Microtubule Reformation Research Articles

Acetyl-CoA carboxylase inhibition regulates microtubule dynamics and intracellular transport in cystic fibrosis epithelial cells.

The use of high-dose ibuprofen as an anti-inflammatory therapy in cystic fibrosis (CF) has been shown to be an effective intervention although use is limited due to potential adverse events. Identifying the mechanism of ibuprofen efficacy would aid in the development of new therapies that avoid these adverse events. Previous findings demonstrated that ibuprofen treatment restores the regulation of microtubule dynamics in CF epithelial cells through a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent mechanism. The goal of this study is to define the AMPK pathway that leads to microtubule regulation. Here, it is identified that inhibition of acetyl-CoA carboxylase (ACC) is the key step in mediating the AMPK effect. ACC inhibition with 5-(tetradecyloxy)-2-furoic acid (TOFA) increases microtubule reformation rates in cultured and primary CF epithelial cells to wild-type (WT) rates. TOFA treatment also restores microtubule-dependent distribution of cholesterol and Rab7-positive organelles, as well as reduces expression of the proinflammatory signaling molecule RhoA to WT levels. ACC activation with citrate replicates these CF phenotypes in WT cells further supporting the role of AMPK signaling through ACC as a key mediator in CF cell signaling. It is concluded that ACC inhibition is the key step in the efficacy of AMPK activation at the cellular level and could represent a novel site of therapeutic intervention to address inflammation in CF.

Ibuprofen regulation of microtubule dynamics in cystic fibrosis epithelial cells

High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared with respective wild-type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule-dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound C. We conclude that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.

The apoptotic microtubule network preserves plasma membrane integrity during the execution phase of apoptosis

It has recently been shown that the microtubule cytoskeleton is reformed during the execution phase of apoptosis. We demonstrate that this microtubule reformation occurs in many cell types and under different apoptotic stimuli. We confirm that the apoptotic microtubule network possesses a novel organization, whose nucleation appears independent of conventional gamma-tubulin ring complex containing structures. Our analysis suggests that microtubules are closely associated with the plasma membrane, forming a cortical ring or cellular "cocoon". Concomitantly other components of the cytoskeleton, such as actin and cytokeratins disassemble. We found that colchicine-mediated disruption of apoptotic microtubule network results in enhanced plasma membrane permeability and secondary necrosis, suggesting that the reformation of a microtubule cytoskeleton plays an important role in preserving plasma membrane integrity during apoptosis. Significantly, cells induced to enter apoptosis in the presence of the pan-caspase inhibitor z-VAD, nevertheless form microtubule-like structures suggesting that microtubule formation is not dependent on caspase activation. In contrast we found that treatment with EGTA-AM, an intracellular calcium chelator, prevents apoptotic microtubule network formation, suggesting that intracellular calcium may play an essential role in the microtubule reformation. We propose that apoptotic microtubule network is required to maintain plasma membrane integrity during the execution phase of apoptosis.

Microtubule assembly in cultured myoblasts and myotubes following nocodazole induced microtubule depolymerisation.

When myoblasts fuse into myotubes, the organisation of the cytoskeleton changes dramatically. For example, microtubules emanate in a radial array form the centrosome in myoblasts, but form linear arrays not linked to a centrosome in myotubes. It is not clear how these linear arrays are formed and nucleated. They could arise in a number of ways: by nucleation and release from centrosomal like structures, cytoplasmic assembly, breakage/severing or nucleation from non-centrosomal sites. To test which of the above mechanisms or combination of mechanisms are responsible we investigated the re-formation of microtubules after depolymerisation by nocodazole, using antibodies against pericentrin, gamma-tubulin, EB1, and tyrosinated alpha-tubulin. In myoblasts, we found that when microtubules were allowed to recover after complete depolymerisation with nocodazole, microtubule recovery began within 1 min and was complete after 5 min. Microtubules grew out from the centrosome, which was positively stained for gamma-tubulin or pericentrin. In untreated myotubes, microtubules were arranged in linear arrays, with EB1 at their ends. The pericentriolar protein, pericentrin was arranged in a band around the nucleus as well as discrete spots in the cytoplasm. In contrast, the microtubule nucleating protein gamma-tubulin was not found in a band around the nucleus, but was found in several punctuate spots throughout the cytoplasm. Further, when microtubules were allowed to recover, after complete depolymerisation with nocodazole, recovery was not as rapid as that seen in myoblasts, and we found that regrowth began with the formation of short microtubule fragments throughout the cytoplasm. Gamma-tubulin was associated with these fragments. These results suggest that in myotubes, nucleation of microtubules can be non-centrosomal.

Evidence for a direct conversion between two tubulin polymers--microtubules and helical filaments--in the foraminiferan, Allogromia laticollaris.

In Allogromia, tubulin lattices transform between microtubule and helical filament states. Helical filaments are composed of approximately 10-nm-thick tubulin filaments wound into approximately 30-nm diameter coils. The transition pathway between these two lattice states was examined in vitro in detergent-lysed pseudopods. Microtubules represented the majority of the assembled tubulin polymers in the detergent extracted pseudopodia. However, microtubules transformed into helical filaments upon exposure to 10 mM Ca2+ or 50 mM Mg2+. The conversion of microtubules into helical filaments involved fragmentation of the tubulin lattice and reduction in total polymer length. Divalent cations were required for the maintenance of the helical filament state; their removal resulted in the loss of helical filaments and the re-formation of microtubules. The data support a direct transition model in which the tubulin lattice interconverts between the helical filament and microtubule states, independently of tubulin subunit concentration. We propose a structural model for the direct pathway whereby disruption of longitudinal bonds between tandem tubulin dimers in protofilaments causes the microtubule lattice to unwind into helical filaments--continuous ribbons of laterally connected tubulin dimers--from the microtubule end. Helical filaments may rewind into microtubules as longitudinal intersubunit bonds reform.

Exogenous histone H1 injection into mitotic cells disrupts synchronous progression of mitotic events by delaying chromosome decondensation

ABSTRACT At the end of open mitosis, chromosome decondensation, nuclear envelope re-formation and reassembly of interphase microtubules following mitotic spindle dissociation occur coordinately. To determine whether these events progress only synchronously in vivo, we delayed chromosome decondensation by injecting of exogenous proteins into the mitotic rat kangaroo kidney epithelium (PtK2) cells. When histone H1 purified from calf thymus was injected at prometaphase, chromosome condensation was prolonged for several hours, and sister chromatid separation and cytokinesis did not occur. However, interphase microtubules reassembled and lamin B-positive structures re-formed around the condensed chromosomes. Exactly the same results were obtained on injection of bacterially expressed H1. Kinetic experiments showed that there were two types of lamin B-positive structures. One type (type A) was stained uniformly with anti-lamin B antibodies. The other (type B) showed peripheral lamin B staining; that is, the normal interphase staining pattern, and was found to be competent for nuclear protein transport. As the chromosomes decondensed, the amount of type A decreased and that of type B increased. However, even cells containing highly condensed chromosomes had both type A and type B. From these results, we conclude that the re-formation of microtubules and reassembly of a nuclear transport-competent envelope do not depend on chromosome decondensation.

The role of cytoskeleton in organizing growth cones: a microfilament-associated growth cone component depends upon microtubules for its localization.

We are interested in the relationship between the cytoskeleton and the organization of polarized cell morphology. We show here that the growth cones of hippocampal neurons in culture are specifically stained by a monoclonal antibody called 13H9. In other systems, the antigen recognized by 13H9 is associated with marginal bands of chicken erythrocytes and shows properties of both microtubule-and microfilament-associated proteins (Birgbauer, E., and F. Solomon. 1989 J. Cell Biol. 109:1609-1620). This dual nature is manifest in hippocampal neurons as well. At early stages after plating, the antibody stains the circumferential lamellipodia that mediate initial cell spreading. As processes emerge, 13H9 staining is heavily concentrated in the distal regions of growth cones, particularly in lamellipodial fans. In these cells, the 13H9 staining is complementary to the localization of assembled microtubules. It colocalizes partially, but not entirely, with phalloidin staining of assembled actin. Incubation with nocodazole rapidly induces microtubule depolymerization, which proceeds in the distal-to-proximal direction in the processes. At the same time, a rapid and dramatic redistribution of the 13H9 staining occurs; it delocalizes along the axon shaft, becoming clearly distinct from the phalloidin staining and always remaining distal to the receding front of assembled microtubules. After longer times without assembled microtubules, no staining of 13H9 can be detected. Removal of the nocodazole allows the microtubules to reform, in an ordered proximal-to-distal fashion. The 13H9 immunoreactivity also reappears, but only in the growth cones, not in any intermediate positions along the axon, and only after the reformation of microtubules is complete. The results indicate that the antigen recognized by 13H9 is highly concentrated in growth cones, closely associated with polymerized actin, and that its proper localization depends upon intact microtubules.

Re-formation of microtubules in Closterium ehrenbergii Meneghini after cold-induced depolymerization

Immunofluorescence microscopy was used to examine the re-formation of microtubules (MT), after cold-induced depolymerization, in Closterium ehrenbergii. The C. ehrenbergii cells undergo cell division followed by semicell expansion in the dark period of daily light-dark cycles. Five types of MTs, namely the MT ring, hair-like MTs around the nuclei, spindle MTs, radially arranged MTs and transverse wall MTs, appeared and disappeared sequentially during and following cell division. The wall MTs were distributed transversely only in the expanding new semicells. When cells were chilled in ice water, wall MTs in expanding cells were fragmented, and then disappeared as did the other types of MTs, within 5 min. When cells were warmed at 20°C after 2 h chilling, wall MTs and the other types of MTs re-formed. At the early stage of wall-MT re-formation in expanding cells, small, star-like MTs were formed, and then randomly oriented MTs developed in both the expanding new and the old semicells. The MT ring was also re-formed at the boundary between the new and old semicells. There were no obvious MT-organizing centers in the random arrangement. As time passed, the randomly oriented wall MTs in the old semicells disappeared and those in the expanding new semicells gradually assumed a transverse orientation. These results indicate that wall MTs can be rearranged transversely after they have been re-formed and that nucleation of wall MTs is separable from the mechanism for ordering them.

Augmentation of macrophage antibody-dependent killing of tumor targets by microtubule inhibitors

Antibody-dependent cellular cytotoxicity (ADCC) to tumor targets was studied using murine resident peritoneal macrophages and a macrophage cell line RAW264.10A, both having low inherent cytolytic activity. The target was 125I-labeled pre-B lymphoma 18-8. Pretreatment of both macrophage populations with 0.5 – 2 μ M concentrations of the microtubule-stabilizing drug taxol greatly increased their antibody-dependent cytotoxicity with no stimulation of nonspecific killing. Taxol present only during the 18-hr cytolytic assays had no effect on target killing. Optimal killing activity was obtained by treating macrophages 2 days with taxol, similar to previously described cytokine stimulation of ADCC. This concentration completely blocked growth of RAW264 cells. Other microtubule inhibitors, lidocaine and colchicine, also augmented peritoneal and cell line macrophage ADCC at cytostatic concentrations. In contrast, the microfilament-disrupting agent, cytochalasin B, caused little or no stimulation of ADCC. These results show that microtubule reformation is not necessary for the development of cytotoxicity. Since microtubule inhibitors block lysosomal discharge, they may stimulate macrophage ADCC by causing accumulation of toxic molecules involved in cytotoxicity.

Some effects of colchicine on microtubules and cell division in roots ofAzolla pinnata

Removal and subsequent reformation of microtubules in cells of the root-tips ofAzolla pinnata R. Br. was achieved by short pulse treatments with the drug colchicine. Loss of microtubules led to the formation of multinucleate cells more frequently than to the arrest of mitosis at metaphase, and primary and secondary wall formation was also disrupted. Recovery of root development was limited. Growth of all roots ceased 5–6 days after the pulse treatment. Following the reappearance of microtubules, renewed deposition of normal wall thickenings occurred in developing xylem elements. Multinucleate cells became subdivided by walls in the apparent absence of a phragmoplast. The plane in which the new wall was formed was often located as it would have been in an untreated root, but in a number of cases abnormal or precious positioning of new walls was observed. Clusters of microtubules, matrix material, and vesicles or particles, taken to indicate microtubule initiation, were observed during the recovery from treatment.

Microtubules in protozoan cells. III. Ultrastructural changes during disintegration and reformation of heliozoan microtubules.

In the heliozoan, Echinosphaerium nucleofilum strain MA, cold temperature (2 degrees C) induced axopodial retraction to about 36% of the initial length after 3 h. By electron microscopy, it was found that such axopodial shortening is accompanied by degradation of axonemal microtubules (25 nm in diameter), followed by the appearance of macrotubles (37 nm in diameter) and filamentous structures (14 nm in diameter) of a tubular and twisted appearance. All of these structures (microtubules, macrotubules and filamentous structures) were found to be depolymerized completely by applying 10 mM colchicine for 1-2 h, and to be replaced by regions with low electron density. Axopodial re-extension was induced rapidly by returning the cold-treated organisms to room temperature (20 degrees C). At a very early stage of axopodial re-extension, the filamentous structures were often observed to be continuous with the macrotubules. At a late stage of axopodial re-extension, the reforming axoneme was composed only of normal microtubules, while the macrotubules and filamentous structures had disappeared. On the basis of these results, the processes of disintegration and reformation of microtubules are discussed.

Electron microscopy of mitosis in amebae. 3. Cold and urea treatments: a basis for tests of direct effects of mitotic inhibitors on microtubule formation.

The mitotic apparatus (MA) of the giant ameba, Chaos carolinensis, has characteristic sequences of microtubule arrays and deployment of nuclear envelope fragments. If mitotic organisms are subjected to 2 degrees C for 5 min, the MA microtubules are completely degraded, and the envelope fragments are released from the chromosomes which remain condensed but lose their metaphase-plate orientation. On warming, microtubules reform but show partial loss of their parallel alignment; displacement of the envelope fragments persists or is increased by microtubule reformation. This study demonstrates that cooling causes destruction of microtubules and intermicrotubular cross-bonds and further shows that such controlled dissolution and reformation can provide an in vivo test sequence for studies on the effects of inhibitor-compounds on microtubule subunit aggregation. Urea, at the comparatively low concentration of 0.8 M, inhibited reformation following cooling and rewarming but was ineffective in altering microtubules that had formed before treatment.