Joe A. Connolly

The distribution of tau and HMW microtubule-associated proteins in different cell types

Abstract To investigate the distribution of the tau and HMW microtubule-associated proteins (MAPS) and their relationship to microtubules in vivo, we have examined a wide variety of avian and mammalian cell types by immunofluorescence with antisera to these two proteins. Anti-HMW serum stains cytoplasmic microtubules in all mammalian cell types so far examined. However, anti-tau serum did not stain cytoplasmic microtubules in rat glial cells or in pig kidney cells. In mammalian neurons, fibroblasts and neuroblastoma cells, the staining of microtubules with both sera was similar. Anti-HMW serum did not stain primary cilia or cilia on isolated tracheal epithelial cells, whereas anti-tau serum did stain these ciliary microtubules. We believe these results indicate that some types of microtubules may be associated with only the tau or the HMW protein, whereas others may be associated with both tau and HMW protein. With respect to avian cells, anti-HMW serum did not stain microtubules in any of the three cell types examined, whereas the anti-tau serum stained them in two cell types. Furthermore, double diffusion tests indicated that anti-pig tau serum will precipitate both pig brain tau and tau protein isolated from chick brain, whereas anti-HMW serum will precipitate only pig brain and not chick brain HMW protein. We believe tau protein is antigenically similar in both avian and mammalian cells, whereas the HMW protein from these two sources is antigenically distinct.

Pattern formation in the mammalian forebrain: patch neurons from the rat striatum selectively reassociate in vitro.

Mechanisms involved in the developmental organization of the rat striatum were investigated in vitro. The neurons of the patch and matrix compartments were preferentially labeled in vivo with a [3H]thymidine injection on embryonic day (E) 13 or 18, respectively. Two or 7 days later the striatum was removed, dissociated into a single cell suspension and plated on a collagen-coated substrate. After 5 days in culture the neurons had migrated into aggregates. Within an individual aggregate, neurons labeled on E13 tended to clump together, whereas neurons labeled on E18 were randomly dispersed. Comparing between aggregates, [3H]thymidine-labeled E13 cells were located in aggregates containing numerous other labeled E13 cells, whereas [3H]thymidine-labeled E18 cells were dispersed randomly between aggregates. These results suggest that early born striatal neurons (primarily patch cells) selectively associate with each other, and that this process may be crucial to the developmental compartmentalization of the rat striatum.

The disappearance of a cyclin-like protein and the appearance of statin is correlated with the onset of differentiation during myogenesis in vitro.

We have used monoclonal antibodies to statin (S-44) and a cyclin-like protein (S-132) to examine the distribution of these two antigens in proliferating and in nonproliferating populations of cells. We have found that this cyclin-like protein is present in proliferating fibroblasts, whereas statin is absent from these same cell populations; in contrast, in senescent populations of fibroblasts the cyclin-like antigen disappears and statin labeling of nuclei appears. During myogenesis in rat muscle cell cultures, S-132 labeling is present in proliferating myoblasts and disappears after cells fuse and differentiate as multinucleated myotubes. In contrast, statin is absent from proliferating myoblasts, but appears when these cells become postmitotic and begin to differentiate. Similar results were seen during chick myogenesis. We have also found similar results during serum-starvation-induced differentiation in neuroblastoma cells. These results indicate that the cyclin-like protein disappears and statin appears upon commitment to differentiation in vitro, and the presence or the absence of these proteins appears to provide cellular markers for the transition from the proliferative to the nonproliferative state during differentiation.

Inhibition of axonal transport ‘in vivo’ by a tubulin-specific antibody

We have used antibodies against the major proteins of the cytoskeleton-tubulin, the neurofilament triplet proteins and actin-as in vivo probes to determine the contribution of separate components of the cytoskeleton in axonal transport. The injection of either Fast Blue or wheat germ agglutinin conjugated horseradish peroxidase into the caudate nucleus of adult rats resulted in the retrograde transport of these tracers to the neuronal cell bodies in the substantia nigra pars compacta. In experimental animals these tracer injections were immediately preceded by injections of antiserum against tubulin, neurofilament triplet protein or actin, into multiple sites in the caudate. Preimmune serum injection preceded tracer injection as a control in the contralateral caudate of the same animal. One antiserum against electrophoretically purified pig brain tubulin (NS-20) produced a dramatic decrease in the normal retrograde and anterograde transport of both tracers to the SN. Other antisera against tubulin, as well as neurofilament and actin antisera, had no effect on the axonal transport of the tracers. Affinity purified antibodies prepared from the NS-20 antitubulin serum also blocked axonal transport of the tracers. These results provide further support for a critical role of microtubules in axonal transport in vivo. Moreover, an antigenic determinant on tubulin that is uniquely recognized by the NS-20 antibodies may provide us with a way to define the site of association of transfer vesicles with microtubules.

Microtubules in colcemid-resistant mutants of CHO cells

Abstract Cytoplasmic and spindle microtubules in parental and colcemid-resistant (CM R ) mutant clones of Chinese hamster ovary (CHO) cells were examined by immunofluorescence microscopy with antiserum to purified brain tubulin. Both the parental and mutant cells displayed apparently normal arrays of cytoplasmic and spindle microtubules. Furthermore, no differences could be detected in the microtubules at the ultrastructural level. Microtubules in the mutant cells, however, were more resistant to the action of colcemid as 5–7 times as much colcemid was required to break down spindle and cytoplasmic microtubules in mutant as compared to parental cells. In contrast the sensitivity of microtubules in both cell types to griseofulvin was similar. When these drugs were removed, microtubules reassembled in a similar fasion in both mutant and parental cells. In addition in the CM R mutants, vinblastine-induced paracrystals differed markedly in their size and shape from those in parental cells. These results indicate that the in vivo behaviour of microtubules in CM R cells is altered and that this alteration(s) is most clearly observed under conditions where microtubules and certain tubulin-binding drugs interact. They also show that the mutant form of tubulin is incorporated into the structure of microtubules in mutant cells.

Microtubules, microfilaments and the transport of acetylcholine receptors in embryonic myotubes

Both microtubules and microfilaments have been implicated in the exocytotic and endocytotic transport of coated and smooth surfaced membrane vesicles. We have reexamined this question by using specific pharmacological agents to disrupt these filaments and assess the effect on the movement of acetylcholine receptor (AChR) containing membrane vesicles in embryonic chick myotubes. Myotube cultures treated with nocodazole (0.6 microgram/ml) or colcemid (0.5 microgram/ml) (to disrupt microtubules) show only a 20-25% decrease in the number of cell surface AChRs after 48 h. Addition of chick brain extract (CBE) to cultured myotubes causes a significant increase in the total number of cell surface AChRs (measured by [125I]alpha-bungarotoxin (alpha-BGT) binding), thus providing us with a way to manipulate receptor and transport vesicle populations. Cultures treated with CBE plus nocodazole or colcemid show a 1.7-fold increase in AChR number over drug treatment alone, the same increase seen in cultures treated with CBE alone, although the total number remains about 20-25% less than that seen in control cultures. In cultures treated with cytochalasin D (0.2 microgram/ml) or dihydrocytochalasin B (5.0 micrograms/ml) (to disrupt microfilaments), 35 and 65% decreases in cell surface AChR number were seen after 48 h. However, in cultures treated with CBE and cytochalasin D, the same total number of AChRs was found as in cultures treated with CBE alone. No significant effects were seen with any of these drugs on the receptor incorporation rate (the appearance of new alpha-BGT-binding sites) after 6 h. The half-life for AChRs in control cultures was 23.0 h. In cytochalasin D and dihydrocytochalasin B it was 21.9 and 19.0 h, respectively; with colcemid and nocodazole, it increased to 37.1 and 28.1 h. These results suggest that non-myofibrillar microfilament bundles are not involved in the movement of AChR-containing membrane vesicles; further, the small effects seen with microtubule inhibitors tend to rule out a major role for microtubules in this transport.

Application of Immunofluorescence in Studies of Cytoskeletal Antigens

Publisher Summary This chapter gives an overview of the immunofluorescent techniques used for localizing macromolecules at the light microscopic level. To begin with, these techniques localize intracellular antigens by immunofluorescent staining; the cells or tissue sections must first be fixed to stabilize the antigens and to make the cells permeable to antibody molecules, which are then treated with labeled antibody molecules if the direct method is used, or first with unlabeled antibody or antiserum against the antigen to be localized and then by a labeled antibody against the first immunoglobulin if the indirect method is used. Finally, the distribution of the labeled antibody is visualized in the fluorescence microscope. The immunofluorescence provides a simple and powerful tool for the examination of the distribution of a particular macromolecule at the light microscope level while allowing for a quick examination of large populations of intact cells and large regions of tissue. This has been used to study the distribution of different neuropeptides, cell-surface constituents, infectious agents, small molecules such as cyclic AMP and acetyl and n-acetyl serotonin. As a result, the labeled antibody method has become a significant tool with many useful research and clinical applications. The technique has also been extensively used to visualize the cytoskeleton, which is composed of different fiber systems. These fiber systems are thought to be responsible for a wide variety of important cell functions, including organization of the cytoplasm, locomotion, endocytosis and exocytosis, cell division, axonal transport, and the formation and maintenance of characteristic cell shapes.

Membrane glycoproteins are involved in the differentiation of the BC3H1 muscle cell line

The nonfusing muscle cell line BC3H1 expresses a family of muscle-specific proteins when the fetal bovine serum (FBS) concentration is reduced from 20 to 1%. We have used a series of glycosylation inhibitors to assess the role played by glycoproteins in the initiation of differentiation in this cell line. Tunicamycin (TNM) and 2-deoxy-D-glucose, added to cells when the FBS concentration was reduced, blocked creatine phosphokinase (CPK) induction by 70-95%. These effects were dose dependent and reversible. TNM and 2-deoxy-D-glucose also reversed CPK induction in differentiated cells. Leupeptin and N-acetylglucosamine did not reverse these effects. 1-Deoxynojirimycin, 1-deoxymannojirimycin, and swainsonine have no effect on induced CPK expression, whereas castanospermine, a glucosidase I inhibitor, blocked its induction completely. As attempts to use conditioned medium from cells grown in 1 or 20% FBS have no effect on this differentiation process we conclude that high mannose structures, but not complex form glycoproteins, bound to the surface of BC3H1 cells play a role in transducing signals for differentiation and are probable mediators of cell/cell contact.

Is there an endogenous bungarotoxin-like molecule in the vertebrate central nervous system?

It has been postulated that there is an endogenous bungarotoxin-like ligand in rat brain which can inhibit the binding of alpha-bungarotoxin (alpha-BGT) to its target nicotinic acetylcholine (ACh) receptor. We have examined this further by testing the ability of rat and chick brain and spinal cord extracts to inhibit the binding of alpha-BGT to ACh receptors in cultured chick and rat myotubes. We find no evidence for inhibition by any of these extracts, and thus cannot support the hypothesis of an endogenous alpha-BGT-like ligand.

The ras Oncogene and Myogenic Commitment and Differentiation

When proliferating BC3H1 muscle cells are shifted to low serum conditions, they exit from the cell cycle and differentiate, activating a family of muscle-specific genes. Addition of the purified growth factors, fibroblast growth factor (FGF) or thrombin reverses this process and stimulates these cells to reenter the cell cycle. Pertussis toxin (PT) blocks thrombin’s, but not FGF’s, effects on muscle proliferation/differentiation. Thrombin, therefore, requires a G protein to transduce its signal. In addition, PT promoted differentiation in the presence of high concentrations of serum. Serum then contains a mitogen that signals through a PT-sensitive pathway in order to promote proliferation and inhibit muscle gene transcription. Transfection of the activated Ha-ras oncogene into BC3H1 and 10T1/2 cells blocked muscle differentiation in both of these lines. PT could not rescue the ras-mediated inhibition of differentiation. These results suggest that G protein-like molecules play important roles in transducing growth factor signals that control myogenesis.