Biomedical subjects

L M Quarmby

Publications and source records attributed to L M Quarmby.

13 recordsLinked to original sources

PubMed2000

Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas.

A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.

Amino Acid Sequence↗

PubMed1999

Microtubule severing.

The regulation of microtubule stability by severing of the polymer along its length is a newly appreciated and potentially important mechanism for controlling microtubule function. Microtubule severing occurs in living cells, but direct observation of this event is infrequent. The paucity of direct observations leave open to question the significance of regulated microtubule severing in the control of microtubule organization. Nevertheless, several lines of evidence suggest that microtubule severing is an important cellular activity. First, the ATP-dependent microtubule-severing activity of katanin is well documented. Katanin is found in most cell types and is enriched at MTOCs. Although it is possible that katanin does not sever microtubules in vivo, this seems unlikely. Second, a physiological event, deflagellation, has been shown to depend on microtubule severing. The deflagellation system of Chlamydomonas has provided a genetic approach to the problem of microtubule severing. The FA genes are essential for the regulated severing of axonemal microtubules during deflagellation, but whether these genes define new severing proteins or whether they are important for katanin activity remains to be determined. Microtubule severing is a relatively new area of investigation and there are still many more questions than answers. It is anticipated that the recent cloning of katanin and the introduction of a genetic model system will soon lead to significant breakthroughs in this problem.

Adenosine Triphosphatases↗

PubMed1999

Cloning of Chlamydomonas p60 katanin and localization to the site of outer doublet severing during deflagellation.

Katanin, a heterodimeric microtubule-severing protein that localizes to sites of microtubule organization, can mediate in vitro the ATP-dependent disassembly of both taxol-stabilized microtubules and axonemal doublet microtubules. In the unicellular biflagellate alga Chlamydomonas, katanin has been implicated in deflagellation, a highly specific process that involves a Ca(2+)-signal transduction pathway starting at the plasma membrane and culminating in the severing of axonemal outer doublet microtubules and excision of both flagella from the cell body. Previously, we showed that the microtubule severing activity of deflagellation and katanin's 60 kD catalytic subunit (termed p60) purified with the flagellar basal body complex (FBBC). Additional evidence supporting the involvement of katanin in deflagellation came from the observation that an antibody against human p60 katanin significantly inhibited FBBC-associated microtubule-severing activity. Here we report the cloning of p60 katanin from Chlamydomonas reinhardtii. Immunogold electron microscopy places Chlamydomonas p60 at several locations within the basal body apparatus and associated structures. Importantly, we find a dense accumulation of colloidal gold labeling the distal end of the flagellar transition zone, the site of outer doublet severing during deflagellation. These results suggest that, in addition to a potential involvement in the deflagellation pathway, katanin-mediated microtubule-severing may be associated with multiple processes in Chlamydomonas.

Adenosine Triphosphatases↗

PubMed1998

A role for katanin-mediated axonemal severing during Chlamydomonas deflagellation.

Deflagellation of Chlamydomonas reinhardtii, and other flagellated and ciliated cells, is a highly specific process that involves signal-induced severing of the outer doublet microtubules at a precise site in the transition region between the axoneme and basal body. Although the machinery of deflagellation is activated by Ca2+, the mechanism of microtubule severing is unknown. Severing of singlet microtubules has been observed in vitro to be catalyzed by katanin, a heterodimeric adenosine triphosphatase that can remove tubulin subunits from the walls of stable microtubules. We found that purified katanin induced an ATP-dependent severing of the Chlamydomonas axoneme. Using Western blot analysis and indirect immunofluorescence, we demonstrate that Chlamydomonas expresses a protein that is recognized by an anti-human katanin antibody and that this protein is localized, at least in part, to the basal body complex. Using an in vitro severing assay, we show that the protein(s) responsible for Ca2+-activated outer doublet severing purify with the flagellar-basal body complex. Furthermore, deflagellation of purified flagellar-basal body complexes is significantly blocked by the anti-katanin antibody. Taken together, these data suggest that a katanin-like mechanism may mediate the severing of the outer doublet microtubules during Chlamydomonas deflagellation.

Adenosine Triphosphatases↗

PubMed1998

Genetics of the deflagellation pathway in Chlamydomonas.

Signal-induced deflagellation in Chlamydomonas involves Ca2+-activated breakage of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. In this study, we isolated 13 new deflagellation mutants that can be divided into two phenotypic classes, the Adf class and the Fa class. Cells with the Adf deflagellation phenotype are defective in acid-stimulated Ca2+ influx, but can be induced to deflagellate by treatment with nonionic detergent and Ca2+. Genetic analyses show that the five new Adf mutations, as well as the previously identified adf1 mutation, are alleles of the ADF1 gene. Mutants in the second phenotypic class, the Fa mutants, fail to deflagellate in response to any known chemical stimulus and are defective in Ca2+-activated microtubule severing. Genetic analysis of these eight new Fa strains demonstrated that they define two complementation groups, and one of these contains the previously identified fa1 mutation. Diploid analysis showed that five alleles map to the FA1 gene, whereas four alleles define a novel gene that we have named FA2. The isolation of multiple mutant alleles of each gene, generated by either ultraviolet irradiation or insertional mutagenesis, indicates that ADF1, FA1, and FA2 may be the only genes that can be identified in a loss-of-function screen. These alleles should provide a better understanding of the regulation of microtubule severing by Ca2+.

Animals↗

PubMed1996

Ca2+ influx activated by low pH in Chlamydomonas.

Cytosolic acidification stimulates an influx of Ca2+ which results in shedding of the two flagella of Chlamydomonas. Ca2+ influxes are also involved in the photoresponses of this alga, but it is not understood how the acidification-activated Ca2+ influx is distinguished from the Ca2+ influxes which mediate phototaxis and the photophobic response. The present study focuses on the deflagellation-inducing Ca2+ influx pathway. Influx occurs through an ion channel or transporter with low abundance or low permeability to Ca2+ (approximately 500 fmol/s/10(6) cells in 50 microM Ca2+). Ca2+ influx was potently blocked by Cd3+ (EC50 approximately 5 microM), but was insensitive to Cd2+ (Quarmby, L.M., and H.C. Hartzell. 1994. J. Cell Biol. 124:807) and organic blockers of Ca2+ channels including SKF-96365 (up to 100 microM) and flufenamic acid (up to 1 mM). Experiments with a flagella-less mutant (bald-2), isolated flagella, and a blocker of flagellar assembly (colchicine) indicated that the acidification-stimulated Ca2+ influx pathway is not localized to the flagellar membrane. The acid-stimulated influx pathway was transiently inactivated after cells shed their flagella. Inactivation did not occur in the deflagellation mutant, fa-1, although acidification-stimulated Ca2+ influx was normal. This suggests that inactivation of this pathway in wild-type cells is probably not a direct consequence of acidification nor of Ca2+ influx, but may be related to deflagellation. Recovery of deflagellation-inducing Ca2+ influx occurred within 30 min after a 30 s exposure to acid and did not require flagellar assembly. The regulation, drug sensitivity, and subcellular localization identify acidification-stimulated Ca2+ influx as a specific Ca2+ entry pathway distinct from established Ca2+ channels.

Acids↗

PubMed1994

Signal transduction in the sexual life of Chlamydomonas.

Several signal transduction pathways play important roles in the sexual life cycle of Chlamydomonas. Nitrogen deprivation, perhaps sensed as a drop in intracellular [NH4+], triggers a signal transduction pathway that results in altered gene expression and the induction of the gametogenic pathway. Blue light triggers a second signalling cascade which also culminates in gene induction and completion of gametogenesis. New screens have uncovered several mutants in these pathways, but so far we know little about the biochemical events that transduce the environmental signals of nitrogen deprivation and blue light into the changes in gene transcription that produce gametes. Cell-cell contact of mature, complementary gametes elicits a number of responses that prepare the cells for fusion. Contact is sensed by the agglutinin-mediated cross-linking of flagellar membrane proteins. An increase in [cAMP] couples protein cross-linking to the mating responses. In C. reinhardtii the cAMP signal appears to be generated by the sequential stimulation of as many as 3 distinct adenylyl cyclase activities. Although the molecular mechanisms of adenylyl cyclase activations are poorly understood, Ca2+ may play a role. Most of the mating responses appear to be triggered by a cAMP-dependent protein kinase, but here too, Ca2+ may play a role. Numerous mutants are facilitating studies of the signalling pathways that trigger the mating responses. Cell fusion triggers another series of events that culminate in the expression of zygote specific genes. The mature zygote is sensitive to a light signal which stimulates the expression of genes whose products are essential for germination. The signal transduction pathways that trigger zygospore formation and germination are ripe for investigation in this experimentally powerful system.

Animals↗

PubMed1994

Dissection of eukaryotic transmembrane signalling using Chlamydomonas.

Novel insights and surprises are often generated when investigators choose an organism that permits a new approach to a problem. For example, secretory and cell-cycle mutants in yeast have provided quantum leaps in elucidating these processes. Similarly, genetic systems are providing exciting new insights into signal transduction. The 'green yeast' Chlamydomonas has the potential to be a particularly rich organism for genetic analysis of signal transduction because, although unicellular, it has several interesting behaviours, which are discussed in this article by Lynne Quarmby and Criss Hartzell. Phototaxis results from the transduction of a light signal received by the eyespot to changes in flagellar beat. The mating reactions, which culminate in the fusion of gametes, are initiated in response to adhesion of flagellar proteins. Deflagellation, or flagellar shedding, is an acute response to a variety of stimuli. Molecular genetic analysis of behavioural mutants is providing new directions for understanding signal integration and segregation.

Animals↗

PubMed1994

Two distinct, calcium-mediated, signal transduction pathways can trigger deflagellation in Chlamydomonas reinhardtii.

The molecular machinery of deflagellation can be activated in detergent permeabilized Chlamydomonas reinhardtii by the addition of Ca2+ (Sanders, M. A., and J. L. Salisbury, 1989. J. Cell Biol. 108:1751-1760). This suggests that stimuli which induce deflagellation in living cells cause an increase in the intracellular concentration of Ca2+, but this has never been demonstrated. In this paper we report that the wasp venom peptide, mastoparan, and the permeant organic acid, benzoate, activate two different signalling pathways to trigger deflagellation. We have characterized each pathway with respect to: (a) the requirement for extracellular Ca2+; (b) sensitivity to Ca2+ channel blockers; and (c) 45Ca influx. We also report that a new mutant strain of C. reinhardtii, adf-1, is specifically defective in the acid-activated signalling pathway. Both signalling pathways appear normal in another mutant, fa-1, that is defective in the machinery of deflagellation (Lewin, R. and C. Burrascano. 1983. Experientia. 39:1397-1398; Sanders, M. A., and J. L. Salisbury. 1989. J. Cell Biol. 108:1751-1760). We conclude that mastoparan induces the release of an intracellular pool of Ca2+ whereas acid induces an influx of extracellular Ca2+ to activate the machinery of deflagellation.

Animals↗

PubMed1993

Regulation of purified type I and type II adenylylcyclases by G protein beta gamma subunits.

Type I and type II adenylylcyclases have been purified after expression in Sf9 cells, each by application of a two-step purification protocol. The specific activities of the essentially homogeneous enzymes are approximately 7 and 2 mumol.min-1.mg-1, respectively. Each purified enzyme preparation is activated by Gs alpha, but they are regulated in an opposite fashion by G protein beta gamma subunits. Purified beta gamma inhibits Gs alpha-stimulated type I adenylylcyclase directly, while beta gamma activates type II adenylylcyclase and potentiates the Gs alpha-mediated stimulation of the enzyme. This is the first demonstration of the activation of a purified effector molecule by G protein beta gamma subunits.

Adenylyl Cyclases↗

PubMed1993

Mechanisms of flagellar excision. I. The role of intracellular acidification.

It has previously been shown that exposure of Chlamydomonas to low pH induces the cells to shed their flagella. In the present paper we report that a 30-s treatment with 20 mM acetic, carbonic, formic, or benzoic acid at pH 4.0 will induce flagellar excision. In contrast, 20 mM concentrations of the stronger aspartic, phosphoric, citric, and tartaric acids (pH 4) do not induce excision. Further, the excision efficacy of acetate is a function of the concentration of protonated acetate. Thus, excision correlates with the presence of a protonated, membrane-permeant species of acid. Relative to acetate, the more permeant benzoate induces excision at a much lower concentration of protonated acid. We conclude that a flux of acid into the cell is the signal for excision. Previous work has shown that detergent-permeabilized cells excise their flagella in response to calcium but not in response to low pH. This suggests that the acidification of intact cells triggers excision by stimulating an increase in intracellular calcium. We have previously reported that the source of this calcium might be IP3-sensitive. In our model for the mechanism of pH-induced flagellar excision, a flux of acid into the cell activates phospholipase C, leading to IP3 production, the activation of an IP3-gated calcium channel (located on either an intracellular or surface membrane), and an increase in cytosolic calcium, which is the trigger of flagellar excision.

Acids↗

PubMed1992

Inositol phospholipid metabolism may trigger flagellar excision in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii cells shed their flagella in response to environmental stress. Under favorable conditions, flagella are quickly regrown. To learn more about the signals that trigger flagellar excision and regrowth we have investigated inositol phospholipid metabolites, molecules implicated in signal transduction in several other systems. After deflagellation by low pH or mastoparan, a potent activator of G proteins, there was a rapid increase in levels of inositol 1,4,5-trisphosphate measured by use of receptor-binding assays and HPLC. This increase was concomitant with a decrease in levels of phosphatidylinositol 4,5-bisphosphate and was followed by an increase in phosphatidic acid, results consistent with activation of phospholipase C and diacylglycerol kinase. Additional experiments suggest that this activated phospholipase C is not important for flagellar regrowth but plays a role in informing the excision apparatus of the environmental stress. Addition of neomycin (an inhibitor of phospholipase C) before exposure of cells to low pH or mastoparan prevented the increase in inositol 1,4,5-trisphosphate and also prevented deflagellation. Addition of neomycin after deflagellation blocked increases in inositol 1,4,5-trisphosphate that normally followed deflagellation, but did not block flagellar assembly. Furthermore, a flagellar excision-defective mutant, fa-1, did not shed its flagella in response to low pH or mastoparan, yet both of these agents activated phospholipase C in these cells. The results suggest that activation of phospholipase C, possibly via a G protein, is a proximal step in the signal transduction pathway inducing deflagellation in Chlamydomonas.

Animals↗

PubMed1989

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman.

To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an alternative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [(3)H]glycerol, [(3)H]inositol, or [(32)P]orthophosphate. Analysis of the products of enzymic hydrolysis, of chemical deacylation, and of ;fingerprint' methanolysis of these phospholipids confirmed the identifications.

Journal Article↗