Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.     

Organization of tubulin in normal and transformed rat kidney cells

We have carried out a quantitative biochemical and ultrastructural study of tubulin and microtubules in a normal rat kidney (NRK) cell line and its viral transformant (442) in culture. Under equivalent culture conditions, both cell lines contain the same amount of tubulin according to a colchicine-binding assay. The normal and transformed cells differ significantly, however, with respect to the state of organization of their tubulin. Counts of microtubules in sectioned cells indicate that NRK cells have almost twice as many microtubules per unit area of cytoplasm as the 442 cells. Centrifugation studies, on the other hand, show that 442 cells have almost twice as much pelletable tubulin as the NRK cells. We propose, therefore, that the transformed cells contain a large amount of tubulin which is in some alternative aggregate form that is not morphologically detectable as microtubles in the cytoplasm     

Radioimmunoassay for tubulin: a quantitative comparison of the tubulin content of different established tissue culture cells and tissues

A quantitative estimate of the cellular tubulin concentration can be obtained by the use of a radioimmunoassay based upon the competition between tubulin in cell extracts and a known amount of radioactively labeled homogeneous tubulin during binding to a limited amount of antitubulin antibodies. This assay shows that a variety of widely used tissue culture cells (mouse L cells, mouse 3T3 cells, chick embryo fibroblasts) have a tubulin content which corresponds to approximately 2.5-3.3% of their total protein. Transformation of mouse 3T3 cells by the DNA virus SV40, and of chick embryo cells by the RNA Rous sarcoma virus, does not change the intracellular tubulin concentration. Transformed cells of brain origin, such as some glia tumor cell lines and some neuroblastoma cell lines, have a much lower tubulin content than does normal brain tissue.The intracellular concentration of tubulin in mouse 3T3 cells is discussed in relation to the number of microtubules detected during interphase by immunofluorescence microscopy. These results are also discussed in view of a mechanism of microtubule elongation in vivo driven by self-assembly.     

Multiple sites for the initiation of microtubule assembly in mammalian cells.

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.     

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.     

培养的正常细胞和肿瘤细胞内微管变化的研究——Ⅱ.肿瘤细胞内微管的免疫荧光细胞化学观察

本实验用管蛋白抗体间接免疫荧光细胞化学方法,观察了我国建株的人胃低分化粘液腺癌MGc 80-3,人胃腺癌SGC-7901,人鼻咽癌上皮样细胞CNE,人食管癌上皮细胞ECa-109,人肺鳞癌LTEP-78,人啼腺癌LTEP-a_1,人肺小细胞癌LTEP-p七株癌细胞和HeLa细胞,小鼠S_(180)-V肉瘤细胞的微管形态。与人的正常包皮成纤维细胞和食管上皮细胞内精细的CMTC结构对比,肿瘤细胞间期的胞质微管普遍有减少或缺如的现象。参考Brin-kley对微管免疫荧光染色图形的分型方法,我们将观察的各种微管染色图形归纳为四种类型,比较各种细胞群体内微管类型的分布。肿瘤细胞群体内多数为微管缺如型和稀疏型,未见典型的丰满型,而正常细胞群体内都是丰满型。同时,肿瘤细胞的MTOC区面积明显增大。分裂期的肿瘤细胞内,有丝分裂器纺锤体微管荧光形态与正常细胞的没有差别。本文对肿瘤细胞间期胞质微管减少和缺如以及MTOC区明显增大的现象及其可能的意义进行了讨论,认为这是癌变机制研究中值得深入探讨的重要课题之一。     

Modification of the phenotype of murine sarcoma virus-transformed cells by sodium butyrate : Effects on morphology and cytoskeletal elements

The effects of sodium butyrate on cellular morphology and the distribution of cytoskeletal elements were examined using a line of normal rat kidney cells transformed by and producing murine sarcoma and leukemia viruses [NRK (MSV-MLV)]. Untreated cells were predominantly round or fusiform in shape and contained few microfilaments and microtubules. Culturing these cells in medium containing 2 mM sodium butyrate induced the formation of long cytoplasmic processes within 12–24 h followed by a progressive flattening of the cells which was apparent in most cells by 72 h. Reversal of these changes in NRK (MSV-MLV) cells grown for several passages in butyrate medium required at least ten cell divisions. The morphological alterations induced by butyrate were accompanied by a striking elaboration of cytoplasmic microfilaments and microtubules as shown by indirect immunofluorescence and by electron microscopy. Butyrate also enhanced the formation of substrate adhesion plaques and intercellular gap junctions, but not adherens junctions. These results suggest that growth of NRK (MSV-MLV) cells in the presence of butyrate induced specific cellular alterations which counteract some of the effects of transformation of NRK cells by MSV.     

The effects of 5-bromodeoxyuridine on cyclic AMP levels and cytoskeletal organization in malignant melanoma cells

5-Bromodeoxyuridine (BUdR) causes mouse melanoma cells to develop a flattened morphology and simultaneously adhere tenaceously to the substratum on which they are growing. Experiments were done to determine if these events are coupled to increases in cAMP levels and to rearrangements in the cells' cytoskeleton. Cyclic AMP assays revealed that cell flattening and the increase in adhesive properties caused by BUdR is not accompanied by an increase in the cellular concentration of cyclic AMP. However, electron micrographs of cells grown in the presence of BUdR show a striking increase in the number of organized microtubules and microfilaments. Colchicine binding revealed no difference in the amount of tubulin present in untreated or BUdR-treated cells indicating that the increase in the number of microtubules is due to the polymerization of pre-existing tubulin subunits. These results are discussed in light of possible similar mechanisms of action of BUdR and cyclic AMP in regulating the organization of microtubules and microfilaments and the role these structures play in altering cell morphology and adhesive properties.     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

The microtubule stabilizing agent laulimalide does not bind in the taxoid site,kills cells resistant to paclitaxel and epothilones,and may not require its epoxide moiety for activity

Laulimalide is a cytotoxic natural product that stabilizes microtubules. The compound enhances tubulin assembly, and laulimalide is quantitatively comparable to paclitaxel in its effects on the reaction. Laulimalide is also active in P-glycoprotein overexpressing cells, while isolaulimalide, a congener without the drug's epoxide moiety, was reported to have negligible cytotoxic and biochemical activity [Mooberry et al. (1999) Cancer Res. 59, 653-660]. We report here that laulimalide binds at a site on tubulin polymer that is distinct from the taxoid site. We found that laulimalide, while as active as paclitaxel, epothilone A, and eleutherobin in promoting the assembly of cold-stable microtubules, was unable to inhibit the binding of radiolabeled paclitaxel or of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]paclitaxel, a fluorescent paclitaxel derivative, to tubulin. Confirming this observation, we demonstrated that microtubules formed in the presence of both laulimalide and paclitaxel contained near-molar quantities, relative to tubulin, of both drugs. Laulimalide was active against cell lines resistant to paclitaxel or epothilones A and B on the basis of mutations in the M40 human beta-tubulin gene. We also report that a laulimalide analogue lacking the epoxide moiety, while less active than laulimalide in biochemical and cellular systems, is probably more active than isolaulimalide. Further exploration of the role of the epoxide in the interaction of laulimalide with tubulin is therefore justified.     

Tau protein function in living cells

Tau protein from mammalian brain promotes microtubule polymerization in vitro and is induced during nerve cell differentiation. However, the effects of tau or any other microtubule-associated protein on tubulin assembly within cells are presently unknown. We have tested tau protein activity in vivo by microinjection into a cell type that has no endogenous tau protein. Immunofluorescence shows that tau protein microinjected into fibroblast cells associates specifically with microtubules. The injected tau protein increases tubulin polymerization and stabilizes microtubules against depolymerization. This increased polymerization does not, however, cause major changes in cell morphology or microtubule arrangement. Thus, tau protein acts in vivo primarily to induce tubulin assembly and stabilize microtubules, activities that may be necessary, but not sufficient, for neuronal morphogenesis.     

Tubulin and actin in paired nonneoplastic and spontaneously transformed neoplastic cell lines in vitro: fluorescent antibody studies.

Pairs of nonneoplastic and spontaneously transformed neoplastic cells were derived from rat, mouse and hamster embryos. The neoplastic cells of each pair had poorly spread cellular morphology, grew in agarose in vitro and produced invasive sarcomas in vivo; the nonneoplastic cells exhibited none of these properties. The distribution of microtubules and microfilament bundles (stress fibers or actin cables) was examined in five such paired lines and in 3T3 and SV40-transformed 3T3 cells by indirect immunofluorescent microscopy of fixed cells treated with rabbit antibody prepared against bovine brain tubulin or guinea pig smooth muscle actin, respectively. Actin cables in all the neoplastic cells appeared thinner and more sparse than in the paired nonneoplastic cells. These differences were also observed in living cells with polarization microscopy. In contrast, microtubules appeared similar in neoplastic and nonneoplastic cells, both in areas of thin peripheral lamellar cytoplasm which allowed a clear visualization of fine, curving microtubules and in regions of thick, central endoplasm which obsecured individual microtubules. In fact, the main morphological difference between neoplastic and nonneoplastic cells was the relative amount of lamellar cytoplasm or endoplasm, rather than the appearance of microtubles in either region. Thus the distinctive growth properties and retracted cellular morphology of neoplastic cells in this study did not correlate with decreased or disorganized microtubules, but with thin and sparse actin cables.     

Identification of novel microtubule inhibitors effective in fission yeast and human cells and their effects on breast cancer cell lines

Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by βIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Cellular interactions and tubulin detyrosination in fibroblastic and epithelial cells

In mammalian cells most microtubules are enriched in tyrosinated alpha-tubulin (tyr-tubulin). Other subclasses of microtubules are present in variable amounts and some are enriched in detyrosinated alpha-tubulin (glu-tubulin). We examined the effect of cell-cell interactions on the level of glu-tubulin in microtubules. This was studied by quantitative immunofluorescence using antibodies against tyr- and glu-tubulin. We found that in cells which have established cell-cell contacts, the ratio of glu-/tyr-tubulin is higher than in isolated cells. We also examined the effect of cell-cell interactions on the glu-/tyr-tubulin ratio by using the antibody blocking method of Schulze and Kirschner [42]. Microtubules containing mainly tyr-tubulin had been blocked first by a polyclonal antibody against tyr-tubulin and several layers of secondary antibodies. The unblocked microtubules were then labeled by a monoclonal antibody against alpha-tubulin. Since the coating efficiency of microtubules by the anti-tyr tubulin depends on the amount of tyr-tubulin in each microtubule, this procedure allows the visualization of microtubules enriched or depleted in tyr-tubulin in specific domains of each cell. Microtubules were more extensively blocked in subconfluent than in confluent cells and preferentially at the periphery of the cytoplasm. In cells present at the margin of an artificial wound produced in a confluent monolayer, the amount of blocked microtubules increased slowly with time (between 2 and 4 h). These results are consistent with the hypothesis that cell-cell contacts lead to increased tubulin dytyrosination both in fibroblastic and epithelial cells.     

The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding. A possible mechanism for its antiproliferative activity

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6 +/- 0.1 microM. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2 microM, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (> 40 microM), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics.     

Incorporation of 3-nitrotyrosine into the C-terminus of alpha-tubulin is reversible and not detrimental to dividing cells.

The C-terminus of the alpha-chain of tubulin is subject to reversible incorporation of tyrosine by tubulin tyrosine ligase and removal by tubulin carboxypeptidase. Thus, microtubules rich in either tyrosinated or detyrosinated tubulin can coexist in the cell. Substitution of the terminal tyrosine by 3-nitrotyrosine has been claimed to cause microtubule dysfunction and consequent injury of epithelial lung carcinoma A549 cells. Nitrotyrosine is formed in cells by nitration of tyrosine by nitric oxide-derived species. We studied properties of tubulin modified by in vitro nitrotyrosination at the C-terminus of the alpha-subunit, and the consequences for cell functioning. Nitrotyrosinated tubulin was a good substrate of tubulin carboxypeptidase, and showed a similar capability to assemble into microtubules in vitro to that of tyrosinated tubulin. Tubulin of C6 cells cultured in F12K medium in the presence of 500 micro m nitrotyrosine became fully nitrotyrosinated. This nitrotyrosination was shown to be reversible. No changes in morphology, proliferation, or viability were observed during cycles of nitrotyrosination, denitrotyrosination, and re-nitrotyrosination. Similar results were obtained with CHO, COS-7, HeLa, NIH-3T3, NIH-3T3(TTL-), and A549 cells. C6 and A549 cells were subjected to several passages during 45 days or more in the continuous presence of 500 micro m nitrotyrosine without noticeable alteration of morphology, viability, or proliferation. The microtubular networks visualized by immunofluorescence with antibodies to nitrotyrosinated and total tubulin were identical. Furthermore, nitrotyrosination of tubulin in COS cells did not alter the association of tubulin carboxypeptidase with microtubules. Our results demonstrate that substitution of C-terminal tyrosine by 3-nitrotyrosine has no detrimental effect on dividing cells.     

Simian virus 40 large T antigen and p53 are microtubule-associated proteins in transformed cells.

The cellular proteins that interact with simian virus 40 large T antigen (T-ag) must be identified in order to understand T-ag effects on cellular growth control mechanisms. A protein extraction procedure utilizing single-phase concentrations of 1-butanol recovered a complex composed of T-ag, p53, and other Mr 35,000-60,000 proteins from suspension cultures of the simian virus 40-transformed mouse cell line mKSA. Partial protease mapping showed each of the associated proteins to be unique. Automated microsequence analysis of the NH2-terminal 30 amino acids of the Mr 56,000 protein purified after coprecipitating with T-ag and p53 identified it as the beta subunit of mouse tubulin. The existence of a complex containing tubulin, T-ag, and p53 was confirmed by reciprocal immunoblotting experiments. Both T-ag and p53 were coprecipitated by three different monoclonal antibodies directed against tubulin, and conversely, monoclonal antibodies specific for T-ag or p53 coprecipitated tubulin. Mixing experiments and extractions in the presence of purified tubulin indicated that the complex existed in situ prior to cell lysis. Both p53 and T-ag copurified with microtubules through two cycles of temperature-dependent disassembly and assembly. Both T-ag and p53 were localized to microtubules in the cytoplasm of mKSA cells by immunoelectron microscopy. Treatment of mKSA cells with 10 microM colchicine followed by lysis in 0.1% Nonidet P-40 resulted in increased amounts of solubilized T-ag and p53. Both T-ag and p53 were also associated with microtubules in three other simian virus 40-transformed mouse cell lines growing as monolayers, confirming the generality of the association. An interaction of T-ag and p53 with microtubules may be important in the intracellular transport of these proteins and may affect cellular signal transduction or growth control.     

Of Mice and Men

Non-muscle myosin II has diverse functions in cell contractility, morphology, cytokinesis and migration. Mammalian cells have three isoforms of non-muscle myosin II, termed IIA, IIB and IIC, encoded by three different genes. These isoforms share considerable homology and some overlapping functions, yet they exhibit differences in enzymatic properties, subcellular localization, molecular interaction and tissue distribution 1-6. Our studies have focused on the IIA isoform, and they reveal unique regulatory roles in cell-cell adhesion and cell migration that are associated with cross-talk of the actomyosin system with microtubules. In humans, various mutations in the MYH9 gene that encodes the myosin IIA heavy chain cause autosomal dominant disease, whereas in mice, the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. We discuss here the differences between mouse and human phenotypes and how the wealth of mechanistic knowledge about myosin II based on in vitro studies and mouse models can help us understand the molecular and cellular pathophysiology of myosin IIA deficiency in humans.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.