Role Of Cytoplasmic Microtubules Research Articles

The Adherence of Polymorphonuclear Leucocytes to an Albumin‐coated Glass Surface

The Catharantus derivatives are microtubule antagonists employed in immunosuppression and chemotherapy of neoplasms. The role of cytoplasmic microtubules in polymorphonuclear leucocyte (PMN) adherence was studied by means of therapeutic concentrations of the Catharantus derivatives vincristine, vinblastine and vindesine. PMN adherence was measured as retention on an albumin-coated glass surface. PMN adherence was reduced by 4-54% by the Catharantus derivatives, as compared with control values. The suppression of adherence was statistically significant. Since the Catharantus derivatives are microtubule antagonists, it is reasonable to assume that PMN adherence is a partially microtubule-dependent process. It is suggested that reduction of PMN adherence could account for at least part of the immunosuppressive properties of the Catharantus derivatives.

The phagocytosis of yeast cells by blood monocytes. Effects of therapeutic concentrations of Vinca alkaloids.

The role of cytoplasmic microtubules in the phagocytosis of yeast cells by blood monocytes was studied by means of therapeutic concentrations of the Vinca alkaloids vincristine, vinblastine, and vindesine. Phagocytosis was measured in a monolayer of glass-adherent monocytes fed with fluorescein-labelled yeast cells. Phagocytosis was composed of two sequential processes, yeast cell adherence to the monocyte and yeast cell engulfment by the monocyte. Monocyte phagocytosis was significantly inhibited by the Vinca alkaloids, mainly due to inhibition of engulfment but probably also due to inhibition of adherence. Since the Vinca alkaloids are microtubule antagonists it is reasonable to assume that monocyte phagocytosis, both adherence and engulfment, are partially microtubule-dependent processes. It is suggested that Vinca alkaloid inhibition of monocyte phagocytosis is of value in the treatment of type II-III autoimmune disorders, such as idiopathic thrombocytopenic purpura and autoimmune haemolytic anaemia.

The role of cytoplasmic microtubules in the regulation of the activity of peptide growth factors

The enhancement of DNA stimulation by peptide growth factors in colchicine-treated cultures of Swiss 3T3 mouse fibroblasts appears to be associated with disassembly of cytoplasmic microtubules. Other antitubulin agents (colcemid, vinblastine, podophyllotoxin, nocodazole, and maytansine) also potentiate the effects of growth promoting factors such as insulin and EGF. The cytoplasmic microtubules are disrupted by antitubulin agents at concentrations very similar to those required for depolymerization of mitotic spindle microtubules. In the presence of highly radioactive [ 3H]thymidine, DNA synthesis can be stimulated in quiescent mouse fibroblasts without completion of the cell cycle. This makes possible studies of the potentiating effects of colchicine during the prereplicative period of the cycle uncomplicated by effects of the antitubulin agent on the mitotic spindle. The enhancing effects of colchicine can be demonstrated with sparse cultures of 3T3 fibroblasts as well as with 3T6 cells. Maytansine and colchicine need not be present during the initial period of interaction of peptides with the cell to produce enhancement of DNA synthesis. The enhancement can be obtained after transient exposure of cells to fetal bovine serum. The potentiating effects of antitubulin agents such as nocodazole, podophyllotoxin, and maytansine can be easily reversed whereas the enhancement with colchicine persists despite washing of cells. The stimulatory effects of insulin and EGF persist in cultures treated with antitubulin agents. The findings support the premise that after internalization the receptor-mitogen complex may remain active within endocytic vesicles when cells are exposed to colchicine.

Studies on the granulocyte plasma membrane oxidase

The plasma membrane of granulocytes contains an oxidase which catalyses reduction of oxygen to superoxide. With phagocytosis, this oxidase is incorporated into the phagocytic vacuole membrane, facilitating the delivery of cytotoxic superoxide to the ingested micro-organism. We have examined the role of cytoplasmic microtubules (MT) and microfilaments (MF) in the control of the activity of this plasma membrane enzyme. These studies were prompted by reports showing that MT and MF control many plasma membrane phenomena, including the movement of proteins within the plane of the membrane and the topographical organization of the plasma membrane during phagocytosis. These experiments were designed to record changes in the initial rate of oxidase activity after treatment of intact granulocytes with drugs known to disrupt MF (cytochalasin B) or MT (colchicine, vinblastine, vincristine). Control experiments showed that MT and MF were indeed disrupted by these drug treatments. MF disruption produced an enhancement in oxidase activity whereas MT disruption resulted in inhibition except when very low doses of, or brief exposures to the agents were used, when a paradoxical stimulation was seen. Correlative electron microscopic studies showed the sites of oxidase activity to the homogeneously distributed on the surface of untreated, but not drug-treated granulocytes. It was concluded that MF may provide a constraint against plasma membrane oxidase activity. Conversely, MT presumably by controlling the topographical organization of the membrane, seem to be required for plasma membrane oxidase activity. These findings may have some relevance in vivo in that inhibition of the crucial granulocyte plasma membrane oxidase activity may partially explain the increased risk of infection in cancer patients treated with the vinca alkaloids.

Stimulation of Steroid Synthesis by Normal Rat Adrenocortical Cells in Response to Antimicrotubular Agents

The role of cytoplasmic microtubules in the process of steroidogenesis was examined in enzyme-dispersed and mechanically dispersed adrenocortical cells obtained from adult male rats. These cells were maintained in culture for periods of 2 h to 2 days and tested with various antimicrotubular drugs as well as ACTH and N6,O2-dibutyryl adenosine 3',5'-monophosphoric acid [(Bu)2cAMP]. The drugs tested, viz. colchicine, vinblastine, and nocodazole, were found to stimulate steroid synthesis in a dose-dependent fashion; the maximal degree of stimulation was found to be the same as that obtained with either ACTH or (Bu)2cAMP. When a maximal concentration of an antimicrotubular drug was mixed with a maximal concentration of either (Bu)2cAMP or ACTH, no additive effect on steroidogenesis was observed. The mixture of maximal concentrations of two antimicrotubular drugs produced a steroidogenic response that was less than when either drug was used alone. However, when submaximal concentrations of two antimicrotubular drugs were mixed, a synergistic response was noted. These antimicrotubular drugs are known to interfere with microtubular function by binding to specific sites on the tubulin protomer and inhibiting normal polymerization. Lumicolchicine, the photoinactivated form of colchicine, does not bind to tubulin and did not stimulate steroid synthesis. These studies support the notion that microtubules can influence the process of steroidogenesis.

Effect of colchicine on urinary phosphate and regulation by parathyroid hormone

The possible role of cytoplasmic microtubules in the renal handling of phosphate and its regulation by parathyroid hormone (PTH) was evaluated with colchicine, a microtubule-disrupting agent. Colchicine-treated rats were thyroparathyroidectomized (TPTX) and subsequently infused with PTH. Treatment with a total dose of 1 mg colchicine had no effect on glomerular filtration rate or fractional excretions of sodium and potassium. Fractional excretion of phosphate in colchicine-treated TPTX rats was significantly higher compared with TPTX controls. After PTH infusion, control rats responded with increases in fractional excretion of phosphate and urinary cyclic AMP but colchicine-treated rats had variable and insignificant changes in both parameters. Fractional excretion of sodium and potassium did not change significantly after PTH. Renal cortical activities of cyclic AMP phosphodiesterase, soluble alkaline phosphatase, cytochrome oxidase, leucine aminopeptidase, or basal adenylate cyclase were not significantly affected by colchicine treatment. On the other hand, stimulation of adenylate cyclase by a submaximal dose of PTH was markedly decreased in colchicine-treated rats, and the activity of membrane-bound alkaline phosphatase was also significantly decreased. The binding of radioactive colchicine in renal cortical extracts from rats treated with colchicine was significantly diminished. These results suggest that disruption of cytoplasmic microtubules in renal cortical cells interferes with phosphate transport and its regulation by PTH.

Neutrophil Segmentation and Radial Segmentation

The oxalate‐induced nuclear hypersegmentation in neutrophils may be recorded by counting the segments. In heparinized blood, the number of segments remains fairly constant during incubation at 37° C. for at least 6 hours. Temperature depression and demecolcine decrease the number of segments recorded. Under identical experimental conditions, the formation of radial‐segmented (RS) nuclei in mononucleated blood cells is promoted by incubation at room temperature and inhibited by treatment with demecolcine. Cold (+4° C.) and demecolcine at room temperature induce segmental clumping in the nuclei of neutrophils. The possible role of cytoplasmic microtubules in neutrophil segmentation is discussed.

The fine structure of the embryonic mouse neural tube with special reference to cytoplasmic microtubules

The fine structure of undifferentiated interphase and mitotic cells of the 10-day mouse embryo neural tube has been described. Cytoplasmic microtubules (270 Å) were seen in the interphase cells, often connected to the Golgi vesicles near the nucleus and ending in an apical vesicle or parabasal body adjacent to the central canal. Similar microtubules forming spindle fibers and connected with chromosomes were prominent in mitotic cells. The possible role of cytoplasmic microtubules in maintenance of cell shape, nuclear migration, and cerebrospinal fluid secretion is discussed.