Microtubule-depolymerizing Agent Research Articles

Meiotic telomere clustering is inhibited by colchicine but does not require cytoplasmic microtubules.

Telomere clustering, the defining feature of the bouquet, is an almost universal feature of meiotic prophase, yet its mechanism remains unknown. The microtubule-depolymerizing agent colchicine was found to inhibit bouquet formation. Telomeres in colchicine-treated cells remained scattered in the nuclear periphery, whereas untreated cells exhibited a prominent telomere cluster. Colchicine administered after the bouquet had formed did not affect telomere dispersal. The effect of colchicine on bouquet formation appeared to be separable from its effect on cytoplasmic microtubules; amiprophos methyl, a highly effective plant microtubule-depolymerizing drug, did not affect telomere clustering. Inhibition of bouquet formation was limited to colchicine and the related drug podophyllotoxin out of the variety of microtubule-depolymerizing drugs tested, suggesting that the target involved in bouquet formation has a structural specificity.

G(1) and G(2) cell-cycle arrest following microtubule depolymerization in human breast cancer cells.

Microtubule-depolymerizing agents are widely used to synchronize cells, screen for mitotic checkpoint defects, and treat cancer. The present study evaluated the effects of these agents on normal and malignant human breast cell lines. After treatment with 1 microM nocodazole, seven of ten breast cancer lines (type A cells) arrested in mitosis, whereas the other three (type B cells) did not. Similar effects were observed with 100 nM vincristine or colchicine. Among five normal mammary epithelial isolates, four exhibited type A behavior and one exhibited type B behavior. Further experiments revealed that the type B cells exhibited a biphasic dose-response curve, with mitotic arrest at low drug concentrations (100 nM nocodazole or 6 nM vincristine) that failed to depolymerize microtubules and a p53-independent p21(waf1/cip1)-associated G(1) and G(2) arrest at higher concentrations (1 microM nocodazole or 100 nM vincristine) that depolymerized microtubules. Collectively, these observations provide evidence for coupling of premitotic cell-cycle progression to microtubule integrity in some breast cancer cell lines (representing a possible "microtubule integrity checkpoint") and suggest a potential explanation for the recently reported failure of some cancer cell lines to undergo nocodazole-induced mitotic arrest despite intact mitotic checkpoint proteins.

Inhibition of Rho-kinase induces αB-crystallin expression in lens epithelial cells

The small heat shock protein, αB-crystallin, has been shown to interact with actin and intermediate filament proteins. However, little is known regarding the cellular mechanisms regulating such interactions. In this study, we explored the role of the Rho/Rho-kinase pathway in αB-crystallin distribution and expression in porcine lens epithelial cells. αB-crystallin was distributed uniformly throughout the cytoplasm and did not exhibit any unique redistribution in response to actin depolymerization induced by Rho/Rho-kinase inhibitors (C3-exoenzyme or Y-27632) or by overexpression of the dominant negative mutant of Rho-kinase (DNRK) in porcine lens epithelial cells. Interestingly, αB-crystallin levels markedly increased in lens epithelial cells treated with the inhibitors of Rho/Rho-kinase proteins (lovastatin, Y-27632 or DNRK) while a protein kinase C inhibitor (GF109203x) was found to have no effect. Further, Y-27632 showed a dose ( 2–50 μ M) response effect on αB-crystallin induction. Nocodazole, a microtubule-depolymerizing agent, elicited an increase in αB-crystallin levels but latrunculin, an actin depolymerizing agent, did not show any significant effect. Pretreatment with cycloheximide or genistein blocked the Rho-kinase inhibitor-induced increase in αB-crystallin protein levels. Rho-kinase inhibitor-induced increases in αB-crystallin levels were found to be associated with activation of P38 mitogen-activated protein kinase (MAPK). These results suggest that Rho/Rho-kinase negatively regulates αB-crystallin expression, and this response appears to be dependent on tyrosine–protein kinase and P38 MAPK function. Finally, αB-crystallin induction appears to be better correlated with the direct inhibition of Rho/Rho-kinase than with actin depolymerization per se.

Cytoskeleton disruption causes apoptotic degeneration of dentate granule cells in hippocampal slice cultures

Colchicine, a potent microtubule-depolymerizing agent, is well known to selectively kill dentate granule cells in the hippocampal formation in vivo. Using organotypic cultures of rat entorhino-hippocampal slices, we confirmed that in vitro exposure to 1 μM and 10 μM of colchicine reproduced a specific degeneration of the granule cells after 24 h. Similar results were obtained with other types of microtubule-disrupting agents, i.e., nocodazole, vinblastine, and Taxol. Interestingly, the actin-depolymerizing agents cytochalasin D and latrunculin A also elicited selective neurotoxicity in the dentate gyrus without affecting survival of hippocampal pyramidal cells. The selective pattern of degeneration was observable 24 h after a brief treatment with the toxins as short as 5 min, but this delayed neuronal death was unlikely to be a result of excitotoxicity because it was virtually unaffected by glutamate receptor antagonists, tetrodotoxin, or extracellular Ca 2+-free conditions. The damaged tissues contained a large number of TUNEL-positive neurons and exhibited an increased level in caspase-3-like activity, suggesting that cytoskeleton disruption triggers an apoptosis-like process in dentate granule cells. Thus, this study may provide a basis for understanding the distinctive mechanism that supports granule cell survival.

Dynamics of lysosomal cholesterol in Niemann-Pick type C and normal human fibroblasts

The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.—Lange, Y., J. Ye, M. Rigney, and T. L. Steck. Dynamics of lysosomal cholesterol in Niemann-pick type C and normal human fibroblasts.

Nocodazole inhibits insulin-stimulated glucose transport in 3T3-L1 adipocytes via a microtubule-independent mechanism.

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 microm nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 microm nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 microm) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 microm nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 microm nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 degrees C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

Role of microtubules in LPS-induced macrophage inflammatory protein-2 production from rat pneumocytes.

We have recently demonstrated that primary cultured rat pneumocytes produce macrophage inflammatory protein-2 (MIP-2) in response to lipopolysaccharide (LPS) stimulation. In this study, we found that brefeldin A, by blocking anterograde transport from the endoplasmic reticulum (ER) to the Golgi apparatus, decreased LPS-induced MIP-2 in the culture medium and increased its storage in cells. This suggests that MIP-2 is secreted via a pathway from the ER to the Golgi apparatus, a process commonly regulated by microtubules. We further found that LPS induced depolymerization of microtubules as early as 1 min after LPS stimulation, and it lasted at least for 4 h. Preventing depolymerization of microtubules with paclitaxel (Taxol; 10 nM to 10 microM) partially inhibited LPS-induced MIP-2 production, whereas the microtubule-depolymerizing agents colchicine (1-10 microM) and nocodazole (1-100 microM) increased LPS-induced MIP-2 protein production without affecting MIP-2 mRNA expression. These results suggest that in pneumocytes, LPS-induced microtubule depolymerization is involved in LPS-induced MIP-2 production and that secretion of MIP-2 from pneumocytes is via the ER-Golgi pathway.

Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulin-stimulated glucose uptake

Insulin stimulates glucose uptake into adipocytes by promoting the translocation of the glucose transporter isoform 4 (GLUT4) from intracellular vesicles to the plasma membrane. In 3T3-L1 adipocytes GLUT4 resides both in an endosomal pool, together with transferrin receptors, and in a unique pool termed 'GLUT4 storage vesicles' (GSVs), which excludes endosomal proteins. The trafficking of GLUT4 vesicles was studied in living 3T3-L1 adipocytes by time-lapse confocal microscopy of GLUT4 tagged with green fluorescent protein. GLUT4 vesicles exhibited two types of motion: rapid vibrations around a point and short (generally less than 10 microm) linear movements. The linear movements were completely blocked by incubation of the cells in the presence of microtubule-depolymerizing agents. This suggests that a subpopulation of GLUT4 vesicles can exhibit motor-driven movements along microtubules. Upon further examination, microtubule depolymerization inhibited insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane by approx. 40%, but had no effect on insulin-induced translocation of the transferrin receptor to the plasma membrane from endosomes. We propose that an intact microtubule cytoskeleton may be required for optimal trafficking of GLUT4 present in the GSV pool, but not that resident in the endosomal pool.

Different microtubule network alterations induced by pachymatismin, a new marine glycoprotein, on two prostatic cell lines

Pachymatismin is a new cytostatic factor extracted from the marine sponge Pachymatisma johnstonii Bowerbank. To investigate the mechanism of action of pachymatismin, we studied its effects on two human prostate cell lines (DU145 and E4) of tumor origin. Immunocytochemistry demonstrated that the drug caused depolymerization of microtubules in DU145 cells, this effect being similar to that of estramustine, known to be a microtubule-depolymerizing agent. E4 cells, described to be resistant to the microtubule-depolymerizing agent estramustine, were also found resistant to pachymatismin. Pachymatismin at the same dose that destroys microtubule organization in DU145 cells is not able to induce microtubule depolymerization in E4 cells. Compared to the estramustine- and pachymatismin-sensitive DU145 cells, E4 cells revealed an increase of betaI+II, betaIII, betaIV isotypes as well as post-translational modifications of tubulin, such as polyglutamylation and acetylation. In addition, the level of tau protein was also enhanced in E4 cells compared to DU145 cells. The effects of pachymatismin were tested in vitro using calf brain microtubules. It was shown that the drug lowers the capacity of microtubules to reassemble in vitro. Interestingly, pachymatismin has been found to inhibit microtubule assembly less efficiently when the ratio of tau to tubulin is increased. Taken together, pachymastismin has been shown to induce in vivo microtubule depolymerization following binding to microtubule proteins. Changes in microtubule components such as tubulin isoforms or tau may be involved in a decrease of sensitivity to pachymatismin.

Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans.

The spindle-assembly checkpoint ensures that, during mitosis and meiosis, chromosomes do not segregate until they are properly attached to the microtubules of the spindle. Here we show that mdf-1 and mdf-2 are components of the spindle-assembly checkpoint in Caenorhabditis elegans, and are essential for the long-term survival and fertility of this organism. Loss of function of either of these genes leads to the accumulation of a variety of defects, including chromosome abnormalities, X-chromosome non-disjunction or loss, problems in gonad development, and embryonic lethality. Antibodies that recognize the MDF-2 protein localize to nuclei of the cleaving embryo in a cell-cycle-dependent manner. mdf-1, a gene encoding a product that interacts with MDF-2, is required for cell-cycle arrest and proper chromosome segregation in premeiotic germ cells treated with nocodazole, a microtubule-depolymerizing agent. In the absence of mdf gene products, errors in chromosome segregation arise and accumulate, ultimately leading to genetic lethality.

Accumulation of P-Selectin in the Lumen of Irradiated Blood Vessels

Ionizing radiation induces the inflammatory response in part through leukocyte binding to cell adhesion molecules that are expressed on the vascular endothelium. We studied the effects of X radiation on the pattern of immunohistochemical staining of CD62P (P-selectin). P-selectin was localized within cytoplasmic granules in the untreated vascular endothelium. Immunohistochemical staining of P-selectin was observed at the luminal surface of vascular endothelium within 1 h of irradiation. Increased P-selectin staining at the blood-tissue interface occurred primarily in pulmonary and intestinal blood vessels. To determine whether localization of P-selectin at the vascular lumen occurs through exocytosis of endothelial cell stores in addition to platelet aggregation, we removed the vascular endothelium from the circulation and irradiated endothelial cells in vitro. In this system, we studied the mechanisms by which ionizing radiation induced translocation of P-selectin by using immunofluorescence of human umbilical vein endothelial cells (HUVEC) and confocal microscopy. Prior to irradiation, P-selectin is localized in cytoplasmic reservoirs of HUVEC. After irradiation of HUVEC, P-selectin was translocated to the cell membrane, where it remained tethered. The lowest dose at which we could expect translocation of P-selectin to the cell membrane was 2 Gy. To determine whether P-selectin in Weibel-Palade bodies requires microtubule-dependent membrane transport, we added two microtubule-depolymerizing agents, Colcemid and nocodazole. Microtubule-depolymerizing agents prevented radiation-induced trans- location of P-selectin to the cell membrane. Thus P-selectin accumulates in irradiated blood vessels through both platelet aggregation and microtubule-dependent exocytosis of storage reservoirs within the vascular endothelium.

Localization of the Drosophila checkpoint control protein Bub3 to the kinetochore requires Bub1 but not Zw10 or Rod.

We report here the isolation and molecular characterization of the Drosophila homolog of the mitotic checkpoint control protein Bub3. The Drosophila Bub3 protein is associated with the centromere/kinetochore of chromosomes in larval neuroblasts whose spindle assembly checkpoints have been activated by incubation with the microtubule-depolymerizing agent colchicine. Drosophila Bub3 is also found at the kinetochore regions in mitotic larval neuroblasts and in meiotic primary and secondary spermatocytes, with the strong signal seen during prophase and prometaphase becoming increasingly weaker after the chromosomes have aligned at the metaphase plate. We further show that the localization of Bub3 to the kinetochore is disrupted by mutations in the gene encoding the Drosophila homolog of the spindle assembly checkpoint protein Bub1. Combined with recent findings showing that the kinetochore localization of Bub1 conversely depends upon Bub3, these results support the hypothesis that the spindle assembly checkpoint proteins exist as a multiprotein complex recruited as a unit to the kinetochore. In contrast, we demonstrate that the kinetochore constituents Zw10 and Rod are not needed for the binding of Bub3 to the kinetochore. This suggests that the kinetochore is assembled in at least two relatively independent pathways.

Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules.

The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4 degreesC, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.

Actin-dependent anterograde movement of growth-cone-like structures along growing hippocampal axons: a novel form of axonal transport?

In time-lapse video recordings of hippocampal neurons in culture, we have identified previously uncharacterized structures, nicknamed "waves," that exhibit lamellipodial activity closely resembling that of growth cones, but which periodically emerge at the base of axons and travel distally at an average rate of 3 microm/min. In electron micrographs of identified waves, the cortical region of the axon appears expanded to either side, forming lamellipodia like those at growth cones. No other gross differences were noted in the ultrastructural features of the axon shaft at the site of a wave. Immunocytochemistry revealed that waves contain a marked concentration of F-actin, GAP-43, cortactin, and ezrin or a related protein, constituents that are also concentrated in growth cones. Treatment with the actin-disrupting agent cytochalasin B caused a reversible collapse of lamellipodia and cessation of the forward movement of individual waves along the axon, indicating that their anterograde transport is dependent on intact actin filaments. Treatment with the microtubule-depolymerizing agent nocodazole led to a rapid disorganization of wave structure and a subsequent suppression of wave activity that may reflect a role of microtubules in actin organization. The results suggest that actin and other cytoskeletal components concentrated in growth cones may be transported together as growth-cone-like structures from the cell body to the axon tip via an actin-dependent mechanism.

Polyploidization and Functional Maturation Are Two Distinct Processes During Megakaryocytic Differentiation: Involvement of Cyclin-Dependent Kinase Inhibitor p21 in Polyploidization

AbstractThe mechanism of megakaryocytic differentiation was investigated using human megakaryocytic leukemia cell line UT-7. Polyploidization of UT-7 cells was induced by the microtubule-depolymerizing agent, nocodazole, and 12-O-tetradecanoylphorbol-13-acetate (TPA), but the effect was much more striking with nocodazole. By contrast, induction of cytoplasmic maturation, as judged by β-thromboglobulin production and platelet factor 4 expression, was more prominent in TPA-treated cells than in nocodazole-treated cells. Nocodazole and TPA could act synergistically to increase ploidy and to enhance the expression of mature phenotypes. Human thrombopoietin induced functional maturation but not polyploidization in UT-7 cells and also acts synergistically with nocodazole. Cyclin-dependent kinase inhibitor p21 was upregulated at the early stage of megakaryocytic differentiation, and overexpression of p21 resulted in an increase in ploidy of UT-7 cells. This suggests that p21 is implicated in polyploidization via suppression of CDC2 activity at mitosis. UT-7 but not HL-60 cells could incorporate [3H]thymidine in the presence of TPA, indicating the presence of megakaryocyte-specific licensing factor to allow DNA replication during differentiation. Taking these data together, we propose that megakaryocytic differentiation consists of two distinct processes, polyploidization and functional maturation, and that these two processes are independently regulated.

Uptake of human urinary trypsin inhibitor by the kidney epithelial cell line, LLC-PK1.

We investigated the uptake of human urinary trypsin inhibitor (UTI) by the kidney epithelial cells, LLC-PK1. Indirect immunogold techniques with an electron microscope demonstrated the localization of UTI within the cells after an incubation during which UTI was added to the apical side. Immunoreactivities were found in endocytic vesicles, vacuoles and lysosomes. Subsequently, we tried to characterize the property of the uptake of UTI using the fluorescein isothiocyanate-labelled UTI (FITC-UTI). FITC-UTI uptake was decreased by an incubation with an excess of unlabelled UTI and showed concentration-dependent saturation. This process was markedly suppressed during the incubation at 4 degrees C. The uptake was significantly lessened with 2,4-dinitrophenol and antimycin A, inhibitors of oxidative phosphorylation, and colchicine, a microtubule-depolymerizing agent. These results indicate that exogenous UTI is internalized by LLC-PK1 cells through an endocytic pathway. From uptake studies, it is suggested that an adsorptive process is partially involved in the mechanisms of endocytosis.

Activation of newt eggs in the absence of Ca2+ activity by treatment with cycloheximide or D2 O.

Unfertilized eggs of urodeles that exhibit physiological polyspermy are difficult to activate by ordinary egg-activating agents, such as pricking and Ca2+ ionophores, that easily activate monospermic anuran eggs. Therefore, we have tested the effects of other agents that cause egg activation in non-amphibian species in order to investigate the mechanism of egg activation in urodeles. We have found that cycloheximide (a protein synthesis inhibitor), D2 O (that induces microtubule polymerization) and 6-DMAP (a protein kinase inhibitor) caused activation of unfertilized eggs of the newt, Cynops pyrrhogaster. The cell cycle, arrested at meiotic metaphase II, was resumed to form the second polar body accompanied by a loss of maturation promoting factor and cytostatic factor activity. The treated eggs underwent abnormal cleavage. These results indicate that protein synthesis followed by protein phosphorylation is necessary to maintain M phase in unfertilized Cynops eggs. Unfertilized eggs failed to be activated by pricking, but were activated by the ionophore A23187, but only at a concentration 30 times higher than that required to activate Xenopus eggs. Eggs whose intracellular Ca2+ ions had been chelated by BAPTA could also be activated by either cycloheximide or D2 O. Cycloheximide- as well as 6-DMAP-induced egg activations were not inhibited by nocodazole, a microtubule-depolymerizing agent. These results suggest that the inhibition of synthesis and phosphorylation of short-lived proteins acts as an egg activation mechanism, downstream of the site of Ca2+ action and independently of microtubule polymerization.

Taxol stabilizes [Ca 2+] i and protects hippocampal neurons against excitotoxicity

Elevation of intracellular calcium levels [Ca 2+] i induces microtubule depolymerization, a process which plays roles in regulation of cell motility and axonal transport. However, excessive Ca 2+ influx, as occurs in neurons subjected to excitotoxic conditions, can kill neurons. We now provide evidence that the polymerization state of microtubules influences neuronal [Ca 2+] i homeostasis and vulnerability to excitotoxicity. The microtubule-stabilizing agent taxol significantly attenuated glutamate neurotoxicity in cultured rat hippocampal neurons. Experiments in which [Ca 2+] i was monitored using the Ca 2+ indicator dye fura-2 showed that the elevation of [Ca 2+] i induced by glutamate was significantly attenuated in neurons pretreated with taxol. Experiments using selective glutamate receptor agonists suggested that taxol suppressed Ca 2+ influx through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, but not through N-methyl-D-aspartate (NMDA) receptors. Taxol attenuated the neurotoxicity of the microtubule-depolymerizing agent colchicine; colchicine neurotoxicity was, in part, dependent on Ca 2+ influx. These findings suggest that microtobules play a role in the mechanism of excitotoxicity and suggest that taxol and related compounds may be useful as antiexcitotoxic agents.

Inhibition of infection of macrophages with Ehrlichia risticii by cytochalasins, monodansylcadaverine, and taxol.

The requirement of functions of clathrin, microfilaments, and microtubules in binding, internalization, proliferation, and spreading of Ehrlichia risticii in macrophages was studied. Monodansylcadaverine (MDC), which inhibits clustering and internalization of the ligand-receptor complexes into clathrin-coated vesicles; cytochalasin B or D, which depolymerizes microfilaments; and taxol, which binds and stabilizes polymerized microtubules, were found to prevent ehrlichial infection in murine peritoneal macrophages when they were present throughout the infection period. [35S]methionine-labeled ehrlichial binding to the macrophage was reduced by 0 to 22%; therefore, the binding was not the major point of inhibition. However, MDC, cytochalasin D, and taxol inhibited ehrlichial internalization into macrophages by 80, 58, and 32%, respectively. When MDC, cytochalasin B or D, or taxol was added immediately after internalization of E. risticii (3 h postinfection), ehrlichial replication in P388D1 cells was almost completely prevented. Also, all of these agents almost completely prevented ehrlichial spreading from P388D1 cells to THP-1 human monocytes. These agents were not found to be ehrlichiacidal when approximately 40%-infected P388D1 cells were treated for 2 days, although further intracellular proliferation was prevented. Furthermore, none of these compounds directly inhibited the metabolic activity of E. risticii, since 14CO2 production from L-[14C]glutamine by Percoll density gradient-purified host-cell-free E. risticii was not shown to be impaired. The action of taxol was probably due to impairment of the microtubule function since a microtubule-depolymerizing agent, colchicine, also inhibited intracellular proliferation of E. risticii. Neither reactive oxygen intermediates, nitric oxide, nor tumor necrosis factor alpha appeared to be involved in taxol-induced inhibition of E. risticii in macrophages. Thus, our findings indicate that ehrlichial internalization appears to take place by a mechanism that is more dependent on the functions of clathrin and less dependent on the functions of microfilaments or microtubules. Replication within macrophages and intercellular spreading appear to require clathrin, microfilaments, and microtubules. Consequently, alteration of these structures with inhibitors can result in complete prevention of infection.

Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments.

Pathogenic strains of Helicobacter pylori cause progressive vacuolation and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, transferrin, and transferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by affinity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findings indicate that the vacuoles specifically originate from late endosomal compartments.