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R H Gomer

Publications and source records attributed to R H Gomer.

At least 37 records · Page 2Linked to original sources
PubMed1996

RtoA links initial cell type choice to the cell cycle in Dictyostelium.

In Dictyostelium, initial cell type choice is correlated with the cell-cycle phase of the cell at the time of starvation. We have isolated a mutant, ratioA (rtoA), with a defect in this mechanism that results in an abnormally high percentage of prestalk cells. The rtoA gene has been cloned and sequenced and codes for a novel protein. The cell cycle is normal in rtoA. In the wild type, prestalk cells differentiate from those cells in S or early G2 phase at starvation and prespore cells from cells in late G2 or M phase at starvation. In rtoA mutants, both prestalk and prespore cells originate randomly from cells in any phase of the cell cycle at starvation.

Amino Acid Sequence↗
PubMed1996

A Dictystelium mutant with defective aggregate size determination.

Starved Dictyostelium cells aggregate into groups of roughly 10(5) cells. We have identified a gene which, when repressed by antisense transformation or homologous recombination, causes starved cells to form large numbers of small aggregates. We call the gene smlA for small aggregates. A roughly 1.0 kb smlA mRNA is expressed in vegetative and early developing cells, and the mRNA level then decreases at about 10 hours of development. The sequence of the cDNA and the derived amino acid sequence of the SmlA protein show no significant similarity to any known sequence. There are no obvious motifs in the protein or large regions of hydrophobicity or charge. Immunofluorescence and staining of Western blots of cell fractions indicates that SmlA is a 35x10(3) Mr cytosolic protein present in all vegetative and developing cells and is absent from smlA cells. The absence of SmlA does not affect the growth rate, cell cycle, motility, differentiation, or developmental speed of cells. Synergy experiments indicate that mixing 5% smlA cells with wild-type cells will cause the wild-type cells to form smaller fruiting bodies and aggregates. Although there is no detectable SmlA protein secreted from cells, starvation medium conditioned by smlA cells will cause wild-type cells to form large numbers of small aggregates. The component in the smlA-conditioned media that affects aggregate size is a molecule with a molecular mass greater than 100x10(3) Mr that is not conditioned media factor, phosphodiesterase or the phosphodiesterase inhibitor. The data thus suggest that the cytosolic protein SmlA regulates the secretion or processing of a secreted factor that regulates aggregate size.

Amino Acid Sequence↗
PubMed1995

PSF and CMF, autocrine factors that regulate gene expression during growth and early development of Dictyostelium.

Throughout growth and development, Dictyostelium cells secrete autocrine factors that accumulate in proportion to cell density. At sufficient concentration, these factors cause changes in gene expression. Vegetative Dictyostelium cells continuously secrete prestarvation factor (PSF). The bacteria upon which the cells feed inhibit their response to PSF, allowing the cells to monitor their own density in relation to that of their food supply. At high PSF/bacteria ratios, which occur during late exponential growth, PSF induces the expression of several genes whose products are needed for cell aggregation. When the food supply has been depleted, PSF production declines, and a second density-sensing pathway is activated. Starving cells secrete conditioned medium factor (CMF), a glycoprotein of Mr 80 kDa that is essential for the development of differentiated cell types. Antisense mutagenesis has shown that cells lacking CMF cannot aggregate, and preliminary data suggest that CMF regulates cAMP signal transduction. Calculations indicate that a mechanism of simultaneously secreting and recognizing a signal molecule, as used by Dictyostelium to monitor cell density, could also be used to determine the total number of cells in a tissue.

Animals↗
PubMed1995

Initial cell-type choice in a simple eukaryote: cell-autonomous or morphogen-gradient dependent?

Dictyostelium discoideum is a simple eukaryote that lives as an amoeba until starvation triggers aggregation. The aggregate forms a slug which then develops into a fruiting body with two main cell types, stalk and spore cells. Two mechanisms have been proposed to explain cell-type differentiation. Studies using expression of the ecmA gene as a prestalk cell marker indicated that gradients of morphogens determine cell fate in the slug. However, studies using dyes or the cysteine proteinase 2 (CP2) gene product as a prestalk cell marker indicated that cell autonomous factors such as cell-cycle phase at the time of starvation cause an initial choice of cell fate. To help resolve these differences, we have used transformed cells containing the promoter of the prestalk gene ecmA fused to beta-galactosidase (Jermyn and Williams, 1991) to study the differentiation of Dictyostelium cells at low cell density, at which cell-to-cell interactions and morphogen gradients are minimal. We find that under all conditions of low cell density in which express ion the ecmA fusion gene occurs, it is invariably detected in less than 25% of the cells from a clonal population. This suggests that a cell-autonomous mechanism is involved in ecmA expression. We then used double-labeled immunofluorescence to examine the ontogeny of the CP2-positive and the ecmA-positive cells. In developing aggregates, 9 to 12% of the cells are CP2-positive from 12 to 24 hr of development. The ecmA-positive cells are first detected at 16 hr as a subset of the CP2-positive cells and then increase in number. At approximately 20 hr, the CP2-positive cells and the ecmA-positive cells are almost completely overlapping sets. By late development, all of the CP2-positive cells are ecmA-positive and an additional 10% of the CP2-negative cells are also ecmA-positive. This indicates that up to 20 hr development, ecmA is expressed only in CP2-positive cells. The data thus suggest that cell-cycle phase at the time of starvation causes an initial choice of cell type and that during later development other factors influence cell fate.

Animals↗
PubMed1995

A density-sensing factor regulates signal transduction in Dictyostelium.

Dictyostelium discoideum initiates development when cells overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation. The activations of Ca2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.

Animals↗
PubMed1994

Cell density-sensing in Dictyostelium by means of the accumulation rate, diffusion coefficient and activity threshold of a protein secreted by starved cells.

The simple eukaryote Dictyostelium discoideum grows as an amoeba on leaf and soil surfaces. When starved, the amoebae aggregate and differentiate. The amoebae can also be induced to differentiate as isolated cells submerged in buffer, if the buffer contains a sufficiently high concentration of a protein (CMF) secreted by starved cells. CMF is also necessary for aggregation and differentiation on surfaces. This indicated that CMF has either an autocrine function or is part of a density-sensing system. To distinguish between these two possible functions, we first examined the rate at which CMF is accumulated and the activity threshold of cells for CMF, since both parameters will affect whether a cell can provide enough CMF to self-stimulate. We find that CMF potentiates its own accumulation, and that otherwise the accumulation rate and activity threshold are affected very little by a variety of physiological conditions. We then use diffusion calculations to show that even after many hours of continuous secretion, the CMF concentration adjacent to an isolated starved cell on a leaf or soil surface will be too low to allow differentiation, whereas an extracellular concentration of CMF sufficiently high to allow differentiation will occur when starved cells are at high densities. We find a close match between the predicted and experimentally observed density necessary for differentiation. The theoretical and observed behavior of cells at different cell densities suggests that due to its accumulation rate, diffusion coefficient, and activation threshold, CMF can function as part of a cell density-sensing system which allows Dictyostelium cells in the wild to co-ordinate their development.

Animals↗
PubMed1994

A developmentally regulated cell surface receptor for a density-sensing factor in Dictyostelium.

Conditioned medium factor (CMF) is an 80-kDa glycoprotein which is the signal in a cell density-sensing system used by developing Dictyostelium cells. CMF is slowly secreted by cells when they starve, and the extracellular level of CMF then becomes an indicator of the density of starving cells. To examine how CMF is sensed, we have made bacterially synthesized recombinant CMF and found that it has as much activity as native CMF, indicating that glycosylation is not part of the active site of CMF. Expression of recombinant fragments of CMF indicates that the active site lies within an 88-amino acid region near the N terminus. To determine whether CMF is sensed by cell surface receptors, we examined binding of iodinated recombinant CMF to live cells. We found saturable binding to 6-h starved cells at 3.9 x 10(4) molecules/cell with a KD of 2.1 nM. The binding saturates in 30 min, and a Scatchard plot indicates that there is only one class of receptor. The binding is competed off by the addition of either the native or recombinant CMF, or the 88-amino acid active fragment region; no binding competition is seen from the nonactive regions or other proteins. Very little binding to vegetative cells is seen, with maximal binding seen in cells starved for 6-8 h. The amount of cell surface CMF binding then decreases during later development. Normal levels of CMF binding are seen to CMF- cells, indicating that CMF is not required for the accumulation of its own receptor.

Animals↗
PubMed1994

Different temporal patterns of expression result in the same type, amount, and distribution of filamin (ABP) in cardiac and skeletal myofibrils.

The morphogenesis of functional myofibrils in chick skeletal and cardiac muscle occurs in greatly different time spans, in about 7 and 2 days, respectively. In chick skeletal myogenic cells, one isoform of the 250 kD actin-binding protein (ABP) filamin is associated with stress fiber-like structures of myoblasts and early myotubes, then disappears for approximately 4 days, whereupon a second filamin isoform reappears at the Z-disc periphery. We sought to determine if cardiac myogenesis involves this sequence of appearance, disappearance, and reappearance of a new filamin isoform in a compressed time scale. It was known that in mature heart, filamin is localized at the Z-disc periphery as in mature (fast) skeletal muscle, and is also associated with intercalated discs. We find that myocardial filamin has an apparent molecular weight similar to that of adult skeletal muscle filamin and lower than that of smooth muscle filamin, and that both skeletal and cardiac muscle contain roughly 200 filamin monomers per sarcomere. Two-dimensional peptide mapping shows that myocardial filamin is very similar to skeletal muscle filamin. Myocardial, slow skeletal, and fast skeletal muscle filamins are all phosphorylated, as previously shown for filamin of non-striated muscle. Using immunofluorescence, we found that filamin could not be detected in the developing heart until the 14-somite stage, when functional myofibrils exist and the heart has been beating for 3 to 4 hours. We conclude that in cardiac and skeletal myogenesis, different sequences of filamin gene expression result in myofibrils with similar filamin distributions and isoforms.

Animals↗
PubMed1993

Skelemin, a cytoskeletal M-disc periphery protein, contains motifs of adhesion/recognition and intermediate filament proteins.

In striated muscle, myofibrils are anchored to an interconnecting cytoskeleton of desmin intermediate filaments. Skelemin (195 kDa) may be a link between myofibrils and the intermediate filament cytoskeleton. Skelemin partitions with desmin to the insoluble cytoskeleton, and increases the thickness of reconstituted intermediate filaments. Concentrated at the M-disc periphery, skelemin may also contact myosin filaments. We used immunoscreening to isolate a mouse muscle cDNA which encodes a protein with a calculated molecular mass of 185 kDa. Anti-skelemin antibodies bound to the protein products of each of three nonoverlapping regions of the open reading frame. Antibodies directed against the protein products of each one-third of the cDNA react with a 195-kDa muscle protein and stain the M-disc indistinguishably from the original anti-skelemin antibodies, suggesting that the cDNA encodes skelemin. A single skelemin mRNA is detected in muscle but not non-muscle tissues, consistent with immunostaining results. Skelemin is a member of a family of myosin-associated proteins containing fibronectin type III and immunoglobulin superfamily C2 motifs. Skelemin is unique in this family in having intermediate filament core-like motifs, one near each terminus. We hypothesize that skelemin could interact with myosin or myosin-associated proteins through its fibronectin and/or immunoglobulin motifs, and with intermediate filaments through intermediate filament-like motifs.

Amino Acid Sequence↗
PubMed1993

Characterization of the signal transduction pathways and cis-acting DNA sequence responsible for the transcriptional induction during growth and development of the lysosomal alpha-mannosidase gene in Dictyostelium discoideum.

The lysosomal alpha-mannosidase gene in Dictyostelium discoideum is representative of a small group of genes that are expressed under two different conditions: 1) immediately upon removal of the bacterial food source from exponentially growing cells at < 5 x 10(5) cells/ml (which also initiates the developmental cycle), and 2) when the concentration of a secreted glycoprotein termed the prestarvation response factor (PSF) reaches a critical threshold in cultures growing at densities > 5 x 10(5) cells/ml. In this report we show that transcription of the alpha-mannosidase gene induced by starvation did not require protein synthesis in axenic wild-type strains, whereas protein synthesis was required for the transcriptional induction observed in response to PSF. Northern blot analysis was also done using mRNA from G alpha 1 and G alpha 2 gene disruption mutants. These genes encode subunits of heterotrimeric G proteins found at the cell surface in growing cells and cells early in development. The pattern of alpha-mannosidase gene expression was normal in these mutants as well as in mutants unable to produce the secreted glycoprotein conditioned medium factor or the cAMP receptor-1 protein. These genes have been shown to regulate the expression of many genes during early development. Promoter analysis studies identified a 145-base pair sequence element containing a TTG box which was required for alpha-mannosidase transcriptional induction under both starvation conditions and in response to PSF. The TTG box identified is an important regulatory element in the promoter of another prestarvation response gene, the discoidin I gamma gene. A ts mutant was found to misregulate the expression of both discoidin I and alpha-mannosidase expression at restrictive temperatures. Taken together these results suggest that the prestarvation response genes may be coordinately regulated possibly through the TTG box.

Animals↗
PubMed1992

Growing and starving Dictyostelium cells produce distinct density-sensing factors.

Prestarvation factor (PSF) and conditioned medium factor (CMF) are two autocrine factors produced by Dictyostelium cells. Although secreted at different times in the Dictyostelium life cycle (PSF by growing cells and CMF by starving cells), both factors are glycoproteins that are used by cells to measure their own density, and both are important in cell aggregation. To examine the relationship between PSF and CMF, a CMF antisense transformant was tested for the production of PSF during growth. Although this transformant produced extremely low levels of CMF, its production of PSF was essentially normal. We conclude that these two factors are not products of the same gene.

Animals↗
PubMed1992

A density-sensing factor controls development in Dictyostelium.

For an unknown reason, several genes expressed during Dictyostelium development are regulated by cell density. This is mediated by an 80-kD glycoprotein, conditioned medium factor (CMF), which is slowly secreted and simultaneously sensed by starved cells. To examine further this eukaryotic cell density-sensing mechanism, we have isolated a cDNA encoding CMF. The derived amino acid sequence of CMF shows no obvious similarity to any known protein and thus may represent a new class of eukaryotic intercellular signal. CMF antisense transformants do not aggregate, whereas normal development is restored by the addition of purified CMF protein. This suggests that CMF might synchronize the onset of development in Dictyostelium by triggering aggregation when a majority of the cells in a given area have starved, as signaled by CMF secretion.

Amino Acid Sequence↗
PubMed1991

A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium.

In submerged monolayer culture, Dictyostelium cells can differentiate into prespore and prestalk cells at high cell densities in response to cAMP but not at low cell densities. However, cells at low densities will differentiate in medium taken from developing cells starved at a high density. The putative factor in the medium was designated CMF for conditioned medium factor (Mehdy and Firtel, Molec. cell. Biology 5, 705-713, 1985). In this report, we size-fractionate conditioned medium and show that the activity that allows low density cells to differentiate can be separated into high and low Mr (relative molecular mass) fractions. Interestingly, the two fractions both have the same activity and do not need to be combined to allow differentiation. The large conditioned medium factor is a protein, as determined by trypsin sensitivity, that can be purified to a single 80 x 10(3) Mr band on a silver-stained SDS-polyacrylamide gel, and has CMF activity at a concentration of approximately 4 pM (0.3 ng ml-1). Our results suggest that CMF is a secreted factor that functions in vivo as an indicator of cell density in starved cells. At high cell densities, the concentration of CMF is sufficient to enable cells to enter the multicellular stage of the developmental cycle. When present below a threshold concentration, cells do not initiate the expression of genes required for early development. This factor plays an essential role in the regulatory pathway necessary for cells to obtain the developmental competence to induce prestalk and prespore gene expression in response to cAMP.

Animals↗
PubMed1991

Regulation and processing of a secreted protein that mediates sensing of cell density in Dictyostelium.

During Dictyostelium development, the expression of some genes is dependent on cell density. This effect is mediated by soluble factors referred to as conditioned medium factors (CMFs) which the developing cells secrete at very low rates and simultaneously sense. There are at least two classes of CMFs: one is an 80 x 10(3) Mr glycoprotein and the other is a heterogeneous group of molecules, with relative molecular masses between 6.5 x 10(3) and 0.65 x 10(3). Interestingly, the two classes of molecules do not need to be combined for activity. We find that the 80 x 10(3) Mr CMF but not the small CMF is sequestered in vegetative cells. The 80 x 10(3) Mr CMF is then secreted by cells during early development, while the small CMF appears only during late development. Like the 80 x 10(3) Mr CMF, the small CMFs are trypsin-sensitive and contain N- and O-linked glycosylation. The breakdown products of a fraction containing 80 x 10(3) Mr CMF cochromatographed from a Sephadex G-50 column and a reverse-phase HPLC column with small CMFs. The specific activity of CMF increases roughtly 100-fold upon breakdown. The results suggest that, during differentiation, the slowly diffusing 80 x 10(3) Mr CMF is first produced from a precursor pool already present in vegetative cells, allowing differentiation of only those cells in the immediate vicinity of the aggregation center. The breakdown of 80 x 10(3) Mr CMF to a faster-diffusing, higher specific activity form then might enable cells farther from the aggregation center to differentiate.

Animals↗
PubMed1990

Paralog chromatography.

As a mixed mode ligand, a small peptide can mimic an antibody's paratope (antigen recognition site). This report describes the construction of a representative set of paratope analogs, or "paralogs," which can be conjugated to a chromatographic sorbent to combine desirable characteristics of traditional high-performance liquid chromatography columns with the specificity of a moderate affinity antibody. The broad utility of this novel set of protein separatory reagents is illustrated on the complex mixture of proteins in a yeast lysate.

Antibodies↗
PubMed1989

Mitoskelin: a mitochondrial protein found in cytoskeletal preparations.

A 70 kD protein, which we have named mitoskelin, is highly enriched in cytoskeletal preparations from bovine cardiac muscle. Mitoskelin has three main variants with isoelectric points between 5.6 and 5.8. Immunoblotting with polyclonal antibodies directed against mitoskelin shows that, like intermediate filament proteins, the majority of mitoskelin resists solubilization from a myocardial homogenate by a series of extraction solutions ranging from very low salt to 0.6 M KI buffers and by 0.1-1% Nonidet P-40 detergent. By double-label immunofluorescence on cells and tissues, mitoskelin is colocalized with the mitochondrial marker cytochrome c oxidase. Mitoskelin is associated with the inner membranes of mitochondria as shown by immunoelectron microscopy and immunoblotting. Immunological cross-reactivity and similarities of molecular weight, pI, distribution, and chromatographic properties indicate that mitoskelin is the 70 kD component of complex I (NADH: ubiquinone oxidoreductase), a portion of the mitochondrial oxidative phosphorylation system. No function or activity has yet been demonstrated for the 70 kD component of the 25-polypeptide complex I. Dialysis against physiological buffers allows purified, urea-solubilized mitoskelin to form 10 nm wide filamentous structures that do not closely resemble intermediate filaments. These results suggest the exciting possibility that mitochondria may contain a membrane-associated filamentous skeleton.

Animals↗