Microtubule assembly and conanavalin A capping in lymphocytes: reappraisal using normal and abnormal human peripheral blood cells.

We have analyzed the assembly of microtubules and the distribution of concanavalin A(Con A)-receptor complexes in the same populations of human peripheral blood T and B lymphocytes. We hoped to resolve the prolonged controversy over the relationship of microtubules to Con A cap formation in lymphocytes and to explain the abnormally high spontaneous and colchicine-induced Con A capping that was observed recently in lymphocytes from a patient with an inherited form of severe combined immunodeficiency disease (SCID) characterized by total immunologic dysfunction despite normal numbers and distribution of T and B cells. The data establish that (i) microtubule disassembly is correlated with enhanced Con A cap formation on normal human lymphocytes; (ii) T and B cells differ significantly from each other and from circulating polymorphonuclear leukocytes with respect to their capping responses after exposure to colchicine; and (iii) there is an abnormal relationship of microtubule assembly to surface topography in the functionally defective SCID cells.     

Aberrant capping of membrane proteins on neutrophils from patients with leukocyte adhesion deficiency

Several functional defects have been found in neutrophils from leukocyte adhesion deficiency (LAD) patients who fail to express the CD11/CD18 leukoadhesins: Mo1, LFA-1, and p150,95. To better understand the functional defects of LAD neutrophils, we have performed capping experiments. Purified normal or LAD neutrophils were labeled with fluorochrome-conjugated concanavalin A (Con A) or F(ab')2 fragments of antiurokinase-type plasminogen activator receptor (uPAR), anti-Fc gamma RIII (CD16), anti-Mo5, and anti-CD14 antibodies. F(ab')2-labeled cells were capped using a second-step F(ab')2 fragment of an antimurine Fab antiserum. Cells were capped for 30 minutes at 37 degrees C, then observed by fluorescence microscopy. LAD neutrophils were found to be deficient in capping, but not clustering of all of the reagents tested to date. The percent of cells exhibiting capping of Con A, Fc gamma RIII, urokinase receptor, CD14, and Mo5 were 52%, 67%, 70%, 25%, and 64% for normal neutrophils but were only 10%, 5%, 2%, 3%, and 1%, respectively, for LAD neutrophils. Capping of this panel of membrane components in LAD or normal neutrophils was not augmented by the addition of either 10(-5) mol/L colchicine or 10(-7) mol/L FMLP. Because capping requires membrane-to-cytosol communication and an intact microfilament linkage, we suggest that leukoadhesins may play a broad role in promoting the redistribution of membrane components including adherence-related receptors such as Fc gamma RIII and the urokinase receptor.     

Colchicine permeation is required for inhibition of concanavalin A capping in Chinese hamster ovary cells.

Several antimitotic, tubulin-binding agents, such as colchicine, colcemid, and podophyllotoxin, inhibit the capping of fluorescent-labeled concanavalin A in Chinese hamster ovary cells. By comparing the effects of these agents on parental cell lines on several independently selected colchicine-resistant mutants with decreased drug permeability, we have demonstrated that permeation of these drugs in required for inhibition of capping. These data support the hypothesis that these antimitotic agents interact with an intracellular component, probably microtubules, to prevent the directional movement of concanavalin A receptors on the surface membranes of Chinese hamster ovary cells.     

Inhibitory effect of colchicine and vinblastine on transport of glucagon receptors to the plasma membrane in cultured rat hepatocytes

The role of microtubules in the regulation of glucagon receptors on cultured rat hepatocytes was studied. Antimicrotubular reagents, colchicine and vinblastine, did not affect the binding of 125I-labelled glucagon to hepatocytes at 4 degrees C. At 20 and 37 degrees C, however, the reagents reduced the binding after 60 or 90 min of incubation. Scatchard analysis indicated that the reduction in the binding was due to loss of glucagon-receptor populations. If hepatocytes were preincubated with both unlabelled glucagon and the reagents at 37 degrees C, the binding of the ligand to the cells decreased markedly after a certain delay. The reagents did not inhibit the internalization of the ligand in the cells until 30 min of incubation at 37 degrees C. The results suggest that the microtubule system plays a role in the transport of glucagon receptors to the plasma membrane, which is followed by their internalization.     

Local anesthetics affect transmembrane cytoskeletal control of mobility and distribution of cell surface receptors.

Tertiary amine local anesthetics facilitated concanavalin A-induced redistribution of lectin receptors on murine BALB/3T3 cells and enhanced the susceptibility of these cells to agglutination by concanavalin A. In contrast, these drugs at similar concentrations inhibited ligand-induced capping of immunoglobulin receptors on mouse lymphocytes. We propose that these differing effects of local anesthetics on membrane receptor mobility in fibroblasts and lymphocytes result from the action of anesthetics on membrane-associated microtubules and microfilaments involved in the transmembrane control of receptor mobility. We present electron microscopic evidence of structural alterations in microtubule and microfilament organization in anesthetic-treated cells, together with data on changes in the responsiveness of anesthetic-treated cells to drugs that act on microtubules and/or microfilaments. This evidence supports the proposal that anesthetics affect the organization of cytoskeletal components or their plasma membrane attachment points. The effects of local anesthetics on ligand-induced redistribution of membrane receptors in both 3T3 cells and lymphocytes can be duplicated by treating cells with colchicine (or Vinca alkaloids) together with cytochalasin B. We propose that the participation of membrane-associated microtubules and microfilaments in the transmembrane control of receptor mobility is such that microtubules and microfilaments play opposing roles in regulating the mobility and topography of cell surface receptors.     

Possible role of nucleus-membrane interaction in capping of surface membrane receptors.

Interaction of multivalent ligands and cell surface receptors can induce redistribution of these receptors to form patches and caps. In this study, we have investigated the role of nucleus-membrane interaction in the capping of membrane components. Mouse L cells and leukemia EL4 cells were enucleated with the aid of cytochalasin B, yielding cytoplasts and karyoplasts. Capping of surface receptors was induced by allo- and hetero-immune sera followed by fluorescein-conjugated antiglobulin serum, or by the plant lectin concanavalin A. Capping could easily be induced in intact cells, but virtually no capping was detected in the nucleus-free cytoplasts. Interestingly, karyoplasts, which posses cell-membrane components but very little cytoplasm, could be easily induced to cap their surface antigens. Hence, cap formation of membrane components seems not to be an autonomous membrane process. The data suggest that interaction of surface membranes and inner cell components associated with the nucleus is involved in the movement of surface membrane receptors.     

Succinyl concanavalin A stimulates and antimicrotubular drugs inhibit the synthesis of a brain-specific protein in rat glial cells.

The relative synthesis of the brain-specific s100 protein increased as clonal rat glial cells, C6, progressed from logarithmic to stationary growth in monolayer culture. Drugs that disrupt microtubules, such as colchicine, vinblastine, Colcemid, and podophyllotoxin, inhibited the relative synthesis of S100 protein in stationary cultures. Colchicine (0.1 muM) caused a 50% inhibition of the relative synthesis of S100 protein whereas lumicolchicine,an isomer of colchicine that does not disrupt the microtubular system, had no effect. Succinylated concanavalin A (500 mug/ml) increased relative synthesis in logarithmic but not stationary cultures. These results suggest that signals inducing an increase in the relative synthesis of S100 protein in stationary cultures are transmitted intracellularly from the cell membrane by the microtubular network.     

The role of the microtubular system in LH/hCG receptor downregulation in rat Leydig cells

The mechanism by which hCG-induced LH/hCG receptor 'downregulation' occurs in rat Leydig cells is unknown. To study the role of microtubules in this process we used the microtubule inhibitors vinblastine and colchicine. 200 IU hCG causes a 92-95% decrease in [125I]hCG binding to testis homogenates within 6 h after injection. Both vinblastine and colchicine prevent this loss of hCG binding. Vinblastine or colchicine did not depress the raised serum testosterone levels following hCG injection, suggesting that these agents were not having a toxic effect on the Leydig cells or on the circulation to the testis such that access of the injected hCG was impeded. The morphological observation of vinblastine-induced bundles of filamentous material in Leydig cells is evidence of a direct action on the microtubular-microfilamentous system. This data strongly implicates a role for microtubules in the process of LH/hCG receptor downregulation that occurs in the Leydig cell following large injections of hCG.     

Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression

Previous studies indicated that the vaccinia virus D10 protein, which is conserved in all sequenced poxviruses, participates in the rapid turnover of host and viral mRNAs. D10 contains a motif present in the family of Nudix/MutT enzymes, a subset of which has been shown to enhance mRNA turnover in eukaryotic cells through cleavage of the 5' cap (m7GpppNm-). Here, we demonstrate that a purified recombinant D10 fusion protein possesses an intrinsic activity that liberates m7GDP from capped RNA substrates. Furthermore, point mutations in the Nudix/MutT motif abolished decapping activity. D10 has a strong affinity for capped RNA substrates (Km approximately 3 nm). RNAs of 24-309 nt were decapped to comparable extents, whereas the cap of a 12-nt RNA was uncleaved. At large molar ratios relative to capped RNA substrate, competitor m7GpppG, m7GTP, or m7GDP inhibited decapping, whereas even higher concentrations of unmethylated analogs did not. High concentrations of uncapped RNA were also inhibitory, suggesting that D10 recognizes its substrate through interaction with both cap and RNA moieties. Thus far, poxviruses represent the only virus family shown to encode a Nudix hydrolase-decapping enzyme. Although it may seem self-destructive for a virus to encode a decapping and a capping enzyme, accelerated mRNA turnover helps eliminate competing host mRNAs and allows stage-specific synthesis of viral proteins.     

Anomalous function of vimentin in chronic lymphocytic leukemia lymphocytes

Chronic lymphocytic leukemia (CLL) lymphocytes manifest anomalous motility and cap formation. Since these processes involve cytoskeletal proteins, vimentin from intermediate filaments of normal and CLL lymphocytes was investigated using hetero- and monoclonal antisera. The antisera reacted predominantly with a 60-kD polypeptide, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total lymphocyte proteins. When lymphocytes were stained by indirect immunofluorescence, normal lymphocytes demonstrated well defined cytoplasmic fibrils that capped spontaneously after contact with a glass surface and incubation at 37 degrees C. This capping was dependent on energy and intact microfilaments. Lymphocytes from patients with CLL showed several patterns. In one group, the initial staining was weak, and few capped cells were present after incubation. Lymphocytes from other patients had either normal or aberrantly organized fibrils in which capping was diminished. In another group, a fibrillar pattern with normal or increased capping was seen. In total, 47% +/- 5.1% (mean +/- SE) of normal lymphocytes capped after a 1-hr incubation at 37 degrees C (n = 12) compared to 21% +/- 5.1% for CLL lymphocytes (n = 20, p less than 0.002). Purified subpopulations of normal B and T cells did not differ from unfractionated normal lymphocyte populations. These results demonstrate an anomalous vimentin capping response in CLL lymphocytes. They also show that the arrangement of vimentin in these cells differs from that of normal lymphocytes.     

Effects of antitubulins on the redistribution of crosslinked receptors on the surface of fibroblasts and epithelial cells

Substrate-attached normal mouse fibroblasts, transformed mouse fibroblasts (L strain), and epithelial cells (MPTR strain) were incubated with two ligands crosslinking different groups of the surface receptors: concanavalin A and cationic ferritin. Surface-attached ligands were revealed by an indirect immunofluorescence method. Incubation of control cells with these ligands induced the patching of corresponding surface receptors and the clearing of these receptors from the surface zones located on the lamellar cytoplasm near cell edges actively protruding pseudopodia.Effects of three antitubulins (microtubule-destroying drugs: Colcemid, colchicine, and vinblastine) on the ligand-induced redistribution of receptors were investigated and compared with the previously described effects of these drugs on the distribution of active cell edges. Incubation of normal and transformed fibroblasts with these antitubulins led to the disappearance of nonactive cell edges; the whole cell perimeter became active. Correspondingly, the clearing pattern of the surface receptors of fibroblasts was altered by antitubulins: the cleared area in antitubulin-treated cells formed a circular band along the whole peripheral cell edge. In epithelial cultures, in contrast to fibroblastic ones, antitubulins changed neither the distribution of the active sites of the surface nor the distribution of the areas cleared of crosslinked receptors. Thus, the specific ability of the surface areas located near the active cell edges to become cleared of crosslinked receptors is characteristic not only for the cells with intact microtubules, but also for the cells with microtubules destroyed with antitubulins.     

Membrane receptors and their redistribution in lymphoproliferative disorders.

Lymphoid cells from 20 patients with lymphoproliferative disorders, including chronic lymphocytic leukemia, hairy cell leukemia, Sezary syndrome, lymphoma, and lymphadenitis, were studied for redistribution of surface membrane immunoglobulins (SmIg) and concanavalin A (Con-A) receptors. Fluorescein-labeled polyvalent goat anti-human immunoglobulin and fluoresceinated concanavalin A were used as ligands. Results were similar with both ligands. The highest percentage of capping of ligand-membrane receptors was noted in mononuclear cells from patients with "hairy" cell leukemia: from 24% to 90%. These cells showed moderate to marked fluorescein activity and were able to cap within 15 min at 4 degrees C. Chronic lymphocytic leukemia cells showed a weak fluorescein stain with a very low percentage of cells (0%--16%) capping. Lymph node cells from patients with lymphoma demonstrated moderate to strong fluorescein activity with only an average of 3% of the cells capping; while lymphoid cells from patients with lymphaedenitis showed an average of 27.5% capping and moderate fluorescein activity. Capping of Con-A receptors in mononuclear cells from patients with Sezary syndrome was poor (0%--14%) with moderate fluorescein intensity. This report demonstrates difference in density and mobility of binding sites for SmIg and Con-A on the surface membrane of lymphoid cells from various subclasses of lymphoproliferative disorders. These differences may assist in the differential diagnosis and classification of these conditions.     

Differences in capping behavior between immunological phenotypes in childhood acute lymphoblastic leukemia. A study with ConA and an anti-HLA-ABC backbone

Capping with concanavalin A (ConA) and monoclonal anti-HLA-ABC backbone was studied in childhood acute lymphoblastic leukemia (ALL). Capping with ConA and HLA gave quite different results, both in common ALL and T-ALL. With ConA most cases capped poorly, comparable to results described in chronic lymphatic leukemia and lymphoma, but in several cases capping was comparable to that of normal lymphocytes. In HLA capping T-ALL cells capped better than common ALL cells. HLA capping of T-ALL cells is comparable to that of normal lymphocytes. HLA capping results in handmirror cell formation giving support to the hypothesis that capping and motility are associated events.     

Human lymphocyte motility: normal characteristics and anomalous behavior of chronic lymphocytic leukemia cells.

The characteristics of human lymphocyte motility and its relationship to the redistribution of surface membrane antigens (capping) are poorly defined. Since chronic lymphocytic leukemia (CLL) cells cap poorly when compared with normal human lymphocytes, this study was undertaken to compare the motility of these two cell types. A modification of the Boyden chamber system was employed to quantify lymphocyte motility by placing lymphocyte suspensions on 8-mum convoluted-pore nitrocellulose filters and measuring the depth of migration of the cells into the filter at 37 degrees C. After 3 hr of incubation, CLL cells migrated significantly less into the filter than normal cells. Incubation in the presence of sodium azide or at 4 degrees C abolished all motility, indicating the active nature of the process. The relative motility of individual CLL patients' cells correlated best with the proportion of abnormal cells present as determined by surface receptor assays. The possibility that decreased cell motility in CLL was a reflection of enrichment by a "bone marrow-derived" (B cell) population was eliminated by the finding that normal B cells purified by gradient separation of rosetted cells migrated faster than normal T cells and considerably faster than CLL cells. Motility of normal and CLL lymphocytes was decreased by cytochalasin B and increased by colchicine, vincristine, and vinblastine. Thus, human lymphocyte motility appears to be dependent on microfilament integrity but not to require the colchicine-sensitive cytoskeleton. The decreased motility of CLL cells is the result of an intrinsic cell abnormality, but this finding cannot fully explain the decreased capping, since in human lymphocytes the latter is not prevented by an inhibitor of motility.     

Morphology, Motility, and Surface Behavior of Lymphocytes Bound to Nylon Fibers

Mouse B lymphocytes that were specifically bound to dinitrophenylated bovine serum albumin on nylon fibers exhibited continuous morphological changes, whereas bound T lymphocytes remained more or less spherical. Cinematomicrographic studies showed that the shape changes were associated with local and global movements, although the attached cells did not translocate along the fiber. Cap formation induced by anti-immunoglobulin was always found to be opposite to the point of attachment. The movements and the shape changes were prevented by cytochalasin B and colchicine. Treatment with these agents did not prevent cap formation but led to randomization of the position of the caps with respect to the fiber. Exposure to concanavalin A or attachment of cells to concanavalin A fibers prevented both movement and patch and cap formation, suggesting that cellular structures regulating the mobility of various receptors are altered by binding to concanavalin A fibers. These observations also indicate that interactions of local areas of the lymphocyte surface with certain ligands and substrates can strongly affect the movement and morphology of the entire cell.     

Stabilization of microtubule dynamics by estramustine by binding to a novel site in tubulin: A possible mechanistic basis for its antitumor action

The cellular targets for estramustine, an antitumor drug used in the treatment of hormone-refractory prostate cancer, are believed to be the spindle microtubules responsible for chromosome separation at mitosis. Estramustine only weakly inhibits polymerization of purified tubulin into microtubules by binding to tubulin (K_d, ≈30 μM) at a site distinct from the colchicine or the vinblastine binding sites. However, by video microscopy, we find that estramustine strongly stabilizes growing and shortening dynamics at plus ends of bovine brain microtubules devoid of microtubule-associated proteins at concentrations substantially below those required to inhibit polymerization of the microtubules. Estramustine strongly reduced the rate and extent both of shortening and growing, increased the percentage of time the microtubules spent in an attenuated state, neither growing nor shortening detectably, and reduced the overall dynamicity of the microtubules. Significantly, the combined suppressive effects of vinblastine and estramustine on the rate and extent of shortening and dynamicity were additive. Thus, like the antimitotic mechanisms of action of the antitumor drugs vinblastine and taxol, the antimitotic mechanism of action of estramustine may be due to kinetic stabilization of spindle microtubule dynamics. The results may explain the mechanistic basis for the benefit derived from combined use of estramustine with vinblastine or taxol, two other drugs that target microtubules, in the treatment of hormone-refractory prostate cancer.     

Role of microtubules in the distribution of the Golgi apparatus: effect of taxol and microinjected anti-alpha-tubulin antibodies.

Immunofluorescence microscopy reveals that both microtubule organizing center (MTOC) and Golgi apparatus are contained in the same perinuclear area of A549 cells in interphase. The cells display long microtubules stretching radially from the MTOC to the plasma membrane. Treatment of cells with taxol results in polymerization of microtubules without relation to the MTOC and formation of microtubule bundles predominantly localized in the cell periphery. After incubation with taxol, the Golgi apparatus is fragmented and is conspicuously present in areas of the cytoplasm enriched in microtubules. Incubation of cells with Colcemid results in complete depolymerization of microtubules and fragmentation of the Golgi into elements randomly distributed throughout the cytoplasm. Cells treated with taxol before being incubated with Colcemid contain large numbers of Golgi-derived elements in close association with Colcemid-resistant microtubules. Microtubule depolymerization by vinblastine also is followed by fragmentation of the Golgi apparatus. These Golgi-derived elements show no association with the atypical polymers of tubulin induced by vinblastine. The codistribution of Golgi-derived elements with taxol-induced microtubule bundles can be reversed by microinjection of a monoclonal (YL 1/2) antibody reacting specifically with the tyrosylated form of alpha-tubulin.     

Capping of leukemic cells with monoclonal anti-HLA-A,B,C related to prognosis in childhood acute lymphoblastic leukemia

Capping of leukemic cells with a monoclonal antibody against HLA A,B,C determinants was studied in 53 cases of childhood acute lymphoblastic leukemia (ALL). Determination of the percentages of capped cells after different times of incubation with anti-HLA A,B,C show that T ALL and common ALL do have quite different kinetics of HLA capping. In T ALL all cases reach levels of percentage of capped cells above 30%, in common ALL only 11 of 31 cases cap well. Dilution of the antiserum in 6 common ALL cases results in an increase of capped cells, but the original kinetics of the common ALL capping remain. ALL cases with capping curves above 30% have a worse prognosis (shorter continuous complete remission) than cases with capping curves below 30% in the total group as well as in the non-high-risk group.     

Involvement of microtubules in lipoprotein degradation and utilization for steroidogenesis in cultured rat luteal cells

Cells isolated from superovulated rat ovaries metabolize low density lipoprotein (LDL) and high density lipoprotein (HDL) of human or rat origin and use the lipoprotein-derived cholesterol as a precursor for progesterone production. Under in vitro conditions, both lipoproteins are internalized and degraded in the lysosomes, although degradation of HDL is of lower magnitude than that of LDL. In this report we have examined the role of cellular microtubules in the internalization and degradation of human LDL and HDL in cultured rat luteal cells. The microtubule depolymerizing agents colchicine, podophyllotoxin, vinblastine, and nocodazole as well as taxol, deuterium oxide, and dimethyl sulfoxide, which are known to rapidly polymerize cellular tubulin into microtubules, were used to block the function of microtubules. When these antimicrotubule agents were included in the incubations, degradation of the apolipoproteins of [125I]iodo-LDL and [125I]iodo-HDL by the luteal cells was inhibited by 50-85% compared to untreated control values. Maximum inhibitory effects were observed when the cells were preincubated with the inhibitor for at least 4 h at 37 C before treatment with the labeled lipoprotein. Lipoprotein-stimulated progesterone production by luteal cells was also inhibited by 50% or more in the presence of antimicrotubule agents. However, basal and hCG-stimulated progesterone production were unaffected by these inhibitors. The binding of [125I]iodo-LDL and [125I]iodo-HDL to luteal cell plasma membrane receptors was not affected by the microtubule inhibitors. Although binding was unaffected and degradation was impaired in the presence of the inhibitors, there was no detectable accumulation of undegraded lipoprotein within the cells during the 24 h of study. From this study we conclude that the uptake and utilization of LDL and HDL by cultured rat luteal cells are mediated by cellular microtubules.     

Effects of Phagocytosis and Colchicine on the Distribution of Lectin-Binding Sites on Cell Surfaces

The effect of phagocytosis on lectin binding to plasma membranes of polymorphonuclear leukocytes was examined. The specific activities of binding sites of concanavalin A and Ricinus communis agglutinin (defined as mug of lectin bound per 100 mug of membrane protein) were measured on isolated membranes; they decreased in parallel with phagocytosis. Our data suggest that this removal occurs by concentration of binding sites into internalized membrane. Colchicine and vinblastine, which did not inhibit phagocytosis, prevented the selective removal of lectin-binding sites from the surface. It was also shown that at 37 degrees lectins induce their own internalization. This property was used to define operationally three classes of lectin receptors, one of which is most extensively removed from plasma membrane during phagocytosis. Based on other morphological studies in which it is shown that before phagocytosis the surface distribution of concanavalin-binding sites is random, it is inferred that phagocytosis alters surface topography by inducing the selective movement of binding sites into membrane undergoing internalization and that colchicine-sensitive proteins are essential for this imposed topographical reorganization.