Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells.

We have examined the effects of microtubule- and microfilament-disrupting drugs on the stability, formation, and removal of acetylcholine (ACh) receptors and ACh receptor clusters on the surface of aneurally cultured chick embryonic myotubes. (a) In muscle cell cultures, cytochalasin D (0.2 microgram/ml) or B (2.0 micrograms/ml) causes the dispersal of 50-60% of the existing clusters over a 24-h period (visualized with rhodamine-conjugated alpha-bungarotoxin); Colcemid (0.5 micrograms/ml) has no affect on these clusters. The total number of cell surface ACh receptors does not decline during this period (measured by [125I]alpha-bungarotoxin binding) in the presence of either drug. (b) When cells are treated with biotinylated alpha-bungarotoxin and fluorescent avidin, ACh receptors are cross-linked and rapidly internalized (Axelrod, D., 1980, Proc. Natl. Acad. Sci. USA., 77: 4823-4827). Within 6 h, I have found that 0-15% of the existing large clusters remain. Cytochalasin D or B had no effect on this removal of clusters; however, Colcemid completely prevented the removal of clusters from the cell surface. (c) Addition of chick brain extract to chick myotubes causes an increase in the synthesis and clustering of ACh receptors (Jessell et al., 1979, Proc. Natl. Acad. Sci. USA. 76: 5397-5401). Cytochalasin D caused a slight increase in the number of receptors synthesized in the presence of brain extract whereas Colcemid had no effect on the synthesis and insertion of new receptors into the plasma membrane induced by the brain extract. However, both drugs prevented the increase in the number of receptor clusters. These results are consistent with the hypothesis that receptor clusters are stabilized by actin-containing filaments, but that the movement of receptors in the plane of the membrane requires Colcemid-sensitive microtubules.

Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera.

Several sera from nonimmunized rabbits have been found which stain centrioles and basal bodies by indirect immunofluorescence in a wide variety of cell types. So far, approximately 10% of the rabbit sera that we have examined gave strong positive staining of centrioles and basal bodies. Cytoplasmic networks, mitotic spindles, and ciliary axonemes, however, remain unstained. This specific fluorescent staining of centrioles and basal bodies could not be abolished by absorption of sera with purified brain tubulin. This technique is superior to previous methods for the visualization of basal bodies and centrioles at the light microscopic level and should be useful for rapid and convenient detection of these organelles in large populations of cells.

Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. II. Two signaling pathways distinguished by pertussis toxin and a potential role for the ras oncogene.

In the preceding report (Kelvin, D.J., G. Simard, H.H. Tai, T.P. Yamaguchi, and J.A. Connolly. 1989. J. Cell Biol. 108:159-167) we demonstrated that pertussis toxin (PT) blocked proliferation and induced differentiation in BC3H1 muscle cells. In the present study, we have used PT to examine specific growth factor signaling pathways that may regulate these processes. Inhibition of [3H]thymidine by PT in 20% FBS was reversed in a dose-dependent fashion by purified fibroblast growth factor (FGF). In 0.5% FBS, the normally induced increase in creatine kinase (CK) activity was blocked by FGF in both the presence and absence of PT. Similar results were obtained with purified epidermal growth factor (EGF). We subsequently examined the effect of a family of growth factors linked to inositol lipid hydrolysis and found that thrombin, like FGF, would increase [3H]thymidine incorporation and block CK synthesis. However, PT blocked thymidine incorporation induced by thrombin, and blocked the inhibition of CK turn-on in 0.5% FBS by thrombin. The ras oncogene, a G protein homologue, has previously been shown to block muscle cell differentiation in C2 muscle cells (Olson, E.N., G. Spizz, and M.A. Tainsky. 1987. Mol. Cell. Biol. 7:2104-2111); we have characterized a BC3H1 cell line, BCT31, which we transfected with the val12 oncogenic Harvey ras gene. This cell line did not express CK in response to serum deprivation. Whereas [3H]thymidine incorporation was inhibited by 70-80% by increasing doses of PT in control cells, BCT31 cells were only inhibited by 15-20%. ADP ribosylation studies indicate this PT-insensitivity is not because of the lack of a PT substrate in this cell line. Furthermore, PT could not induce CK expression in BCT31 cells as it did in parental cells. We conclude that there are at least two distinct growth factor pathways that play a key role in regulating proliferation and differentiation in BC3H1 muscle cells, one of which is PT sensitive, and postulate that a G protein is involved in transducing signals from the thrombin receptor. We believe that ras functions in the transduction of growth factor signals in the nonPT-sensitive pathway or downstream from the PT substrate in the second pathway.

Growth factors, signaling pathways, and the regulation of proliferation and differentiation in BC3H1 muscle cells. I. A pertussis toxin-sensitive pathway is involved.

Cells of the nonfusing muscle cell line BC3H1 stop proliferating and express a family of muscle-specific proteins when the FBS concentration is reduced from 20 to 0.5% (Munson, R., K.L. Caldwell, and L. Glaser. 1982. J. Cell Biol. 92:350-356). Several growth factors have been shown to block differentiation in this cell line. To begin to investigate the potential role of G proteins in signal transducing pathways from these receptors, we have examined the effects of cholera toxin (CT) and pertussis toxin (PT) on proliferation and differentiation in BC3H1 cells. PT specifically ADP ribosylates a protein with an apparent molecular mass of 40 kD in BC3H1 cell membranes, whereas CT specifically ADP ribosylates three proteins of 35-43 kD. When added to exponentially growing cells in 20% FBS, CT and PT inhibited [3H]thymidine incorporation by up to 75% in a dose-dependent fashion. We found the synthesis of creatine kinase (CK) and skeletal muscle myosin light chain was reversibly induced in cells in 20% FBS treated with PT, but no increased synthesis was seen in cells treated with CT or in control cells; Northern analysis indicated this induction was at the level of mRNA. In cells shifted to 0.5% FBS, CT inhibited the normally induced synthesis of CK whereas PT potentiated it by approximately 50%. Forskolin also inhibited growth in 20% FBS and differentiation in 0.5% FBS medium in a dose-dependent fashion. both forskolin and CT elevated cAMP levels compared with control or PT-treated cells, suggesting that CT is blocking proliferation and differentiation by elevating cAMP levels. These results establish that a PT-sensitive pathway is involved in regulating proliferation and differentiation in BC3H1 cells, and we postulate that PT functions by ADP ribosylating a G protein that transduces signals from growth factor receptors in these cells.

Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence.

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.

Cytoplasmic microtubule organizing centers isolated from Polytomella agilis.

Basal body rootlets in Polytomella function as organizing centers for cytoplasmic microtbules in vivo. A method is described to isolate intact basal body-rootlet complexes. The integrity of the isolated complexes is confirmed by electron microscopy, and the rootlets are shown to be competent as initiation sites for the in vitro polymerization of brain microtubule protein.

Subducting serpentinites release reduced, not oxidized, aqueous fluids.

The observation that primitive arc magmas are more oxidized than mid-ocean-ridge basalts has led to the paradigm that slab-derived fluids carry SO2 and CO2 that metasomatize and oxidize the sub-arc mantle wedge. We combine petrography and thermodynamic modelling to quantify the oxygen fugacity (fO2) and speciation of the fluids generated by serpentinite dehydration during subduction. Silicate-magnetite assemblages maintain fO2 conditions similar to the quartz-fayalite-magnetite (QFM) buffer at fore-arc conditions. Sulphides are stable under such conditions and aqueous fluids contain minor S. At sub-arc depth, dehydration occurs under more reducing conditions producing aqueous fluids carrying H2S. This finding brings into question current models in which serpentinite-derived fluids are the cause of oxidized arc magmatism and has major implications for the global volatile cycle, as well as for redox processes controlling subduction zone geodynamics.

The BPV-4 co-carcinogen quercetin induces cell cycle arrest and up-regulates transcription from the LCR of BPV-4.

Bracken fern is the environmental co-carcinogen of BPV-4 in the induction of neoplasias of the upper alimentary canal of cattle. The flavonoid quercetin is one of the most potent and best characterised mutagens present in the fern. We have shown that transfection with BPV-4 DNA and exposure to a single dose of quercetin leads to tumorigenic transformation of primary bovine cells. We now show that quercetin induces cell cycle arrest and up-regulates transcription from the BPV-4 long control region (LCR). This up-regulation is mediated by a 21 nucleotide-long cis-element in the LCR, designated QRE-1, which is located immediately downstream of the TATA box. Cellular proteins bind to QRE-1 and removal or substitution of QRE-1 lead to the abrogation of the response to quercetin. As expression of the viral oncogenes is controlled by the LCR, perturbation in this control and increased oncoprotein expression are likely to contribute to fully malignant cell transformation by overcoming the cell cycle arrest induced by quercetin, thus forcing damaged cells to proliferate.

Microtubules and the formation of acetylcholine receptor clusters in chick embryonic muscle cells.

We have used the microtubule-stabilizing drug taxol to examine the relationship between microtubules and the appearance and cell surface distribution of acetylcholine receptors (AChRs) in primary cultures of chick embryonic muscle cells. Taxol at a 5-microM concentration induced the large scale polymerization of tubulin in muscle cells that was most obvious as intermittent bundles of microtubules along the myotube. Prominent bundles of microtubules were also clearly visible in the fibroblasts. This concentration of taxol had no significant effect on the incorporation rate, increased synthesis induced by brain extract or the total cell surface number of AChRs measured over a 24-h period. Thus, excess polymerization of microtubules does not affect the movement of receptors to the cell surface. However, when cell surface AChR distribution was examined using rhodamine-conjugated alpha-bungarotoxin, taxol treatment of myotubes was shown to induce the aggregation of receptors. If receptors were labeled before taxol addition, aggregation of these prelabeled receptors was also seen, a result indicating that taxol can induce the movement of receptors already in the membrane. We believe this evidence further implicates microtubules as being involved in the movement of these cell surface receptors in the plane of the myotube membrane.

Actin filaments and acetylcholine receptor clusters in embryonic chick myotubes.

The tumor-promoting drug 4 beta-phorbol 12-myristate 13-acetate (TPA) causes the loss of myofibrils in primary cultures of chick embryonic myotubes [9]. When myofibrils in chick myotubes are dispersed by TPA treatment (5 X 10(-8) M), there remains a class of non-myofibrillar actin filaments that are sensitive to subsequent breakdown by cytochalasin D. Microfilament bundles in fibroblasts in the same cultures seem unaffected by this TPA treatment, but are also broken down by cytochalasin D (0.2 micrograms/ml); this dose has little effect on myofibrils. We have previously shown that treatment of chick myotubes with cytochalasins would destabilize clusters of acetylcholine receptors (AChRs) [6]. In order to further examine the relationship between actin filaments and cell surface AChRs, we have used the receptor-specific ligand alpha bungarotoxin (a-BGT) to study the fate of AChR clusters in drug-treated and control myotubes. Cells treated with TPA showed no loss in the number of receptor clusters present on their surface. However, if these cells were also treated with cytochalasin D, cluster number was reduced to approximately the same value as seen for cytochalasin treatment alone (50% of the control value). These results suggest that the cytoskeletal link to these cell surface receptors is not mediated by attachment to the alpha actin-containing myofibrils, but rather clustered AChRs are stabilized by a class of non-myofibrillar actin filaments.

Calmodulin and acetylcholine receptor clustering in embryonic chick myotubes.

We have used the calmodulin antagonists, trifluoperazine (TFP) and calmidazolium, to study the potential role of this protein in the movement of acetylcholine receptors (AChRs) to and from the myotube membrane, as well as in the formation of clusters of AChRs within the plasma membrane. Neither calmidazolium (up to 10(-6) M) nor TFP (10(-5) M) inhibited receptor degradation or the incorporation of new receptors (12 to 24 h). In addition, neither drug blocked the increased synthesis of receptors induced by chick brain extract, nor significantly affected AChR clusters already in the plane of the membrane at the time of drug addition. However, both drugs blocked new receptor clusters (induced by a basement membrane extract from Torpedo electric organ) from forming. These results indicate that receptors can move to and from the cell membrane in a calmodulin-independent fashion, but movement in the plane of the membrane to form a cluster requires the participation of calmodulin.

Tau and HMW microtubule-associated proteins have different microtubule binding sites in vivo.

We have previously demonstrated the immunofluorescent localization of the tau and HMW MAPS on microtubules in vivo. When cells were treated with the non-ioni detergent triton before fixation, however treated with the non-ionic detergent triton before fixation, however, the immunofluorescent staining of microtubules with antiserum to HMW protein disappears, while the staining with anti-tubulin of anti-tau serum remains unaffected. Because of these differences in sensitivity to triton, we conclude that tau protein and HMW protein have different binding sites on microtubules. Parallel ultrastructural studies indicate that this loss of staining correlates with a loss of filamentous projections from the outer wall of cytoplasmic microtubules. These results indicate that in vivo the HMW protein is most probably distributed along the outer surface of the microtubule as filamentous projections similar to those observed in vitro. The in vivo distribution of the HMW protein suggests that it may form part of the microtrabecular system and be involved in the interaction of microtubules with other cell organelles.

Centrioles are lost as embryonic myoblasts fuse into myotubes in vitro.

Embryonic chick myoblasts possess an extensive network of cytoplasmic microtubules which emanate from a single, perinuclear centrosome containing a microtubule-organizing center (MTOC) and the centrioles. However, after myoblasts fuse into myotubes the centrosome is no longer apparent, and instead long parallel arrays of microtubules are seen. From ultrastructural studies on developing muscle tissue, it has been proposed that centrioles are present in myoblasts but are absent from fused muscle fibers. We have examined this hypothesis in vitro in cultures of chick embryonic muscle cells using sera which specifically label centrioles. Almost all (90-97%) mononucleated cells in these cultures, including myoblasts aligned just prior to fusion, contain a pair of centrioles in close proximity to the nucleus. However, in newly fused multinucleated myotubes as well as in older myotubes that had developed myofibrils, centrioles were rarely found (1-10% positive cells). This study thus provides direct evidence for a loss of centrioles from muscle cells soon after they fuse to form myotubes.

A unique tubulin antiserum attenuates the rate of poleward chromosome movement in anaphase.

An antiserum against tubulin, NS20, was previously shown to specifically attenuate both fast axonal transport in vivo (Johnston, K. M. et al., Brain Res. 385, 38-45 (1986)) and in vitro (Johnston, K. M. et al., Cell Motil. Cytoskel. 7, 110-115 (1987)) and flagellar motility (Goldsmith, M. et al., Cell Motil. Cytoskel. 20, 249-262 (1991)). We hypothesized that NS20 blocked motility by binding to a multifunctional motor binding domain on the microtubules (MTs), or axonemes. Here we have examined the effect of microinjecting NS20, at metaphase, into dividing PtK2 cells. Plotting chromosome separation (CS) as a function of time, we report here that CS rates for anaphase A (chromosome-to-pole movement) were reduced by approximately 50% relative to uninjected controls. CS rates for anaphase B (spindle pole elongation) were unaffected by the NS20 antiserum. The inhibition of CS rate during anaphase A by NS20 was significantly greater than the inhibition caused by a control antitubulin serum (PC5). Two possible mechanisms underlying NS20's inhibition of CS during anaphase A were considered. NS20 could block the binding of a kinetochore-associated motor to kinetochore MTs (kMTs) or, alternatively, NS20 could stabilize kMTs against depolymerization. Our results favor the first alternative. In a cold-induced depolymerization assay, NS20 had no selective stabilizing effect on MTs. Moreover, we show that NS20 can selectively block the binding of a well characterized MT-associated motor (kinesin) to MTs, in vitro. These results suggest that NS20 may be defining a unique tubulin binding domain common to the motors underlying vesicle transport, flagellar motility, and chromosome movements during anaphase A.

Local recurrence after radical prostatectomy: characteristics in size, location, and relationship to prostate-specific antigen and surgical margins.

OBJECTIVES: To define the sonographic characteristics of local cancer recurrence after radical prostatectomy. METHODS: in 114 patients with an elevated prostate-specific antigen (PSA) and negative bone scan, 156 ultrasound-guided prostate fossa biopsies were carried out. RESULTS: in 53.5%, biopsy proved local recurrence. More than one ultrasound-guided biopsy session was required to make the diagnosis in 33% of patients. Local recurrence was seen on ultrasound at the anastomotic site (66%), the bladder neck (16%), and posterior to the trigone (13%). in 5% of patients there was a normal-appearing anastomotic site. Transrectal ultrasound was greater than 90% sensitive in detecting local recurrence, but lacked specificity. Examination of the radical prostatectomy specimens in patients with local recurrence showed positive surgical margins in 66% and organ-confined disease in 20%. CONCLUSIONS: Transrectal ultrasonography is a useful adjunct to PSA and digital rectal examination in the detection of local recurrences following radical prostatectomy.

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.

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.

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.

Management of yolk sac tumors in children.

The current trend toward minimizing treatment morbidity in children with yolk sac carcinomas is laudable. As most children will present with stage I disease and be cured by radical orchiectomy alone, careful surveillance is adequate with the knowledge that effective chemotherapy is available should tumor recur. All new yolk sac carcinomas in children should be reported to the Prepubertal Testicular Tumor Registry and should ideally be treated at institutions that have experience with surveillance protocols and pediatric oncology. In this way, prospective studies on treatment options can be initiated, and management controversies may ultimately be ironed out. In particular, more information is needed on patients more than 2 years old, as the literature to date is contradictory regarding the prognosis for these patients and the need for adjuvant therapy when the older child presents with stage I disease.

Should cryosurgery be considered a therapeutic option in localized prostate cancer?

Cryosurgical ablation of the prostate currently is under investigation as a potential treatment for localized prostate cancer. Results to date indicate that the majority of patients have negative biopsies and a marked reduction in prostate-specific antigen levels following cryotherapy. This treatment, however, is associated with significant side effects, notably bladder outflow obstruction, impotence, and incontinence, and its long-term durability is still unknown.