Possible Role Of Microtubules Research Articles

Prediction of Tubulin Resonant Frequencies Using the Resonant Recognition Model (RRM).

Tubulin proteins were analyzed using the Resonant Recognition Model to predict possible electromagnetic resonances in tubulin and microtubules. We propose that these electromagnetic resonances are caused by charge transfer through the protein molecule. The frequencies of these electromagnetic resonances depend on charge velocity. Using different velocities of charge transfer, we predicted resonant frequencies in different frequency ranges from KHz to THz. These resonant frequencies could be relevant for taxol binding as well as a possible role of microtubules as a macromolecular computer.

Microtubular disrupter nocodazole and vesicular transport inhibitor brefeldin A attenuate the glutoxim effect on Na+ transport in frog skin

Using the voltage-clamp technique, a possible role of microtubules and vesicular transport in the effect of pharmacological analogue of oxidized glutathione, drug glutoxim, on Na+ transport in the frog Rana temporaria skin was investigated. It was shown for the first time that the disrupter of microtubules nocodazole or inhibitor of vesicular transport brefeldin A similarly modulate (completely inhibit) the stimulatory effect of glutoxim on Na+ transport. The data suggest the involvement of reorganization of microtubules and vesicular transport in the regulatory effect of glutoxim on Na+ transport. .

Disappearance of Microtubules, Nuclei and Starch During Cell Death of Ray Parenchyma in Abies Sachalinensis

Morphological changes and the timing of disappearance of individual organelles provide key information for understanding the mechanism of cell death. The disappearance of microtubules, nuclei and starch grains was monitored during the death of long-lived ray parenchyma cells in the conifer Abies sachalinensis. From the eighth to the tenth annual ring from the cambium, morphological changes occurred in ray parenchyma cells and organelles disappeared. Morphological changes in nuclei became apparent first. Then microtubules disappeared and, finally, nuclei disappeared. Therefore, microtubules might play an important role during the death of ray parenchyma cells. The timing of the disappearance of starch grains differed among individual ray parenchyma cells. This result indicates that the timing of the loss of storage function in ray parenchyma cells might not depend on the progress of cell death. The possible role of microtubules during cell death of long-lived ray parenchyma cells that might differ from cell death of short-lived ray tracheids is discussed.

Research note: Deposition patterns of cellulose microfibrils in flange wall ingrowths of transfer cells indicate clear parallels with those of secondary wall thickenings.

The arrangement of cellulose microfibrils and cortical microtubules in transfer cells depositing flange wall ingrowths have been determined with field emission scanning electron microscopy and immunofluorescence confocal microscopy. In xylem transfer cells of wheat (Triticum aestivum) stem nodes and transfer cells of corn (Zea mays) endosperm tissue, cellulose microfibrils were aligned in parallel bundles to form the linear wall ingrowths characteristic of flange ingrowth morphology. In both cell types, linear bundles of cellulose microfibrils were deposited over an underlying wall composed of randomly arranged microfibrils. Acid extraction of wheat xylem transfer cells established that flange ingrowths were composed of crystalline cellulose. Immunofluorescence labelling of microtubules in wheat xylem transfer cells showed that bundles of microtubules were positioned directly below and parallel with developing flange ingrowths, whereas more mature ingrowths were flanked by bundles of microtubules. These results show that the parallel organisation of cellulose microfibrils in flange wall ingrowths is similar to those in secondary wall thickenings in xylem elements, and that deposition of these structures in transfer cells is also likely to involve bundling of parallel arrays of microtubules. Our observations are discussed in terms of the possible role of microtubules in building flange-type wall ingrowths and the consequences in terms of predicted mechanisms required to build the fundamentally different reticulate-type wall ingrowths.

Position and cell type‐dependent microtubule reorientation characterizes the early response of the Arabidopsis root epidermis to ethylene

The involvement of cortical microtubules in the control of plant cell expansion was studied in the Arabidopsis root epidermis. In the zone of fast elongation microtubules were transverse to the root axis in all epidermal cells. However when cells entered the differentiation zone cell type‐specific microtubule reorientation took place. In the trichoblasts that were then approximately 130 µm long and formed the root hair bulge, the microtubules switched to a random distribution. In the adjoining atrichoblasts microtubules adopted a slightly oblique orientation. In more proximal parts of the differentiation zone atrichoblast microtubules were found in a more oblique and finally in a longitudinal orientation. Upon exposure to ethylene or 1‐aminocyclopropane‐1‐carboxylic acid (ACC – the precursor of ethylene) at a saturating dose, cell elongation abruptly stopped. From then on trichoblast cells reached only a length of about 35 µm, and developed root hairs. Cortical microtubules changed orientation within 10 min. In trichoblasts they adopted the typical random orientation, in atrichoblasts however, they took up a longitudinal orientation. Microtubule reorientation was complete within 60 min. The possible role of microtubules in the control of cell elongation is discussed.

Hypertonic activation of the renal betaine/GABA transporter is microtubule dependent

Epithelial cells in the renal inner medulla accumulate osmolytes such as betaine to maintain normal cell volume during prolonged extracellular hypertonic stress. Betaine accumulation is the result of activation of transcription of the BGT1 transporter gene followed by increased betaine transport. We studied the possible role of microtubules in this adaptive mechanism using renal cells in culture. RESULTS.: In cultured renal cell lines [Madin-Darby canine kidney (MDCK) and mouse inner medullary collecting duct (mIMCD-3)], up-regulation of BGT1 activity was maximal after 24 to 30 hours in growth medium made hypertonic (510 mOsm/kg) by the addition of sucrose or NaCl. Up-regulation was reversed within 24 to 36 hours after returning cells to isotonic medium. Both cycloheximide (20 micromol/L) and nocodazole (20 micromol/L) blocked the hypertonic up-regulation of BGT1. Nocodazole was partially effective even when added 16 to 20 hours after the switch to hypertonic medium. Recovery from nocodazole action was rapid, and there was full activation of BGT1 transport within three to six hours after nocodazole removal, suggesting rapid trafficking to the cell surface once microtubules repolymerized. Hypertonic activation of BGT1 transport was detected in an isolated membrane fraction and was blocked by cycloheximide but not by nocodazole. Confocal microscopy confirmed the increased abundance of BGT1 proteins in the plasma membrane of hypertonic cells and showed that BGT1 remained intracellular during nocodazole treatment. Hypertonic activation of BGT1 in renal cells requires de novo protein synthesis and microtubule-dependent trafficking of additional transporters to the cell surface. The apparent resistance of membrane BGT1 to nocodazole blockade is likely due to the presence in the membrane fraction of an increased intracellular pool of active BGT1 transporters.

Tubulin expression in the developing and adult gerbil organ of Corti

In the late stages of inner ear development, the relatively undifferentiated cells of Kollicker’s organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to β-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.

Stacking of Golgi cisternae in Schizosaccharomyces pombe requires intact microtubules

Fission yeast was treated with the anti-microtubule agent, thiabendazole. Cytoplasmic microtubules broke down with a half-time of less than 10 minutes followed closely by the unstacking of Golgi cisternae. The final product appeared to be single Golgi cisternae. No other organelle seemed to be affected by this treatment, which was completely reversible. The nda3 mutant strain has an altered beta-tubulin and its cytoplasmic microtubules are resistant to thiabendazole. The Golgi in this cold-sensitive mutant was unaffected by treatment at the permissive temperature but unstacked at the non-permissive temperature even in the absence of thiabendazole. Taken together these data show that disruption of the microtubular network can cause dissociation of Golgi cisternae. Newly synthesised acid phosphatase was transported and secreted to the same extent and with the same kinetics whether or not the Golgi was unstacked. The possible role of microtubules in Golgi stacking and the lack of effect on secretion are discussed.

Root contraction in hyacinth. II. Changes in tubulin levels, microtubule number and orientation associated with differential cell expansion

Root contraction in hyacinth (Hyacinthus orientalis L.) is marked by reoriented cell growth in the cortex of the contractile region. Cellular volume of the inner cortex enlarges fourfold during root contraction. This is associated with large increases in the radial and tangential dimensions and decreases in the longitudinal dimension of the cells. In order to determine the possible role of microtubules (MTs) in these changes we compared tubulin levels and MT numbers and orientation in contracted and non-contracted regions of hyacinth roots. Tubulin content was analysed by a radioimmunoassay; MT numbers and orientation were analyzed by counting profiles in sectioned material using transmission electron microscopy. Contracted tissue was found to have significantly higher levels of tubulin on a per-cell basis than non-contracted tissue, and also increased tubulin levels relative to total protein. The spatial MT frequencies were the same in contracted and non-contracted tissues, indicating a proportional increase in MT numbers in the expanded cells. Although the absolute spatial frequency of MTs was constant, the orientation, as determined by morphometric analysis of MT profiles, was not. While in the longitudinal section plane 42% of the MTs in the non-contracted cells were oblique, in the contracted cells the percentage of MTs presenting oblique profiles increased to 87%. Additionally, a qualitative difference in MTs was observed in contracted cells; electron-opaque material was seen peripherally associated with the MTs of the inner cortex. The changes in tubulin levels and in MT numbers as well as the qualitative differences in the MTs of contracted and non-contracted root regions indicate that, in hyacinth, reoriented cellular enlargement associated with root contraction cannot be explained simply by shifts in the arrangement of preexisting cortical MT arrays, but involves more complex changes in the cytoskeleton.

Effect of colchicine on the ultrastructure of secretory-state smooth muscle cells from the rabbit artery wall

Little is known about the possible role of microtubules and related structures with respect to the structural and functional heterogeneity of arterial smooth muscle cells (SMC). This study demonstrates that a short-term treatment with an antitubulin, colchicine, resulted in two major structural changes in arterial SMC in the aortic arch, the thoracic aorta and the pulmonary trunk of 20-d-old rabbits. The SMC of control rabbits showed well-developed rough endoplasmic reticulum and Golgi complexes, and numerous microtubules. Colchicine treatment affected (a) microtubules in a monotypic way (all SMC contained no microtubules after colchicine), and (b) Golgi complexes and rough endoplasmic reticulum in a pleiotypic way. In effect two major structural subpopulations of SMC were obtained; the first subpopulation displayed markedly altered Golgi complexes, whereas the rough endoplasmic reticulum was unaltered; the second subpopulation showed a vacuolar dilation of rough endoplasmic reticulum (or extensively-developed smooth sarcoplasmic reticulum), but a disappearance of Golgi complex. These data suggest that an organelle-specific sensitivity to colchicine (most likely to its antitubulin action) may contribute to the heterogeneity of SMC. They also suggest that within control SMC two subpopulations might exist which can only be detected under special conditions like colchicine treatment. The disassembly of microtubules detected in parallel may be one trigger, but not the main mechanism in the heterogeneous changes of SMC in reaction to colchicine.

Growth polarity and cytokinesis in fission yeast: the role of the cytoskeleton.

The distribution of F-actin in the fission yeast Schizosaccharomyces pombe was investigated by fluorescence microscopy using rhodamine-conjugated phalloidin. Fluorescence was seen either at the ends of the cell or at the cell equator. End staining was predominantly in the form of dots whilst equatorial actin was resolved as a filamentous band. The different staining patterns showed a close correlation with the known pattern of cell wall deposition through the cell cycle. In small, newly divided cells actin was localized at the single growing cell end whilst initiation of bipolar cell growth was coincident with the appearance of actin at both ends of the cell. As cells ceased to grow and entered cell division, a ring of actin was seen to anticipate the deposition of the septum at cytokinesis. The relationship between actin and cell wall deposition was further confirmed in three temperature-sensitive cell division cycle (cdc) mutants; cdc10, cdc11 and cdc13. Immunofluorescence microscopy of S. pombe with an anti-tubulin antibody revealed a system of cytoplasmic microtubules extending between the cell ends. The function of these was investigated in the cold-sensitive, benomyl-resistant mutant ben4. In cold-grown cells actin was seen to form conspicuous filamentous rings around the nucleus. The origin of these and the possible role of microtubules in the cell-cycle-dependent rearrangements of F-actin are discussed.

Possible role of microtubules in tumor cell surface membrane shedding, permeability, and lympholysis

Low temperature (0 °C/1 hr) induced changes in the mastocytoma P815 cell surface morphology which mimicked those induced by alloimmune T-lymphocytes during the cytolytic process. These morphological changes consist of the formation and shedding of membrane vesicles (MV) from the P815 cell surface. When the low-temperature-induced MV shedding process takes place at 22 °C, it occurs without changes in membrane permeability to 51Cr, whereas at 37 °C, the kinetics of 51Cr release resemble those induced by alloimmune T lymphocytes. The effects of two microtubule-stabilizing agents, deuterium oxide and hexylene glycol, were investigated on the low-temperature-induced MV shedding process as well as on the lymphocyte-mediated cytolysis (LMC) of target cells. These agents were found to inhibit (a) the low-temperature-induced MV shedding, (b) the low-temperature-induced membrane permeability changes to 51Cr, and (c) the LMC process. These data indicate that disruption of microtubules may be involved in all these events.

Parallel arrays of microtubules formed in electric and magnetic fields.

The influence of electric and magnetic fields on microtubule assembly in vitro was studied. Both types of field caused alignment of microtubules in parallel arrays, as demonstrated by electron micrographs. These findings suggest a possible role of microtubules in the biological effects of exogenous as well as endogenous fields.

Possible role of microtubules and associated proteases in organophosphorus ester-induced delayed neurotoxicity

Organophosphorus delayed neurotoxicants (phenyl saligenin cyclic phosphate and diisopropyl phosphorofluoridate) altered cyclic AMP (cAMP)-dependent phosphorylation and several other processes in brain homogenates and cytoplasmic microtubules. Phenyl saligenin cyclic phosphate slightly stimulated in vitro cAMP-dependent phosphorylation in brain homogenates of three species (rat, mouse and rabbit) that have been reported to be insensitive to delayed neurotoxicity, whereas it slightly decreased this phosphorylation in brain homogenates of three sensitive species (chicken, cow and pig) and in brain microtubules of chicken and pig. The microtubule-associated processes that were moderately inhibited by phenyl saligenin cyclic phosphate in sensitive species were: in vitro [ 3H]cAMP binding to protein kinase, in vitro assembly when tubulin rings were absent, and cAMP-dependent phosphorylation of microtubule-associated proteins (MAPs) both in vitro and on intracerebral administration of 32P i. The endogenous proteases that degrade the high molecular weight MAPs were strongly inhibited in vitro by phenyl saligenin cyclic phosphate and diisopropyl phosphorofluoridate. In contrast, treatment of chickens with diisopropyl phosphorofluoridate remarkably decreased the in vitro stability of their brain cytoplasmic high molecular weight MAPs, perhaps by enhancing the MAPs-degrading protease activity. These findings indicate that the MAPs-protease system is a possible target for organophosphorus delayed neurotoxicants.

Autoradiographic studies of protein and polysaccharide synthesis during vitellogenesis in Drosophila

Quantitative light- and electron-microscopic autoradiography was used to evaluate metabolic processes that occur during late developmental stages (10-14) of oogenesis in Drosophila melanogaster. Major differences in radiolabelling patterns were found after in vivo (10-45 min) uptake of [3H]-monosaccharides and [3H]-L-lysine. Several different methods of data analysis were required to facilitate interpretation of these patterns. [3H]-L-lysine produced extensive cytoplasmic labelling at all developmental stages. In addition, about 15% of alpha yolk spheres were intensely labelled at stage 10, reflecting the incorporation of radiolabelled vitellogenins synthesized during the incubation period. Subsequent stages showed low silver grain density over alpha yolk spheres until stage 14, when a burst of [3H]-L-lysine incorporation by most alpha spheres was observed, possibly indicative of a maturation process for embryogenesis. [3H]-D-glucose and [3H]-D-galactose (10 min, in vivo) both induced intense labelling of the beta yolk spheres in a manner suggesting in situ assembly beginning at early stage 13. Inasmuch as the polysaccharide of beta yolk spheres has the properties of glycogen (e.g., rosette structure digested by alpha-amylase) and the radiolabelled monosaccharides were introduced intra-abdominally, it is evident that transport systems as well as enzymes utilizing glucose and galactose for glycogenesis must be readily available. It is notable that wide-spread labelling of egg chambers was elicited by [3H]-D-glucose and [3H]-D-galactose (e.g., nurse cells, follicle cells, chorion, vitelline membrane), but the labelling induced by [3H]-N-acetylmannosamine was restricted mainly to the endochorion. A possible role of microtubules in distribution and assembly of yolk spheres was inferred when colchicine, admixed to the culture medium (2-5 ppm), produced abnormal distribution and diminution in number of both alpha and beta yolk spheres. In addition to revealing previously unknown metabolic events of vitellogenesis, the results provide additional criteria for stage characterization as well as a means to specifically label certain macromolecules for purposes of isolation.

Effect of methylmercury and inorganic mercury on the nerve growth factor-induced neurite outgrowth in chick embryonic sensory ganglia

Methylmercury inhibited the fiber outgrowth in chick embryonic sensory ganglia in the presence of nerve growth factor (NGF) by 50% at 2.10 −6 M and completely depressed it at 5.10 −6 M. Whereas inorganic mercury completely inhibited the fiber outgrowth at 10 −4 M. The possible role of microtubules is discussed.

Effect of microtubule-disrupting drugs on protein and RNA synthesis in Physarum polycephalum amoebae.

The effects of the microtubule-disrupting drugs, colchicine, vinblastine, podophyllotoxin, griseofulvin, and lumicolchicine (10(-5) M), on protein and RNA synthesis were studied in Physarum polycephalum amoebae in culture. All, except lumicolchicine, were found to simultaneously reduce the rate of protein synthesis and stimulate RNA synthesis. These results parallel the effects seen in cells exposed to heat shock. Treatment of cells with a microfilament-disrupting drug, cytochalasin B (10 micrograms/ml in ethanol), resulted in a reduced rate of protein synthesis after 2 h compared to a similar effect by vinblastine in 5--15 min. A morphological abnormality, microtubule paracrystals, were seen associated with centrioles in vinblastine-treated cells in which protein synthesis had been reduced by 50%. Vinblastine and podophyllotoxin were shown to interfere with the recovery of protein synthesis after inhibition by low or elevated temperatures. The possible role of microtubules in regulating the translational response of a cell to an external environmental stimulus is discussed.

Implication of microtubules and microfilaments in the cyclic GMP response of rat exocrine pancreas to secret agogues

Microtubules and microfilaments have been shown to be involved in the secretion processes of many glands: thyroid gland [ 11, pancreatic islets [2], adrenal medulla [3], anterior pituitary [4], parotid gland [ 51 and exocrine pancreas [6,7]. Most of these studies used drugs known to alter the structure of these cellular organelles. On the other hand, microtubules as well as microfilaments have been implicated in the control of the redistribution and motility of membrane surface components [8-l 21. Furthermore, recent studies suggested that microtubules and/or microfilaments are probably involved in the process of adenylate cyclase stimulation by hormones [ 13,141. In pancreatic acinar cells the initial step in the sequence of events mediating the action of secretagogues is an increase in intracellular guanosine 3’:5’ cyclic monophosphate (cyclic GMP) [ 15 ,161. Further, microtubules and microfilaments were shown to be widely distributed in these cells [7]. Thus the present study was undertaken in order to investigate the possible role of microtubules and microfilaments in the secretagogue-induced cyclic GMP accumulation by rat exocrine pancreas. We report that both vinblastine which causes the disappearance of microtubules [ 7,171, and cytochalasin B which impairs the function of microfilaments [7,18] inhibit the cyclic GMP response of rat exocrine pancreas to peptide hormones (caerulein), cholinergic agents (carbachol) or the calcium ionophore A 23 187.

Effect of colchicine and cytochalasin B on the secretion of proteochondroitin sulfate and protein-free chondroitin sulfate synthesized in cartilage slices.

A possible role of microtubules and microfilaments in the secretion process of proteoglycan and protein-free glycosaminoglycan synthesized in slices of bovine nasal cartilage was examined by using colchicine as an antimicrotubular agent, cytochalasin B as a microfilament-modifying agent, and 4-methylumbelliferyl β-D-xyloside as an artificial initiator of glycosaminoglycan. The present study suggested that the microtubule system was partly involved in the secretion of proteochondroitin sultate and protein-tree chondroitin sultate synthesized in chondrocytes. The microfilament system did not have any direct effect on this secretion process.

Transport of glial smooth endoplasmic reticulum acid phosphatase into damaged axons: possible role of microtubules

Earlier observations indicated that cutting the photoreceptor (R) cell perikarya of the flies Musca domestica and Calliphora erythrocephala in the retina led to clear cut degenerative changes, occurring within minutes, in the R axons in the optic neuropiles. Biochemical assays of the lysosomal marker enzyme acid phosphatase (AcPase) in the optic ganglia at various times following the lesion showed a highly significant increase in activity concomitant with the morphological changes. Cytochemical assay of AcPase activity using the Gomori method indicated that the increase in AcPase activity was exclusively associated with one specific glial cell type - the satellite glia (SG). These cells are contiguous with the R cell axons only in the distal lamina, directly beneath the basement membrane of the retina. Reaction product indicating AcPase activity appeared sequentially in the SG cytoplasm (2-4 mins), extracellularly between glial and axon membranes (3-5 mins) and then in the axon cytoplasm (>4 mins).