Identified taste bud cell proliferation in the perihatching chick [published erratum appears in Chem Senses 1998 Aug;23(4):459]

Developing taste buds in the anterior mandibular floor of perihatching chicks were studied by high voltage electron microscopic autoradiography in order to identify proliferating gemmal cell types. Montaged profiles of 29 taste buds in five cases euthanized between embryonic day 21 and posthatching day 2 were analyzed after a single [3H]thymidine injection administered on embryonic day 16, 17 or 18. Results showed that dark cells comprised 55% of identified (n = 900 cells) and 62% of labeled (n = 568 cells) gemmal cells as compared with light, intermediate, basal or perigemmal bud cells. Dark cells had both a greater (P < 0.05) number of labeled cells and a greater amount of label (grains/nucleus) than the other four bud cell types, irrespective of injection day. The nuclear area (micron 2) of dark cells was not significantly larger (P > 0.05) than that of the other gemmal cell types and therefore cannot account for the greater amount for label in the dark cells. Interestingly, only dark cells showed a positive correlation (P < 0.003) between amount of label and nuclear area. Results suggest that, during the perihatching period of robust cell proliferation, dividing dark cells may give rise primarily, but not exclusively, to dark cell progeny.      

Neurotrophin-3 induced by tri-iodothyronine in cerebellar granule cells promotes Purkinje cell differentiation [published erratum appears in J Cell Biol 1998 Dec 28;143(7):following 2090]

Thyroid hormones play an important role in brain development, but the mechanism(s) by which triiodothyronine (T3) mediates neuronal differentiation is poorly understood. Here we demonstrate that T3 regulates the neurotrophic factor, neurotrophin-3 (NT-3), in developing rat cerebellar granule cells both in cell culture and in vivo. In situ hybridization experiments showed that developing Purkinje cells do not express NT-3 mRNA but do express trkC, the putative neuronal receptor for NT-3. Addition of recombinant NT-3 to cerebellar cultures from embryonic rat brain induces hypertrophy and neurite sprouting of Purkinje cells, and upregulates the mRNA encoding the calcium-binding protein, calbindin-28 kD. The present study demonstrates a novel interaction between cerebellar granule neurons and developing Purkinje cells in which NT-3 induced by T3 in the granule cells promotes Purkinje cell differentiation.     

Midzone microtubule bundles are continuously required for cytokinesis in cultured epithelial cells [published erratum appears in J Cell Biol 1996 Dec;135(6 Pt 1):1679]

The current model of cytokinesis proposes that spindle poles and associated microtubules determine the cleavage plane, and, once the signal has been delivered to the cortex, the entire mitotic apparatus can be removed without affecting cell division. While supported by compelling data from Echinoderm embryos, recent observations suggest that the model may not be universally applicable. In this study, we have examined the relationship(s) among microtubules, chromosomes, and cleavage activity in living normal rat kidney (NRK) cells with multipolar mitotic figures. We found that cleavage activity correlated with the distribution of midzone microtubule bundles and Telophase Disc 60 protein (TD60) rather than the position of spindle poles. In addition, reduction of midzone microtubules near the cortex, by either nocodazole treatment or spontaneous reorganization in tripolar cells, caused inhibition or regression of furrowing. These results demonstrate that continuous interaction between midzone microtubule bundles and the cortex is required for successful cleavage in tissue culture cells.     

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 x 10(-7) cm2/S, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 x 10(-7) cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 x 10(-8) cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITC-dextran indicated that FITC-dextran was relatively excluded excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.     

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes [published erratum appears in J Cell Biol 1990 Dec;111(6 Pt 1):following 2800]

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.     

A 13-A map of the actin-scruin filament from the limulus acrosomal process [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1621]

We have determined the structure of the actin-scruin filament to 13-A resolution using a combination of low-dose EM and image analysis. The three-dimensional map reveals four actin-actin contacts: two within each strand and two between strands. The conformation of the actin subunit is different from that in the Holmes et al. (1990) model as refined by Lorenz et al. (1993). In particular, subdomain II is tilted in a similar way to that seen by Orlova and Egelman (1993) in F-Mg2(+)- ADP actin filaments in the absence of Ca2+. Scruin appears to consist of two domains of approximately equal volume. Each scruin subunit cross- links the two strands in the actin filament. Domain I of scruin contacts subdomain I of actin and makes a second contact at the junction of subdomains III and IV. Domain II of scruin contacts actin at subdomains I and II of a neighboring actin subunit. The two scruin domains thus bind differently to actin.     

Detyrosination of alpha tubulin does not stabilize microtubules in vivo [published erratum appears in J Cell Biol 1990 Sep;111(3):1325-6]

The relationship between alpha tubulin detyrosination and microtubule (MT) stability was examined directly in cultured fibroblasts by experimentally converting the predominantly tyrosinated MT array to a detyrosinated (Glu) array and then assaying MT stability. MTs in mouse Swiss 3T3 cells displayed an increase in Glu immunostaining fluorescence approximately 1 h after microinjecting antibodies to the tyrosinating enzyme, tubulin tyrosine ligase. Detyrosination progressed to virtual completion after 12 h and persisted for 30-35 h before tyrosinated subunits within MTs were again detected. The stability of these experimentally detyrosinated MTs was tested by first injecting either biotinylated or Xrhodamine-labeled tubulin and then measuring bulk turnover by hapten-mediated immunocytochemistry or fluorescence recovery after photobleaching, respectively. By both methods, turnover was found to be similarly rapid, possessing a half time of approximately 3 min. As a final test of MT stability, the level of acetylated tubulin staining in antibody-injected cells was compared with that observed in adjacent, uninjected cells and also with the staining observed in cells whose MTs had been stabilized with taxol. Although intense Glu staining was observed in both injected and taxol-treated cells, increased acetylated tubulin staining was observed only in the taxol-stabilized MTs, indicating that the MTs were not stabilized by detyrosination. Together, these results demonstrated clearly that detyrosination does not directly confer stability on MTs. Therefore, the stable MTs observed in these and other cell lines must have arisen by another mechanism, and may have become posttranslationally modified after their stabilization.     

Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p [published erratum appears in J Cell Biol 1996 Dec;135(6 Pt 2):1925]

The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small- budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal- Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.     

Signal transduction in Dictyostelium fgd A mutants with a defective interaction between surface cAMP receptors and a GTP-binding regulatory protein [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Transmembrane signal transduction was investigated in four Dictyostelium discoideum mutants that belong to the fgd A complementation group. The results show the following. (a) Cell surface cAMP receptors are present in fgd A mutants, but cAMP does not induce any of the intracellular responses, including the activation of adenylate or guanylate cyclase and chemotaxis. (b) cAMP induces down-regulation and the covalent modification (presumably phosphorylation) of the cAMP receptor. (c) The inhibitory effects of GTP gamma S and GDP beta S on cAMP binding are reduced; the stimulatory effect of cAMP on GTP gamma S binding is lost in fgd A mutants. (d) Basal high-affinity GTPase activity is reduced 40% and the stimulatory effect of cAMP is decreased from 40% in wild type to 30% in fgd A. (e) GTP-mediated stimulation and inhibition of adenylate cyclase is normal in mutant membranes. The results suggest a defective interaction between cell surface cAMP receptors and a specific G-protein in fgd A mutants. This interaction appears to be essential for nearly all signal transduction pathways in Dictyostelium discoideum.     

Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1623]

The long arm of laminin, which binds heparin and cells, consists of three polypeptides (A, B1, and B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). Previously, we found that recombinant globular domain (rG) supported heparin and myoblast binding (Yurchenco, P. D., U. Sung, M. D. Ward, Y. Yamada, and J. J. O'Rear. 1993. J. Biol. Chem. 268:8356-8365). To further analyze long arm functions, we expressed the distal moiety of the mouse laminin A chain extending from the middle of the rod to the carboxyl terminus (rAiG). This larger glycoprotein, secreted by Sf9 insect cells infected with recombinant baculovirus, was intercalated in vitro into the corresponding disulfide-linked B chain segments of laminin fragment E8 (distal long arm rod and proximal globule). The hybrid molecule (B- rAiG) possessed a structure similar to laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG with E8-B chains, the affinity of G domain for heparin decreased from that observed with rAiG and rG to one similar to native protein. HT1080 cells adhered to E8, rAiG, and B-rAiG, less well to rG, and not to denatured E8/B-rAiG, the A and B chain moieties of E8, or to a mixture of rG and E8-B chains. Cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with antibodies specific for alpha 6 and beta 1 integrin chains. Since intercalation (a) restored a conformationally dependent alpha 6 beta 1 integrin recognition site present in native protein, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site present in proximal G domain, we conclude that biological activities of laminin are different from that of its isolated subunits.