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G Gerisch

Publications and source records attributed to G Gerisch.

At least 55 records · Page 3Linked to original sources
PubMed1995

Coactosin interferes with the capping of actin filaments.

Coactosin, a 16 kDa protein associated with the actin cytoskeleton from Dictyostelium discoideum, was purified by an improved method, in which other components of the cytoskeleton were removed. The highly purified coactosin had no effect on the time course of actin polymerization, but when added to actin in presence of capping proteins, coactosin counteracted the capping activity of these proteins. The capping proteins cap32/34 and severin domain 1 retarded actin polymerization, on addition of coactosin to samples containing one of these capping proteins the time course of actin polymerization became close to controls without capping proteins.

Actins↗
PubMed1995

pDcsA vectors for strictly regulated protein synthesis during early development of Dictyostelium discoideum.

Two expression vectors have been constructed to express proteins exclusively in developing cells of Dictyostellium discoideum. In these Escherichia coli/D. discoideum shuttle vectors, proteins are synthesized under control of the promoter of the contact site A (csA) gene, which is efficiently suppressed during growth and becomes strongly activated during early development of D. discoideum. The pDcsA vectors appear to be valuable tools for the production of proteins that are not compatible with growth of D. discoideum cells.

Ampicillin Resistance↗
PubMed1995

Cell-substrate interactions and locomotion of Dictyostelium wild-type and mutants defective in three cytoskeletal proteins: a study using quantitative reflection interference contrast microscopy.

Reflection interference contrast microscopy combined with digital image processing was applied to study the motion of Dictyostelium discoideum cells in their pre-aggregative state on substrata of different adhesiveness (glass, albumin-covered glass, and freshly cleaved mica). The temporal variations of the size and shape of the cell/substratum contact area and the time course of advancement of pseudopods protruding in contact with the substratum were analyzed. The major goal was to study differences between the locomotion of wild-type cells and strains of triple mutants deficient in two F-actin cross-linking proteins (alpha-actinin and the 120-kDa gelation factor) and one F-actin fragmenting protein (severin). The size of contact area, AC, of both wild-type and mutant cells fluctuates between minimum and maximum values on the order of minutes, pointing toward an intrinsic switching mechanism associated with the mechanochemical control system. The fluctuation amplitudes are much larger on freshly cleaved mica than on glass. Wild-type and mutant cells exhibit remarkable differences on mica but not on glass. These differences comprise the population median of AC and alterations in pseudopod protrusion. AC is smaller by a factor of two or more for all mutants. Pseudopods protrude slower and shorter in the mutants. It is concluded that cell shape and pseudopods are destabilized by defects in the actin-skeleton, which can be overcompensated by strongly adhesive substrata. Several features of amoeboid cell locomotion on substrata can be understood on the basis of the minimum bending energy concept of soft adhering shells and by assuming that adhesion induces local alterations of the composite membrane consisting of the protein/lipid bilayer on the cell surface and the underlying actin-cortex.

Animals↗
PubMed1995

A talin homologue of Dictyostelium rapidly assembles at the leading edge of cells in response to chemoattractant.

In an attempt to identify unknown actin-binding proteins in cells of Dictyostelium discoideum that may be involved in the control of cell motility and chemotaxis, monoclonal antibodies were raised against proteins that had been enriched on an F-actin affinity matrix. One antibody recognized a protein distinguished by its strong accumulation at the tips of filopods. These cell-surface extensions containing a core of bundled actin filaments are rapidly protruded and retracted by cells in the growth-phase stage. The protein of 269 kD turned out to resemble mouse fibroblast talin (Rees et al., 1990) in its primary structure. The fit is best among the first 400-amino acid residues of the NH2-terminal region where identity between the two proteins is 44% and the last 200-amino acid residues of the COOH-terminal region with 36% identity. In the elongated cells of the aggregation stage the Dictyostelium talin is accumulated at the entire front where also F-actin is enriched. Since this protein exists in a soluble state in the cytoplasm, mechanisms are predicted that cause accumulation at sites of the cell where a front is established. Evidence for receptor-mediated accumulation was obtained by local stimulation of cells with cAMP. When a new front was induced by the chemoattractant, the talin accumulated there within half a minute, indicating a signal cascade in Dictyostelium responsible for assembly of the talin beneath sites of the plasma membrane where chemoattractant receptors are strongly activated. The ordered assembly of the talin homologue together with actin and a series of other proteins is considered to play a key role in chemotactic orientation.

Actins↗
PubMed1995

Motility and substratum adhesion of Dictyostelium wild-type and cytoskeletal mutant cells: a study by RICM/bright-field double-view image analysis.

To investigate the dynamics of cell-substratum adhesion during locomotion, a double-view optical technique and computer-assisted image analysis has been developed which combines reflection interference contrast microscopy (RICM) with bright-field imaging. The simultaneous recording of cell-substratum contact and cell body contour has been applied to aggregation-competent cells of Dictyostelium discoideum. These cells are distinguished from cells at earlier stages of development by small areas of contact to a substratum. Three questions have been addressed in analysing the locomotion of aggregation-competent cells. (1) What is the relationship between changes in the shape of cells and their contact to a substratum during a chemotactic response? (2) What is the relationship between protrusion and retraction of the cell body, and between local attachment and detachment? (3) Are there differences between wild-type and mutant cells that lack certain cytoskeletal proteins? During a chemotactic response the front region of the amoeba can bend towards the gradient of attractant without being supported by its contact with a surface, which excludes the necessity for gradients of adhesion for the response. The finding that in locomoting cells protrusion of the leading edge often precedes retraction establishes a pioneer role for the front region. The finding that gain of contact area precedes loss provides evidence for the coordination of interactions between the cell surface and a substratum. For comparison with wild-type, aggregation-competent triple mutant cells have been used that lack two F-actin crosslinking proteins, alpha-actinin and 120 kDa gelation factor, and an actin filament fragmenting protein, severin. Disturbances in the spatial and temporal control of cytoskeletal activities have been unravelled in the mutant by RICM and quantified by cross-correlation analysis of attachment and detachment vectors. In order to detect these disturbances, it was essential to analyse cell locomotion on the weakly adhesive surface of freshly cleaved mica.

Animals↗
PubMed1994

Replacement of the phospholipid-anchor in the contact site A glycoprotein of D. discoideum by a transmembrane region does not impede cell adhesion but reduces residence time on the cell surface.

The contact site A (csA) glycoprotein of Dictyostelium discoideum, a cell adhesion molecule expressed in aggregating cells, is inserted into the plasma membrane by a ceramide-based phospholipid (PL) anchor. A carboxyterminal sequence of 25 amino acids of the primary csA translation product proved to contain the signal required for PL modification. CsA is known to be responsible for rapid, EDTA-resistant cohesion of cells in agitated suspensions. To investigate the role of the PL modification of this protein, the anchor was replaced by the transmembrane region and short cytoplasmic tail of another plasma membrane protein of D. discoideum. In cells transformed with appropriate vectors, PL-anchored or transmembrane csA was expressed under the control of an actin promoter during growth and development. The transmembrane form enabled the cells to agglutinate in the presence of shear forces, similar to the PL-anchored wild-type form. However, the transmembrane form was much more rapidly internalized and degraded. In comparison to other cell-surface glycoproteins of D. discoideum the internalization rate of the PL-anchored csA was extremely slow, most likely because of its exclusion from the clathrin-mediated pathway of pinocytosis. Thus, our results indicate that the phospholipid modification is not essential for the csA-mediated fast type of cell adhesion but guarantees long persistence of the protein on the cell surface.

Amino Acid Sequence↗
PubMed1994

Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells.

When oxidative phosphorylation is inhibited in cells of Dictyostelium discoideum, the phosphorylation of tyrosine residues on actin is strongly increased. This increase is fully reversible. Under the same conditions the amoeboid cells undergo a series of shape changes. Within three minutes the pseudopods are withdrawn and replaced by cell surface blebs. Subsequently, the cells are rounding up to become immobile. In parallel with the changes in cell shape, the distribution of actin filaments is grossly altered within the cells. The cortical network of actin filaments of normal cells is broken down, and the F-actin forms large, irregular clusters deep within the cytoplasm. In these clusters the actin is associated with myosin II and with the heterodimeric F-actin capping protein cap32/34. After restoration of oxidative phosphorylation the actin returns within less than four minutes to its normal cortical position. A causal relationship between tyrosine phosphorylation and changes in the distribution of actin remains to be established. The rearrangements in the actin system that result from the inhibition of oxidative phosphorylation indicate that the organisation of this system and its maintenance in a functional state depend on the continuous supply of energy by ATP.

Actins↗
PubMed1993

Coactosin, a 17 kDa F-actin binding protein from Dictyostelium discoideum.

A 17 kDa protein, designated as coactosin, has been purified from an actin-myosin complex reconstituted in vitro from a soluble fraction of Dictyostelium discoideum cells. The protein binds to F-actin in vitro without significantly altering its viscosity. Immunoblots labeled with monoclonal antibodies indicate that part of the protein is associated with the detergent-insoluble cytoskeleton. cDNA clones comprising the entire coding region of coactosin have been isolated from an expression library. The cDNA-derived amino-acid sequence reveals similarities of coactosin to the drebrins identified in neurons and to actin-binding proteins from other organisms, including yeast ABP1p, and yeast and vertebrate cofilins.

Actomyosin↗
PubMed1993

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility.

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.

Actins↗
PubMed1993

Actin-associated proteins in motility and chemotaxis of Dictyostelium cells.

The amoeboid cells of Dictyostelium discoideum are amenable to a combined biochemical, genetic, and cell biological approach that can be focussed to the study of molecular interactions underlying the chemotactic responses of eukaryotic cells. In these responses the actin-based motility system is involved. This system is characterised in Dictyostelium cells by a large number and variety of regulatory proteins. Most of these proteins belong to families that are likewise represented in the cytoskeletons of higher eukaryotes including man. Elimination of some of these actin-binding proteins by chemical mutagenesis or gene disruption is being used to simplify the system by separating essential proteins from non-essential ones. These studies are complemented by the selection and analysis of mutants with altered motility or chemotaxis. Quantitative motion analysis of mutants is employed to establish a link between defects on the molecular level and alterations in cell behaviour. Dictyostelium cells respond to local stimulation by extending a newly formed leading edge towards a chemoattractant within less than a minute, thereby changing their polarity. The leading edge is formed by the recruitment of soluble proteins from the cytoplasm and their coassembly with actin into a complicated framework of microfilaments. Patterns of assembly are shown in this report for two proteins, the talin-like filopodin and coronin. Elucidation of the control mechanisms of this ordered assembly will provide the key for understanding the molecular processes responsible for a chemotactic response.

Actins↗
PubMed1992

Purification, functional characterization, and cDNA sequencing of mitochondrial porin from Dictyostelium discoideum.

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was purified by hydroxyapatite and CM-cellulose chromatography. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The channel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochondrial porin from D. discoideum was determined. It showed little sequence similarities to other known mitochondrial porins. The functional similarity, however, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by immunogold labeling of cryosections of fixed cells.

Amino Acid Sequence↗
PubMed1992

Overexpression of the csA cell adhesion molecule under its own cAMP-regulated promoter impairs morphogenesis in Dictyostelium.

The contact site A (csA) glycoprotein is a strictly developmentally regulated plasma membrane component responsible for the EDTA-stable (Ca(2+)-independent) form of intercellular adhesion in Dictyostelium discoideum. Using inverse polymerase chain reaction and a terminator vector we have isolated a 1.6 kb genomic fragment carrying a 1.1 kb upstream region of the csA gene. This fragment had promoter activity in D. discoideum cells, giving rise to a 3'-truncated csA RNA that was regulated like the mRNA of the endogenous gene. Cyclic AMP pulses strongly enhanced transcription from the cloned csA promoter. These findings provide evidence that the cloned region of the csA gene comprises the complete promoter. It contains a G/C-rich octamer motif similar to other cAMP-regulated D. discoideum promoters. When the csA protein was strongly overexpressed under the developmental control of the csA promoter, morphogenesis was substantially altered. Aggregation was delayed, and secondary centres were formed along aggregation streams that led to fragmentation of the aggregates and multiple slug formation. At high cell density a substantial portion of aggregated cells was left behind on the substratum when slugs and fruiting bodies were built. The transformation vector was also employed to rescue a csA-negative mutant, HG1287, from its cell adhesion defect.

Animals↗
PubMed1992

Stage-specific tyrosine phosphorylation of actin in Dictyostelium discoideum cells.

A 45 kDa protein in Dictyostelium discoideum cells that was recognized by a phosphotyrosine-specific antibody was identified by its binding activity to DNase I and its 2D-electrophoretic behavior as actin. The reactivity of actin with the antibody was transiently enhanced for about 30 minutes shortly after starving cells were reintroduced into nutrient medium. This effect indicates a modification of actin that is regulated under physiological conditions. A similar effect was obtained when growing cells were treated with phenylarsine oxide (PAO), an inhibitor of phosphotyrosine phosphatases. This effect was reversed and the cells fully recovered upon addition of the PAO antagonist 2,3-dimercaptopropanol. Starved cells did not show this enhancement of antibody labelling, which indicates that the response to PAO depends on the developmental stage. Phosphorylated amino acid residues were identified after in vivo labelling with [32P]phosphate in the presence of PAO. Part of the radioactivity in the actin band was recovered as phosphotyrosine, another part as phosphoserine. PAO caused the cells to form elongated blebs, to round up and finally to become immobilized. Fluorescence labelling with phalloidin of cells that were fixed at different times of PAO treatment revealed a progressive decrease in the staining for actin filaments and showed that these alterations in cytoskeleton organization were readily reversible, in accordance with the reversal of tyrosine phosphorylation at actin.

Actins↗
PubMed1991

cDNA sequence of cyclophilin from Dictyostelium discoideum.

A cDNA encoding a protein homologous to cyclophilins from other species has been isolated from a Dictyostelium discoideum cDNA library. From the deduced amino acid sequence a protein with a molecular mass of 19 kD and 64% identity with human cyclophilin is predicted. Southern blot analysis indicates that there is one cyclophilin gene in the D. discoideum genome. The mRNA is present in all developmental stages.

Amino Acid Isomerases↗
PubMed1991

Coronin, an actin binding protein of Dictyostelium discoideum localized to cell surface projections, has sequence similarities to G protein beta subunits.

A soluble actin binding protein of Dictyostelium discoideum cells has been extracted and purified from precipitated actin-myosin complexes. This protein with a relative molecular mass of 55 kDa has been named coronin because of its association with crown-shaped cell surface projections of growth-phase D. discoideum cells. In aggregating cells, which respond most sensitively to the chemoattractant cyclic AMP, coronin is accumulated at the front where surface projections are directed towards a cAMP source. Since these cells can quickly change shape and polarity, it follows that coronin is rapidly reshuffled within the cells during motion and chemotactic orientation. The cDNA derived sequence of coronin indicates a protein of 49 kDa, consisting of an amino-terminal domain with similarities to the beta subunits of G proteins and a carboxy-terminal domain with a high tendency for alpha-helical structure. It is hypothesized that coronin is implicated in the transmission of chemotactic signals from cAMP receptors in the plasma membrane through G proteins to the cortical cytoskeleton, whose structure and activity is locally modulated.

Amino Acid Sequence↗
PubMed1990

Replacement of threonine residues by serine and alanine in a phosphorylatable heavy chain fragment of Dictyostelium myosin II.

The target sites of soluble myosin heavy chain kinases partially purified from growth phase or aggregation competent cells of Dictyostelium discoideum were identified by the use of normal and mutated fragments of the myosin heavy chain. The kinases from both developmental stages phosphorylated two previously established threonine residues, as well as an additional one. The newly identified site is located within the putative core region of the coiled-coil formed by the myosin tail. A lysine following the phosphorylated threonine residue is the only common feature of the sequences around these sites. The kinases, which specifically phosphorylate threonine residues in wild-type myosin, did accept serine if it was in the right structural context.

Alanine↗