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J J Otto

Publications and source records attributed to J J Otto.

At least 19 recordsLinked to original sources
PubMed1998

Changes in the F-actin cytoskeleton during neurosensory bristle development in Drosophila: the role of singed and forked proteins.

Drosophila neurosensory bristle development provides an excellent model system to study the role of the actin-based cytoskeleton in polarized cell growth. We used confocal fluorescence microscopy of isolated thoracic tissue to characterize changes in F-actin that occurred during macrochaete development in wild type flies and mutants that have aberrant bristle morphology. At the earliest stages in wild type bristle development, cortical patches of F-actin were present, but no bundles were observed. Actin bundles began to form at 31% of pupal development and became more prominent as development progressed. The F-actin patches gradually disappeared and were no longer present by 38% of pupal development. The distribution of F-actin in singed3 mutant macrochaetae was indistinguishable from wild type bristles until 35% of development when the actin bundles began to splay and appear ribbon-like. In forked36a bristles, the mutant phenotype was evident at earlier stages of development than the singed3 mutant. Wild type tissue stained with antibodies against the forked protein demonstrated that the forked protein colocalized with F-actin structures found in early and late stage developing macrochaetae. Antibodies against the singed protein showed it appeared to localize with F-actin structures only at later stages in development. These data suggested that the forked gene product was required for the initiation of fiber bundle formation and the singed gene product was required for the maintenance of fiber bundle morphology during bristle development. Similar analyses of singed3/forked36a double mutants provided additional genetic evidence that the forked gene product was required before the singed gene product. Further, the analyses suggested that at least one additional crosslinking protein was present in these bundles.

Actins↗
PubMed1997

Vinculin and talin: kinetics of entry and exit from the cytoskeletal pool.

Vinculin and talin, two major components of focal contacts, exist in cytosolic and cytoskeletal pools. The kinetics of entry and exit of the two proteins between the two pools were investigated in normal and transformed cells. In cultured chick embryo fibroblasts, a fraction (2-5%) of the newly synthesized vinculin and talin reached maximal levels in the cytoskeleton in 30-45 min. Both proteins had 2-3 times shorter half-lives in the cytoskeletal pool (t1/2 = 6-7 h) than in the cytosolic pool (t1/2 = 14-15 h), which suggests that the incorporation of cytosolic vinculin and talin into the cytoskeleton does not involve a simple equilibrium between the two pools. However, after disruption of cell-to-substrate adhesion by trypsinization, an equilibrium in the incorporation between the two pools was transiently established, resulting in the use of the preexisting cytosolic pools of the two proteins during re-establishment of cell-to-matrix contacts. Viral transformation did not cause a significant change in the incorporation rates into the cytoskeleton. However, it decreased the half-lives of both proteins in the cytoskeletal pool (t1/2 = approximately 4 h) and in the cytosolic pool (t1/2 = 9-10 h). The increased turn-over rates of vinculin and talin in the cytoskeletal pool in transformed cells may contribute to the enhanced motility of transformed cells.

Animals↗
PubMed1996

Differences in turnover rates of vinculin and talin caused by viral transformation and cell density.

Vinculin and talin are two major components of focal contacts which interact with each other. In order to understand how the relative levels of these proteins are maintained under various conditions, the synthesis rates and half-lives of vinculin and talin in chick embryonic fibroblasts were determined by autoradiography combined with immunoblotting. High cell density and transformation by Rous sarcoma virus decreased the vinculin synthesis rate by 40%. Upon viral transformation, the synthesis rate of talin decreased by 30%. In contrast to vinculin, the synthesis rate of talin was not affected by cell density. The effect of cell density on the synthesis rate of vinculin was retained after viral transformation, suggesting that cell density and viral transformation affect vinculin synthesis by two independent mechanisms. The synthesis rate of vinculin was approximately two to three times greater than that of talin under all conditions tested. The half-lives of vinculin and talin remained constant at different cell densities in untransformed cells (t1/2 = 18-21 h), but transformation slightly decreased half-lives of both proteins (t1/2 = 16-18 h). These results suggest that the decreased expression of vinculin and talin in transformed chick fibroblasts can be attributed mainly to changes in their biosynthesis rates rather than degradation. This may contribute to a decrease in the number of focal contacts in transformed cells.

Animals↗
PubMed1996

Characterization of changes in F-actin during maturation of starfish oocytes.

Major morphological and mechanical changes occur in the starfish oocyte during maturation. Measurements made by quantitative fluorescence microscopy of fixed specimens stained with saturating levels of rhodamine-phalloidin demonstrated that changes in the level of F-actin in intact oocytes, in the endoplasm, and in the cortex may contribute to these changes. The level of F-actin increased transiently after exposure of oocytes to the maturation inducing hormone, 1-methyladenine (1-MA). This increase correlated with the formation of spikes on the cell surface. The level of F-actin decreased at the time of germinal vesicle breakdown (GVBD), which may account for the decrease in stiffness that occurs at this time. No increase in the level of F-actin was observed during formation of polar bodies, suggesting the existence of a secondary mechanism affecting oocyte stiffness. The changes in the amount of F-actin during oocyte maturation were largest in the cortex. The data also suggested that there are two distinct populations of cortical actin that are regulated both spatially and temporally; these are the F-actin in spikes and nonspike cortical F-actin. Changes in either or both of these populations of cortical actin were induced independently of GVBD by short exposures to 1-MA, induction of maturation with dithiothreitol, and pretreatment of immature oocytes with forskolin before adding 1-MA. Stabilization of F-actin by microinjection of phalloidin had no effect on GVBD. These results suggest that polymerization and depolymerization of actin during maturation are responsible for morphological and mechanical changes in the oocyte. In addition, the data suggest that the regulation of actin polymerization and depolymerization can be dissociated from GVBD.

Actins↗
PubMed1996

Relative changes in F-actin during the first cell cycle: evidence for two distinct pools of F-actin in the sea urchin egg.

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.

Actins↗
PubMed1994

Actin-bundling proteins.

Recent studies have greatly expanded our understanding of actin-bundling proteins. A new group of actin-bundling proteins, the fascins, has been recognized. An actin-bundling protein inhibits actin depolymerization even under conditions in which it cannot produce a gel, which suggests that bundling proteins may affect actin filament dynamics. A villin-like protein is present in Dictyostelium, shedding doubt on current ideas on the evolution of villin. Domain mapping continues to be a major thrust of research into most groups of bundling proteins.

Actinin↗
PubMed1993

Immunoblotting.

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Amido Black↗
PubMed1992

Vinculin binding site mapped on talin with an anti-idiotypic antibody.

Vinculin and talin are major adhesion plaque components which interact in vitro and presumably in vivo. The amino acid sequence of talin is now known so details of its domain structure can be mapped. We localized vinculin binding sites in the talin sequence by overlaying peptide maps of talin with an anti-idiotypic vinculin antibody that recognizes talin and with 125I-vinculin. A rabbit injected only twice with vinculin and producing anti-vinculin antibodies spontaneously generated a second antibody that recognizes talin. Vinculin and anti-vinculin antibodies specifically compete with this second antibody for binding to talin as determined by solid-phase binding and overlay assays. The antibody is thus most likely an anti-idiotypic antibody which mimics a region of vinculin that interacts with talin. The binding site of the anti-idiotypic antibody on talin was mapped to the 196 amino acids spanning residues 1653 to 1848. A second vinculin binding site identified with an 125I-vinculin blot overlay technique was located between residues 483 and 1652. The observation that talin has two immunologically distinct vinculin binding sites suggests that vinculin may have two different talin binding sites or one "complex" site with two interacting regions.

Amino Acid Sequence↗
PubMed1991

Snoods: a periodic network containing cytokeratin in the cortex of starfish oocytes.

An extensive fibrous cytoskeletal component in the cortical cytoplasm of oocytes of the starfish Pisaster ochraceus reproducibly stains with anticytokeratin antibody and hence contains cytokeratin. The large-meshed network resembles a snood (hair net). Snood fibers form loops and branches throughout the cortex of a premeiotic oocyte, except at the animal pole where they emanate from a nonstaining zone surrounding the centrosomes. By immunofluorescence microscopy of isolated cortices and electron microscopy of isolated cortices and intact oocytes, snood fibers exhibit complex striations with a periodicity of approximately 0.75 micron. Snoods are not colocalized with the cortical arrays of microtubules and are unaffected by drugs that disrupt microtubules or microfilaments. Stimulation of oocyte maturation by 1-methyladenine causes snoods to disappear, presumably by disassembly, about halfway to the time of germinal vesicle breakdown. They do not reappear during meiosis, fertilization, or development to the two-cell stage, and their functional importance, if any, during oogenesis or development remains to be elucidated.

Actin Cytoskeleton↗
PubMed1990

Purification and characterization of an 85 kDa talin-binding fragment of vinculin.

Vinculin and talin are adhesion plaque proteins which have been shown to interact with each other in vitro. In order to begin to investigate where the talin-binding domain is in vinculin, vinculin was digested with Staphylococcus aureus V8 protease to generate two major fragments of 85 and 30 kDa, and these fragments were purified. Nitrocellulose overlays with 125I-talin and the 125I-85 kDa vinculin fragment and sucrose density gradient centrifugation demonstrated that the talin-binding domain was localized to the 85 kDa vinculin fragment. Quantification of 125I-talin binding in the overlays showed that four times more talin bound to the 85 kDa fragment as compared to intact vinculin. Competitive immunoprecipitation experiments demonstrated that unlabeled 85 kDa fragment was about three-fold more effective at competing for 125I-85 kDa binding to talin than was unlabeled vinculin. These results suggest that the 30 kDa fragment inhibits the vinculin-talin interaction even though the talin-binding domain is localized in the 85 kDa fragment.

Binding Sites↗
PubMed1990

Vinculin.

Vinculin is clearly a key element in the transmembrane assemblages that link cells to each other or to the substrate. However, despite all the studies that have been done on the protein, we still do not know its function within these assemblages. The bulk of the biochemical and cell biological evidence suggests that, in some unknown way, its presence in the junctions may be involved in the stable association of actin with the membrane, yet vinculin by itself does not appear to interact with actin. In the future, identification of additional junctional molecules that interconnect actin and vinculin may resolve this dilemma. Alternatively, studies with vinculin that is phosphorylated or acylated may yield clues to its function. Perhaps the complexity of the protein composition of microfilament-containing junctions suggests that protein assemblages rather than individual proteins provide novel functions. As new proteins belonging to these junctions are discovered, it will be important to assess their interaction with already known components such as vinculin and to ask if the protein combination has a particular function.

Animals↗
PubMed1986

Hemocyanin respiratory pigment in bivalve mollusks.

Hemocyanins, high molecular weight oxygen-binding proteins, were identified in two species of protobranch bivalve mollusks, Acila castrensis and Yoldia limatula. Although hemocyanins have been reported in chitons, gastropods, and cephalopods, they have not been observed in the Class Bivalvia. In A. castrensis the dissociation products of hemocyanin, characterized by gel electrophoresis, had a subunit molecular weight of approximately 250K. Negatively stained preparations of extracted hemocyanin formed protein aggregates in the shape of cylinders measuring 35 by 38 nanometers. X-ray microanalysis of hemocyanin aggregates in thin sections of Y. limatula demonstrated the presence of copper in the molecules. The discovery of hemocyanin in the protobranchs reinforces the primitive nature of the taxon and is further evidence that the major molluscan classes have a common ancestry.

Animals↗
PubMed1986

The lack of interaction between vinculin and actin.

Vinculin was purified from chicken gizzard by a modification of the method of Feramisco and Burridge [1980; J Biol Chem 255:1194]. Vinculin did not alter the viscosity of actin as measured in an Ostwald viscometer, nor did it affect actin polymerization as measured with the fluorescent NBD-actin assay. Sedimentation experiments demonstrated that vinculin did not bind to actin, and electron microscopy of negatively stained specimens indicated that vinculin did not aggregate actin filaments into bundles. These results suggest that vinculin, by itself, does not interact with actin at least under commonly used conditions to assay actin-protein interactions in vitro.

Actin Cytoskeleton↗
PubMed1984

Assembly-disassembly of actin bundles in starfish oocytes: an analysis of actin-associated proteins in the isolated cortex.

One rapid response of starfish oocytes to the maturation-inducing hormone, 1-methyladenine (1-MA), is the formation of transient actin-filled spikes on the cell surface. The presence and distribution of G- and F-actin and several actin-associated proteins were examined in cortices isolated from oocytes before, during, and after spike formation by using antibodies and the F-actin-specific stain, NBD-phallacidin. Before 1-MA addition, staining with antiactin and NBD-phallacidin indicates that most of the actin in the cortex is either G-actin or oligomeric actin, but rather little is F-actin. Application of the hormone results in the conversion and redistribution of this cortical actin into large bundles of F-actin which form the cores of spikes. When the spikes recede, F-actin disappears, and the amount of all forms of actin bound in the cortex appears to decrease. Antibodies to sea urchin egg myosin, fascin and a 220-kDa protein were used to examine these actin-associated proteins during the times that the organization of actin changes. Myosin and the 220-kDa protein are bound to the cortex and uniformly distributed before 1-MA application while fascin appears to be unbound. When spikes appear after 1-MA addition, fascin and the 220-kDa protein are localized coincidently with the spikes, whereas myosin remains uniformly distributed throughout the cortex and is excluded from the spikes. After spike resorption, fascin and the 220-kDa protein appear to lose their cortical binding while myosin retains its localization unchanged. These results indicate that actin, fascin and the 220-kDa protein undergo major organizational changes in the cortex in response to 1-MA.

Actins↗
PubMed1984

Microtubule arrays in the cortex and near the germinal vesicle of immature starfish oocytes.

An extensive array of long, crisscrossing microtubules has been discovered in the cortex of oocytes of the starfish Pisaster ochraceus. The microtubules were visualized in cortex preparations by indirect immunofluorescence microscopy using antibodies to tubulin. The cortical array of microtubules is present in all oocytes before and for about 30 min after the application of 1-methyladenine, the hormone that induces oocyte maturation. The presence of microtubules was confirmed by electron microscopy. The microtubules in this array are depolymerized when oocytes are treated with colchicine or nocodozole and are augmented when oocytes are treated with taxol. Dihydrocytochalasin B treatment of the oocytes causes the microtubules to aggregate, presumably by altering a microfilament network also found in the cortex. The distribution of microtubules was also explored in whole oocytes stained with antitubulin. One or two aster-like structures were observed adjacent to the germinal vesicle of each oocyte.

Adenine↗