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M Schleicher

Publications and source records attributed to M Schleicher.

89 records · Page 5Linked to original sources
PubMed1989

A Dictyostelium mutant deficient in severin, an F-actin fragmenting protein, shows normal motility and chemotaxis.

A severin deficient mutant of Dictyostelium discoideum has been isolated by the use of colony immunoblotting after chemical mutagenesis. In homogenates of wild-type cells, severin is easily detected as a very active F-actin fragmenting protein. Tests for severin in the mutant, HG1132, included viscometry for the assay of F-actin fragmentation in fractions from DEAE-cellulose columns, labeling of blots with monoclonal and polyclonal antibodies, and immunofluorescent-labeling of cryosections. Severin could not be detected in the mutant using these methods. The mutation in HG1132 is recessive and has been mapped to linkage group VII. The mutant failed to produce the normal severin mRNA, but small amounts of a transcript that was approximately 100 bases larger than the wild-type mRNA were detected in the mutant throughout all stages of development. On the DNA level a new Mbo II restriction site was found in the mutant within the coding region of the severin gene. The severin deficient mutant cells grew at an approximately normal rate, aggregated and formed fruiting bodies with viable spores. By the use of an image processing system, speed of cell movement, turning rates, and precision of chemotactic orientation in a stable gradient of cyclic AMP were quantitated, and no significant differences between wild-type and mutant cells were found. Thus, under the culture conditions used, severin proved to be neither essential for growth of D. discoideum nor for any cell function that is important for aggregation or later development.

Actins↗
PubMed1989

The Dictyostelium gelation factor shares a putative actin binding site with alpha-actinins and dystrophin and also has a rod domain containing six 100-residue motifs that appear to have a cross-beta conformation.

The 120-kD gelation factor and alpha-actinin are among the most abundant F-actin cross-linking proteins in Dictyostelium discoideum. Both molecules are homodimers and have extended rod-like configurations that are respectively approximately 35 and 40 nm long. Here we report the complete cDNA sequence of the 120-kD gelation factor which codes for a protein of 857 amino acids. Its calculated molecular mass is 92.2 kD which is considerably smaller than suggested by its mobility in SDS-PAGE. Analysis of the sequence shows a region that is highly homologous to D. discoideum alpha-actinin, chicken fibroblast alpha-actinin, and human dystrophin. This conserved domain probably represents an actin binding site that is connected to the rod-forming part of the molecule via a highly charged stretch of amino acids. Whereas the sequence of alpha-actinin (Noegel, A., W. Witke, and M. Schleicher. 1987. FEBS [Fed. Eur. Biochem. Soc.] Lett. 221:391-396) suggests that the extended rod domain of the molecule is based on four spectrin-like repeats with high alpha-helix potential, the rod domain of the 120-kD gelation factor is constructed from six 100-residue repeats that have a high content of glycine and proline residues and which, in contrast to alpha-actinin, do not appear to have a high alpha-helical content. These repeats show a distinctive pattern of regions that have high beta-sheet potential alternating with short zones rich in residues with a high potential for turns. This observation suggests that each 100-residue motif has a cross-beta conformation with approximately nine sheets arranged perpendicular to the long axis of the molecule. In the high beta-potential zones every second residue is often hydrophobic. In a cross-beta structure, this pattern would result in one side of the domain having a surface rich in hydrophobic side chains which could account for the dimerization of the 120-kD gelation factor subunits.

Actinin↗
PubMed1988

Severin, gelsolin, and villin share a homologous sequence in regions presumed to contain F-actin severing domains.

cDNA clones encoding the actin filament severing protein severin from Dictyostelium discoideum were isolated from a cDNA library in lambda gt 11 using monoclonal antibodies. Comparison of the deduced amino acid sequence with the sequence of a severin peptide indicated that the complete coding region of severin is contained in the isolated clones. Severin, a 39.9-kDa protein, is encoded by one gene in D. discoideum. An mRNA of approximately 1.4 kilobases is present throughout the developmental cycle of D. discoideum. The amino acid sequence of severin contains a region highly homologous to a conserved sequence in villin and gelsolin, two proteins of similar function isolated from vertebrates. This homologous region is believed to participate in the actin filament severing activity of these proteins. Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which gelsolin has been derived by a duplication.

Actins↗
PubMed1988

Actin-binding proteins are conserved from slime molds to man.

DNA clones encoding the actin-binding proteins alpha-actinin and severin from Dictyostelium discoideum were isolated and sequenced. Comparisons of the deduced amino acid sequences with proteins from other species showed striking similarities at distinct regions. The F-actin cross-linking molecule alpha-actinin carries two characteristic EF-hand structures highly homologous to the Ca2+-binding loops of proteins from the calmodulin superfamily. An N-terminal region that is conserved in alpha-actinin from D. discoideum and vertebrates is also related to parts of the dystrophin sequence and might represent the F-actin binding site. Severin, gelsolin, villin, and fragmin share homologous sequences that are believed to participate in the severing activity of these proteins.

Amino Acid Sequence↗
PubMed1988

A Dictyostelium mutant with severe defects in alpha-actinin: its characterization using cDNA probes and monoclonal antibodies.

Cells of a Dictyostelium discoideum mutant deficient in binding a monoclonal antibody to alpha-actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for alpha-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3.1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybridized to all but a 3'-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95 x 10(3) Mr polypeptide of alpha-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the alpha-actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95 x 10(3) Mr and 88 x 10(3) Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95 x 10(3) Mr polypeptide only. The 88 x 10(3) Mr polypeptide most probably lacks the C-terminal portion of alpha-actinin. The binding of an antibody that recognized both polypeptides was quantified by a radio-immuno competition assay using wild-type alpha-actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their partial purification and SDS-polyacrylamide gel electrophoresis the mutant 95 x 10(3) Mr and 88 x 10(3) Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of alpha-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.

Actinin↗
PubMed1987

Calcium-sensitive non-muscle alpha-actinin contains EF-hand structures and highly conserved regions.

The F-actin crosslinking molecule alpha-actinin from the slime mould Dictyostelium discoideum carries two characteristic EF-hand structures at the C-terminus. The calcium-binding loops contain all necessary liganding oxygens and most likely form the structural basis for the calcium sensitivity of strictly calcium-regulated non-muscle alpha-actinins. Furthermore, the sequence exhibits at the N-terminal site of the molecule a high degree of homology to chicken fibroblast alpha-actinin. This stretch of amino acids appears to have remained essentially constant during evolution and might represent the actin-binding site. The findings have led us to propose a model for the inhibitory action of Ca2+ on non-muscle alpha-actinins.

Actinin↗
PubMed1986

Selection of Dictyostelium mutants defective in cytoskeletal proteins: use of an antibody that binds to the ends of alpha-actinin rods.

A monoclonal antibody, mAb 47-19-2, was used to study the subunit topology of the rod-shaped alpha-actinin molecules of Dictyostelium discoideum and to screen for mutants defective in the production of alpha-actinin. Electron microscopy of rotary-shadowed alpha-actinin-antibody complexes showed binding of mAb 47-19-2 to both ends of the alpha-actinin rods and cleavage of the rods into its subunits, indicating that the two subunits of alpha-actinin extend in an anti-parallel mode through the whole length of the rod. The antibody binding sites were located in close proximity to the sites responsible for actin cross-linking, which is consistent with the blocking activity of the antibody. In a mutant, HG1130, no antibody label was detected in colony blots, and by immunoblotting of mutant proteins separated by SDS-PAGE, only trace amounts of alpha-actinin were found. The mutant showed normal binding of antibodies directed against the actin-binding proteins severin and capping protein. The mutation responsible for the alpha-actinin defect was recessive and located on linkage group I of the genetic map of D. discoideum. HG1130 cells grew on bacteria at a normal rate and also axenically like cells of the parent strain AX2. After starvation the mutant cells expressed the contact site A glycoprotein, a marker of the aggregation-competent stage, and reacted chemotactically to cyclic AMP. The aggregation patterns and fruiting bodies of the mutant appeared to be normal. Patching and capping on the surface of HG1130 cells was induced by antibodies against the contact site A glycoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinin↗
PubMed1986

Studies on the transcription, translation, and structure of alpha-actinin in Dictyostelium discoideum.

A clone coding for the F-actin cross-linking protein alpha-actinin was obtained by screening a genomic library of Dictyostelium discoideum DNA in lambda gt11 with monoclonal antibodies specific for Dictyostelium alpha-actinin. The 1.2-kilobase (kb) genomic clone was confirmed as containing part of the alpha-actinin gene by comparing its nucleotide sequence with the amino acid sequence of tryptic peptides from purified alpha-actinin. The clone recognized a 3.0-kb message in a Northern blot. Hybridization to RNA isolated from different developmental stages of several D. discoideum strains indicated that the mRNA content increased during early development. A similar result was obtained when the alpha-actinin content of the cells was followed by Western blot analysis. Hybridization of the clone to DNA from different wild-type strains of D. discoideum indicated a polymorphism on the DNA level that coincided with a polymorphism on the protein level. The data suggest continuous transcription of the alpha-actinin gene throughout the development of D. discoideum, up- and down-regulation of the levels of alpha-actinin mRNA and protein with maximum levels at the onset of aggregation, and a high diversity of alpha-actinin at the DNA and protein level among different D. discoideum strains. The structural data make it conceivable that the highly conserved nature of alpha-actinin resides only at the functional sites, whereas the helical portions of the alpha-actinin molecule allow a higher level of diversity throughout evolution.

Actinin↗
PubMed1984

Isolation and characterization of calmodulin from the motile green alga Chlamydomonas reinhardtii.

Calmodulin, a calcium-binding protein with no known enzymatic activity but multiple, in vitro effector activities, has been purified to apparent homogeneity from the unicellular green alga Chlamydomonas reinhardtii and compared to calmodulin from vertebrates and higher plants. Chlamydomonas calmodulin was characterized in terms of electrophoretic mobility, amino acid composition, limited amino acid sequence analysis, immunoreactivity, and phosphodiesterase activation. Chlamydomonas calmodulin has two histidine residues similar to calmodulin from the protozoan Tetrahymena. However, unlike the protozoan calmodulin, only one of the histidinyl residues of Chlamydomonas calmodulin is found in the COOH-terminal third of the molecule. Chlamydomonas calmodulin lacks trimethyllysine but does have a lysine residue at the amino acid sequence position corresponding to the trimethyllysine residue in bovine brain and spinach calmodulins. The lack of this post-translational modification does not prevent Chlamydomonas calmodulin from quantitatively activating bovine brain phosphodiesterase. These studies also demonstrate that this unique calmodulin from a phylogenetically earlier eukaryote may be as similar to vertebrate calmodulin as it is to higher plant calmodulins, and suggest that Chlamydomonas calmodulin may more closely approximate the characteristics of a putative precursor of the calmodulin family than any calmodulin characterized to date.

Amino Acid Sequence↗
PubMed1984

New actin-binding proteins from Dictyostelium discoideum.

Dictyostelium discoideum contains a soluble actin-binding protein that caps actin filaments at their fast growing ends. The purified protein consists of two subunits with 34 kd and 32 kd apparent mol. wts. Like similar proteins from Acanthamoeba and bovine brain the capping protein from D. discoideum acts in a Ca -independent manner. It lacks severing activity as indicated by its inability to disrupt the stress fibers and the microfilament network in detergent-extracted cells. Two actin-binding proteins from a plasma membrane-enriched fraction were labeled with [I]actin using a gel overlay technique. One of these proteins, with an apparent mol. wt. of 17 kd in SDS-polyacrylamide gels, has been purified from high-salt extracts, the other protein with an apparent mol. wt. of 31 kd has been purified from Triton X-100 extracted membranes. Monoclonal antibodies were raised against D. discoideum severin, alpha-actinin, the larger subunit of the capping protein, and the 17-kd membrane-associated protein. Immunoblotting of proteins from whole cell lysates showed that all these actin-binding proteins were present in both growth phase and aggregation-competent cells.

Journal Article↗
PubMed1984

Structural characterization of a higher plant calmodulin : spinacia oleracea.

Calmodulin is a eukaryotic calcium binding protein which has several calcium-dependent in vitro activities. Presented in this report is a structural characterization of calmodulin from spinach leaves (Spinacia oleracea). Spinach calmodulin may be representative of higher plant calmodulins in general since calmodulin from the monocotyledon barley (Hordeum vulgare) is indistinguishable by a variety of physical, chemical, and functional criteria (Schleicher, Lukas, Watterson 1983 Plant Physiol 73: 666-670). Spinach calmodulin is homologous to bovine brain calmodulin with only 13 identified amino acid sequence differences, excluding a blocked NH(2)-terminal tripeptide whose sequence has not been elucidated. Two extended regions of sequence identity are in the NH(2)-terminal half of the molecule, while nine of the 13 identified differences are in the COOH-terminal half of the molecule. Two of the changes, a cysteine at residue 26 and a glutamine at residue 96, require a minimum of two base changes in the nucleotide codons. Both of these changes occur in the proposed calcium binding loops of the molecule. Five additional amino acid differences found in spinach calmodulin had not been observed previously in a calmodulin. As described in an accompanying report (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798), these limited number of amino acid sequence variations appear to result in differential effects on the activation of calmodulin-dependent enzymes by plant and vertebrate calmodulins.

Journal Article↗
PubMed1984

Towards a molecular and atomic anatomy of calmodulin and calmodulin-binding proteins.

The molecular mechanisms by which calcium regulates cellular processes such as metabolic and mechanochemical events probably involve interactions with a variety of molecules. A large body of evidence suggests that the targets of calcium's regulatory effects inside the cell are calcium-binding proteins. Our work attempts to correlate calcium-binding protein structure with activities. In this chapter we have presented some of our recent studies on these functional domains in calmodulin and calmodulin-binding proteins. Selected chemical modifications of known amino acid sequence positions have demonstrated the presence of multiple functional domains on calmodulin, have allowed the dissociation of calmodulin functions, and have provided the necessary tools for further investigations of the molecular basis of calmodulin action. One of these modifications, iodination of tyrosine-99, has allowed us to develop procedures to reproducibly detect calmodulin-binding proteins by a binding technique. This method is technically simple. It allows us to detect and study calmodulin-binding proteins (e.g., myosin heavy chain and membrane gap junction proteins) that would be difficult to study with immobilized calmodulin. We have developed a library of antibodies to calmodulin and related proteins such as troponin C and S100 beta. Some of these antisera appear to be site-specific gamma globulins. We have demonstrated that reactivity can be contained in an amino acid sequence as short as seven residues. We have demonstrated the feasibility of using an immunochemical mapping approach to study calmodulin and calmodulin-binding proteins. Comparative sequence analyses combined with functional analyses have allowed correlation of function and structure and have suggested logical candidates for functional domains on calmodulin and calmodulin-binding proteins. Although not discussed in detail in this chapter, these calmodulin-binding proteins appear to contain amino acid sequence homologies. This suggests, analogous to the approach used for calmodulin and related proteins, a logical starting point for domain analyses of calmodulin-binding proteins. Interestingly, structural homologies among calmodulin-binding proteins are reminiscent of the different phosphorylation sites found in many of the physiological substrates for protein kinases. Since a number of calmodulin-binding proteins are themselves substrates for protein kinases, these results suggest another possible point of interrelationships between calcium and cyclic nucleotide regulation.

Amino Acid Sequence↗
PubMed1983

Analysis of differences between coomassie blue stain and silver stain procedures in polyacrylamide gels: conditions for the detection of calmodulin and troponin C.

It is reported that the conditions used in some silver stain procedures can fail to detect calmodulin, troponin C, and other proteins with similar physical properties. Conditions are described that allow the reproducible detection of these proteins. Two phenomena are described: (1) lack of protein staining when treatment with glutaraldehyde is omitted from the protocol, and (2) loss of small proteins from the gel matrix during prolonged washing procedures. These data directly demonstrate that the use of some silver staining protocols can result in misleading data in biological studies and provide an explanation for at least one class of proteins of how silver staining and Coomassie blue staining of gels can give different results.

Calcium-Binding Proteins↗
PubMed1983

Analysis of suborganellar fractions from spinach and pea chloroplasts for calmodulin-binding proteins.

Purified chloroplasts from spinach and pea leaves were subfractionated into envelope, thylakoid, and stroma fractions and were analyzed for calmodulin-binding proteins using a 125I-calmodulin gel overlay assay. Calmodulin binding was primarily associated with a major polypeptide (Mr 33,000) in the envelope membrane fraction. In contrast, major calmodulin-binding proteins were not detected in the thylakoid or stroma fractions. Our results provide the first evidence of calmodulin-binding proteins in the chloroplast envelope, and raise the possibility that calmodulin may contribute to the regulation of chloroplast function through its interaction with calmodulin-binding proteins in the chloroplast envelope. In addition, our results combined with those of other investigators support the proposal that subcellular organelles may be a primary site of calmodulin action.

Calmodulin-Binding Proteins↗
PubMed1983

Further Characterization of Calmodulin from the Monocotyledon Barley (Hordeum vulgare).

We report here that calmodulin isolated from the monocotyledon barley is indistinguishable by a variety of criteria from calmodulin isolated from the dicotyledon spinach. In contrast to previous reports, we find that barley (Hordeum vulgare) calmodulin has an amino acid composition similar to that of vertebrate and spinach calmodulins, including the presence of a single trimethyllysinyl residue, and that barley calmodulin quantitatively activates cyclic nucleotide phosphodiesterase. Furthermore, spinach and barley calmodulins are similar in terms of tryptic peptide maps and immunoreactivity with various antisera that differ in their molecular specificities for calmodulins. Limited amino acid sequence analysis demonstrates that the region around the single histidinyl and trimethyllysinyl residues is identical among barley, spinach, and vertebrate calmodulins and that barley calmodulin, like spinach calmodulin, has a novel glutamine residue at position 96. We conclude that calmodulin is highly conserved among higher plants and that detailed sequence analysis is required before significant differences, if any, can be assigned to barley or other higher plant calmodulins. These studies suggest that calmodulin's fundamental importance to the eukaryotic cell may have been established prior to the evolutionary emergence of higher plants.

Journal Article↗