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G Piperno

Publications and source records attributed to G Piperno.

At least 55 records · Page 3Linked to original sources
PubMed1987

Cross-reactivity of antibodies specific for flagellar tektin and intermediate filament subunits.

Monoclonal antibodies specific for each of the flagellar tektins were prepared and used to determine whether structures similar to tektin filaments are present in cells lacking cilia or flagella. This analysis was performed by double-label immunofluorescence microscopy of several cell lines and by immunoblots of protein fractions. Two of the four anti-tektin antibodies, the antibodies 3-7-1 and 3-10-1, which bind different epitopes of the C-tektin, label 3T3, HeLa, PtK2, and BHK-21 cells as well as myotubes. The antibody 3-7-1 stains intermediate filament structures in the cells and binds vimentin or desmin in preparations of cytoskeletal proteins; whereas the antibody 3-10-1 stains nuclear envelopes in the cells and binds lamin A and C in preparations of cytoskeletal proteins or nuclear lamina. Structural similarities between the C-tektin and intermediate filament proteins probably are extended to more than two epitopes because polyclonal antibodies anti-vimentin and anti-desmin bind to C-tektin. These polyclonal antibodies also bind to A-tektin. The cross-reaction of monoclonal and polyclonal antibodies binding to epitopes in tektin and intermediate filament components and the existence of a high content of alpha-helical structure in the tektin subunits (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22) indicate that tektin and intermediate filaments are homologous in several parts of their structure.

Animals↗
PubMed1986

Cytoplasmic microtubules containing acetylated alpha-tubulin in Chlamydomonas reinhardtii: spatial arrangement and properties.

A monoclonal antibody, 6-11B-1, specific for acetylated alpha-tubulin (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) was used to study the distribution of this molecule in interphase cells of Chlamydomonas reinhardtii. Double-label immunofluorescence was performed using 6-11B-1, and 3A5, an antibody specific for all alpha-tubulin isoforms. It was found that acetylated alpha-tubulin is not restricted to the axonemes, but is also present in basal bodies and in a subset of cytoplasmic microtubules that radiate from the basal bodies just beneath the plasma membrane. Immunoblotting experiments of basal body polypeptide components using 6-11B-1 as a probe confirmed that basal bodies contain acetylated alpha-tubulin. In the cell body, 6-11B-1 stained an average of 2.2 microtubules/cell, while 3A5 stained an average of 6.5 microtubules. Although exposure to 0 degrees C depolymerized both types of cytoplasmic microtubules, exposure to various concentrations of colchicine or nocodazole showed that the acetylated microtubules are much more resistant to drug-induced depolymerization than nonacetylated microtubules. Axonemes and basal bodies are already known to be colchicine-resistant. All acetylated microtubules appear, therefore, to be more drug-resistant than nonacetylated microtubules. The acetylation of alpha-tubulin may be part of a mechanism that stabilizes microtubules.

Acetylation↗
PubMed1985

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms.

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.

Acetylation↗
PubMed1984

Monoclonal antibodies to dynein subunits reveal the existence of cytoplasmic antigens in sea urchin egg.

Monoclonal antibodies directed against subunits of a sea urchin flagellar dynein were used to test for the presence of cytoplasmic antigens in preparations of fertilized eggs and mitotic apparati . A 9-10 S complex composed of 330,000-, 134,000-, and 126,000-mol-wt subunits was isolated from outer arms of Strongylocentrotus purpuratus sperm flagella and used to characterize the antibodies. Seven monospecific antibodies to the 330,000 subunit and two against the 134,000 subunit of the 9-10 S complex were identified by binding to nitrocellulose blots of electrophoretograms resolving polypeptides from different dynein preparations. The antibodies were applied also to blots of polypeptides from fertilized sea urchin egg at the first metaphase and a cellular fraction of mitotic apparati . Three of the antibodies to the 330,000 subunit bound to a cytoplasmic polypeptide of approximately the same molecular weight and the two antibodies to the smaller subunits recognized a polypeptide of 124,000 apparent molecular weight. Both antigens appeared to be enriched in the fraction containing mitotic apparati . These results indicate that polypeptides similar to two subunits of the 9-10 S complex are present in eggs at metaphase, and they are apparently associated with the mitotic apparatus.

Adenosine Triphosphatases↗
PubMed1981

Inner arm dyneins from flagella of Chlamydomonas reinhardtii.

Two dyneins have been isolated from axonemes of Chlamydomonas flagella by a three step procedure consisting of extraction in a high salt containing buffer, hydroxyapatite chromatography and sedimentation in sucrose gradient. A dynein with Mg+2- dependent ATPase activity 6.0 mumole Pi/min/mg, sedimenting at 12.5S was found associated with a polypeptide of molecular weight 310,000. A second dynein with specific activity of 3.7, sedimenting at 10-11S was found associated with a polypeptide of molecular weight 315,000. In their most purified forms, the two dyneins are complexed with nonstoichiometric amounts of four polypeptides ranging in molecular weight between 42,000 and 19,000. The 42,000 component has been identified previously as an actin-like protein. The high molecular weight subunits of both dyneins and two polypeptides of 28,000 and 19,000 molecular weight were found to be phosphorylated by in vivo pulse-labeling with 32P-phosphoric acid. All components of the 12.5S and 10-11S dynein complexes, with the exception of the 19,000 polypeptide, form a subset of polypeptides found to be deficient in pf-23, a chlamydomonas mutant, which is defective for inner arms.

Adenosine Triphosphatases↗
PubMed1981

Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components.

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S-containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8-7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild-type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.

Adenosine Triphosphatases↗
PubMed1981

Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function.

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes (pf-17, pf-24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes. Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology. Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci (pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components. In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants. In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides.

Chlamydomonas↗
PubMed1981

Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants.

Four mutants of Chlamydomonas reinhardtii representing independent gene loci have been shown to lack totally (pf-18, pf-19, and pf-15) or nearly totally (pf-20) the central microtubular pair complex in isolated axonemal preparations. Analysis of 35S-labeled axonemal proteins, using two methods of electrophoresis, reveals that all four mutants lack or are markedly deficient in 18 polypeptides, ranging in molecular weight from 360,000 to 20,000, that are regularly present in wild-type axonemes. Analyses of axonemal proteins labeled by cellular growth on 32P-labeled medium indicates that a subset of 8 of the 18 polypeptides are phosphorylated. Mutant and wild-type axonemes and flagella have been analyzed for their content of tubulin subunits using a high resolution two-dimensional electrophoresis system combined with agarose gel overlays containing either anti-alpha or anti-beta tubulin sera prepared from Chlamydomonas tubulins. The immunoprecipitates identify two major alpha tubulins, a major beta tubulin, and a minor component which is also precipitated by the anti-beta serum. None of these tubulins shows a specific defect in mutant axonemes, nor do the tubulin polypeptides show altered two-dimensional map positions in the mutant flagella. The 18 polypeptides provide a useful signature for identifying other mutants affecting the central-pair microtubular complex. Such mutants could be useful in defining the structural or functional role of these polypeptides in the central microtubules. Efforts to obtain additional central-pair mutants based on the motility phenotype of the four mutants analyzed here have yielded mutants which are allelic to three of the four mutants.

Chlamydomonas↗
PubMed1980

Similarities and dissimilarities between calmodulin and a Chlamydomonas flagellar protein.

A protein that resembles vertebrate calmodulins and troponin C has been isolated from Chlamydomonas flagella by using a calmodulin purification protocol that included calcium-dependent affinity-based adsorption chromatography on phenothiazine-Sepharose conjugates. The flagellar protein resembled calmodulin in elution from reverse-phase columns, had a peptide map similar to that of calmodulin, and competed with vertebrate calmodulin in a radioimmunoassay using antisera against vertebrate calmodulin. However, this flagellar protein did not activate phosphodiesterase, lacked N epsilon-trimethyllysine, and had an isoelectric point approximately 0.3 pH unit higher than that of vertebrate calmodulin. When analyzed by polyacrylamide gel electrophoresis under various conditions, the Chlamydomonas protein migrated between vertebrate calmodulins and rabbit skeletal muscle troponin C and did not manifest a large calcium-dependent mobility shift. This calmodulin-like protein was identified as one of the approximately 200 35S-labeled components in Chlamydomonas flagella resolved by two-dimensional gel electrophoresis. These studies indicate that calmodulin and a structurally and functionally homologous protein are present in the same cell. These studies also demonstrate that caution is necessary: (i) in identifying a protein as a calmodulin, (ii) in using phenothiazines or antisera directed against vertebrate calmodulins as specific probes for calmodulin, and (iii) in the interpretation of experiments on biological systems in which calmodulin is substituted for the homologous calmodulin-like protein.

Calcium↗
PubMed1979

An actin-like protein is a component of axonemes from Chlamydomonas flagella.

Several lines of evidence indicate that a component of axonemes from Chlamydomonas flagella is similar to actin from rabbit skeletal muscle. The polypeptide has an apparent molecular weight of 42,000 and in a pH gradient has the electrophoretic behavior of beta-actin. It was co-polymerized with rabbit actin and purified by affinity chromatography on DNase I-Sepharose. Incomplete proteolysis of mixed 35S-labeled axonemal protein and cold rabbit actin formed similar sets of peptides as analyzed by one-dimensional gel electrophoresis. The actin-like protein and the tubulin appear to be present in the axoneme in the molar ratio 1:60.

Actins↗
PubMed1977

Microtubular proteins of Chlamydomonas reinhardtii. An immunochemical study based on the use of an antibody specific for the beta-tubulin subunit.

An immunochemical assay for tubulin subunits is described. The method is applied directly to homogenates of Chlamydomonas reinhardtii solubilized in sodium dodecyl sulfate (Na dodecyl-SO4), and it makes use of a two-dimensional electrophoresis system; the first separation is carried out by Na dodecyl-SO4-polyacrylamide gel electrophoresis and the second by electrophoresis into an agarose gel containing antibodies. Tubulin is precipitated in the form of a "rocket" and the method is made quantitative through the use of cells labeled with [35S]sulfate. The antiserum used in this assay was prepared in rabbits using beta subunit of tubulin purified from Chlamydomonas flagella by two preparative Na dodecyl-SO4-polyacrylamide gel electrophoreses. This antiserum and an antiserum to alpha subunit of tubulin from porcine brain, prepared for comparative study, were extensively characterized. Both antisera show specificity for the polypeptide used as antigen and react with the native dimeric tubulin. The antiserum to beta subunit from Chlamydomonas flagella also forms immunoprecipitates with native brain tubulin and its beta subunit when used at high titer. In contrast, the antiserum to alpha subunit from porcine brain does not cross-react with Chlamydomonas tubulin. The immunochemical assay was applied to Chlamydomonas cells synchronized by a 12-h light/dark cycle. In cells collected during the light period (late G1), after removal of flagella, the content of tubulin is estimated to be 0.3% of total protein. As cells enter the dark period there is a striking increase in tubulin content which reaches a maximum just before cell division.

Animals↗
PubMed1977

Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii.

Flagellar polypeptides of Chlamydomonas reinhardtii were analyzed in two-dimensions by isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate. In addition to flagellar tubulin, over 130 polypeptides were resolved and 100 of these were identified as axonemal components in wild-type organisms. Flagella of two nonconditional paralyzed mutants, pf 14 and pf 1, were also analyzed and, at the same time, electron microscopic studies were carried out. pf 14 flagella, which completely lack radial spokes and associated spokeheads, are missing 12 polypeptides. Six of these polypeptides are also missing from pf 1 flagella in which spokes are clearly present but spoke heads appear to be absent.

Cell Movement↗
PubMed1977

Flagellar mutants of Chlamydomonas: studies of radial spoke-defective strains by dikaryon and revertant analysis.

The motility mutant of Chlamydomonas reinhardtii pf14 lacks radial spoke structures in its flagellar axonemes, and 12 proteins present in wild type are missing from a two-dimensional map (isoelectrofocusing/sodium dodecyl sulfate electrophoresis) of its (35)S-labeled flagellar proteins. Six of these same proteins are missing in pf1, which lacks spoke-heads. To determine whether any of the missing proteins represent the mutant gene product two experimental approaches have been applied. The first makes use of the fact that gametes of either mutant strain when fused with wild-type gametes to form quadriflagellate dikaryons undergo recovery of flagellar function. Recovery at the molecular level was monitored by prelabeling the mutant proteins with (35)S and allowing recovery to occur in the absence of protein synthesis. It is to be expected that the mutant gene product would not be restored as a radioactive protein and that recovery would depend on the assembly of the wild-type counterpart that is not labeled. The second technique makes use of revertants induced by UV irradiation. Dikaryon rescue in the case of pf14 leads to restoration of 11 radioactive components; only protein 3 fails to appear as a radioactive spot. For pf1 only two radioactive proteins are restored; proteins 4, 6, 9, and 10 were not radioactive. Analysis of revertants of pf1 gave evidence (altered map positions) that protein 4 is the mutant gene product. In the case of pf14, analysis of 22 revertants has not provided similar positive evidence that protein 3 is the gene product.

Alleles↗