapses around the forming spindle so that it is greatly diminished or nonexistent by the onset of anaphase. The sharply defined nature of the boundary between the clear area and the remaining cytoplasm and the fact that organelles accumulate at its periphery suggest that a structural barrier is present at the boundary that limits organelle migration into the forming spindle. Immunofluorescence and electron microscopy, of cells previously followed in the living state, reveal that the periphery of the clear area contains prominent bundles of keratin filaments but lacks microtubules and actin. From our observations we conclude that keratin filaments form a loosely organized cage that surrounds the forming newt pneumocyte spindle. We propose that this cage functions, in part, to restrict the dispersion of chromosomes during nuclear envelope breakdown and to impede the bulk migration of organelles into the forming spindle.     

Microtubule-mediated and microtubule-independent transport of adenovirus type 5 in HEK293 cells

Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules.