Biomedical subjects

M Schleicher

Publications and source records attributed to M Schleicher.

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

Structure/function studies on cytoskeletal proteins in Dictyostelium amoebae as a paradigm.

The actin cytoskeleton in motile non-muscle cells is being regulated by a large number of actin-binding proteins. A deeper insight into the complex nature of the dynamic rearrangements of the microfilament system during cell movement requires an experimental system that allows the combined application of biochemical, biophysical, cell biological and molecular methods. Dictyostelium amoebae are well suited especially for a genetic approach because they are amenable to gene disruption, antisense and gene tagging techniques. The actin-binding proteins profilin, hisactophilin and protovillin are being described in this context as typical examples that either bind to G-actin, or anchor the actin cytoskeleton to the plasma membrane, or are structurally similar to vertebrate proteins but distinct in their functions.

Animals↗

PubMed1995

Structure of severin domain 2 in solution.

The three-dimensional structure of domain 2 of severin in aqueous solution was determined by nuclear magnetic resonance spectroscopy. Severin is a Ca(2+)-activated actin-binding protein that servers F-actin, nucleates actin assembly, and caps the fast-growing ends of actin filaments. The 114-residue domain consists of a central five-stranded beta-sheet, sandwiched between a parallel four-turn alpha-helix and, on the other face, a roughly perpendicular two-turn alpha-helix. There are two distinct binding sites for Ca2+ located near the N and C termini of the long helix. Conserved residues of the gelsolin-severin family contribute to the apolar core of domain 2 of severin, so that the overall fold of the protein is similar to those of segment 1 of gelsolin and profilins. Together with biochemical experiments, this structure helps to explain how severin interacts with actin.

Actins↗

PubMed1995

The in vivo role of annexin VII (synexin): characterization of an annexin VII-deficient Dictyostelium mutant indicates an involvement in Ca(2+)-regulated processes.

Dictyostelium discoideum cells harbor two annexin VII isoforms of 47 and 51 kDa which are present throughout development. In immunofluorescence and cell fractionation studies annexin VII was found in the cytoplasm and on the plasma membrane. In gene disruption mutants lacking both annexin VII isoforms growth, pinocytosis, phagocytosis, chemotaxis and motility were not significantly impaired under routine laboratory conditions, and the cells were able to complete the developmental cycle on bacterial plates. On non-nutrient agar plates development was delayed by three to four hours and a significant number of aggregates was no longer able to form fruiting bodies. Exocytosis as determined by measuring extracellular cAMP phosphodiesterase, alpha-fucosidase and alpha-mannosidase activity was unaltered, the total amounts of these enzymes were however lower in the mutant than in the wild type. The mutant cells were markedly impaired when they were exposed to low Ca2+ concentrations by adding EGTA to the nutrient medium. Under these conditions growth, motility and chemotaxis were severely affected. The Ca2+ concentrations were similar in mutant and wild-type cells both under normal and Ca2+ limiting conditions; however, the distribution was altered under low Ca2+ conditions in SYN-cells. The data suggest that annexin VII is not required for membrane fusion events but rather contributes to proper Ca2+ homeostasis in the cell.

Animals↗

PubMed1994

Dictyostelium amoebae that lack G-actin-sequestering profilins show defects in F-actin content, cytokinesis, and development.

To study in vivo functions of the ubiquitous actin-binding protein profilin, we generated by antisense and gene disruption techniques Dictyostelium mutants that lack one or both of the profilin isoforms. Whereas the single mutants showed an essentially unchanged phenotype, the behavior of the double mutant was drastically altered. Motility was significantly reduced, single cells were up to 10 times larger than wild-type cells and showed a broad rim of filamentous actin below the plasma membrane, the filamentous actin concentration was increased by about 60%-70%, and development was blocked prior to fruiting body formation. Furthermore, double mutants could not be grown in shaking culture under normal conditions, reflecting an impaired cytokinesis. The aberrant phenotype could be rescued by reintroducing a functional profilin I or profilin II gene. The data in this study suggest that profilin functions in Dictyostelium amoebae primarily as an actin-sequestering protein.

Actins↗

PubMed1993

Conformation of thymosin beta 4 in water determined by NMR spectroscopy.

The conformational preferences of a 43-amino-acid G-actin-binding peptide, thymosin beta 4, in water at 1, 4 and 14 degrees C, and at pH 3.0 and 6.5 were studied by NMR. NMR showed that thymosin beta 4 lacks a uniquely folded conformation in water. However, some preferential alpha-helical conformations of thymosin beta 4 can be observed in aqueous solutions. The segment at residues 5-16 showed characteristic interactions for conformations in both the beta-strand and alpha-helical regions of the phi-psi space, based on strong C alpha H(i)-NH(i+1) interactions and NH-NH, C alpha H(i)-NH(i+3), and C alpha H(i)-C beta H(i+3) interactions, respectively. At 1-4 degrees C, another segment at residues 31-37 also shows both beta and alpha conformations, forming however a less well-defined helix than the segment at residues 5-16. At 14 degrees C, the conformational population of the helix at positions 5-16 is shifted more towards the random and turn-like structures, whereas the segment at positions 31-37 becomes exclusively a random coil.

Amino Acid Sequence↗

PubMed1993

The 100 kDa F-actin capping protein of Dictyostelium amoebae is a villin prototype ('protovillin').

The 100 kDa actin-binding protein from Dictyostelium amoebae is an F-actin capping protein that displays neither severing nor crosslinking nor nucleating activities [Hofmann et al. (1992) Cell Motil. Cytoskel. 23,133-144]. Cloning and sequencing of the gene revealed that the protein is highly homologous to vertebrate villin, a unique component of brush border microvilli and contains six domains fused to a villin-like headpiece domain via a threonine/proline rich neck region. The functional differences and similarities between the 100 kDa protein and villin are reflected in the amino acid sequences. We draw from the data the following conclusions. (i) The presence of a six domain protein in Dictyostelium suggests that in contrast to the current view gene duplications must have happened before Dictyostelium branched off during evolution. (ii) The villin-like molecule in Dictyostelium appears to be a premature villin ('protovillin') which is able to cap actin filaments but still lacks the other villin-type actin-binding activities. This renders capping of actin filaments as the evolutionarily oldest function of an F-actin binding protein.

Actin Depolymerizing Factors↗

PubMed1993

The heat shock cognate protein from Dictyostelium affects actin polymerization through interaction with the actin-binding protein cap32/34.

During isolation of the F-actin capping protein cap32/34 from Dictyostelium discoideum, a 70 kDa protein was copurified which by cloning and sequencing was identified as a heat shock cognate protein (hsc70). This protein exhibited a specific and MgATP-dependent interaction with the heterodimeric capping protein. To investigate the protein-protein interaction in vitro, we expressed all three polypeptides separately in Escherichia coli and performed reconstitution experiments of complete or truncated hsc70 with the 32 and 34 kDa subunits of the capping protein. Viscosity measurements and studies on the polymerization kinetics of pyrene-labeled actin showed that hsc70 increased the capping activity of cap32/34 up to 10-fold, whereas hsc70 alone had no effect on actin polymerization. In addition, hsc70 acted as a molecular chaperone by stimulating the refolding of the denatured 32 and 34 kDa subunits of the capping protein. To study the interaction of the two domains of hsc70 with cap32/34, the N-terminal 42 kDa ATPase region and the C-terminal 30 kDa tail of hsc70 were expressed separately in E. coli. The 32 and 34 kDa subunits were capable of associating with both domains of hsc70. The ATPase domain of hsc70, which is structurally related to actin, proved to be responsible for the increased capping activity of cap32/34, whereas the C-terminal tail of hsc70 was involved in folding of the subunits of cap32/34. Our data indicate a novel linkage between 70 kDa heat shock proteins and the actin cytoskeleton.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1993

The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis.

Dictyostelium alpha-actinin is a Ca(2+)-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca(2+)-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum alpha-actinin but Ca2+ binding by EF hand II was still observed in a 45Ca2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, approximately 500-fold more Ca2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum alpha-actinin: (a) at the level of the EF-hands, the Ca(2+)-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated alpha-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca2+ concentrations for inactivation, it is interesting to note that an unregulated alpha-actinin was able to rescue the mutant phenotype.

Actinin↗

PubMed1993

The actin-binding protein comitin (p24) is a component of the Golgi apparatus.

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.

3T3 Cells↗

PubMed1992

Homonuclear three-dimensional NOE-NOE nuclear magnetic resonance/spectra for structure determination of proteins in solution.

The solution structures of two proteins (CMTI-I, a trypsin inhibitor from Cucurbita maxima, and hisactophilin, an actin binding protein of 118 amino acids) have been determined based on the NOE data derived solely from the homonuclear 3D NOE-NOE magnetic resonance spectroscopy. Two different approaches for extraction of the structural information from the 3D NOE-NOE experiment were tested. One approach was based on the transformation of the 3D intensities into distance constraints. In the second, and more robust approach, the 3D NOE intensities were used directly in structure calculations, without the need to transform them into distance constraints. A new 2D potential function representing the 3D NOE-NOE intensity was developed and used in the simulated annealing protocol. For CMTI-I, a comparison between structures determined with the 3D NOE-NOE method and various 2D NOE approaches was carried out. The 3D data set allowed better definition of the structures than was previously possible with the 2D NOE procedures that used the isolated two-spin approximation to derive distance information.

Carrier Proteins↗

PubMed1992

Structure of hisactophilin is similar to interleukin-1 beta and fibroblast growth factor.

The fast reaction of the actin-based cytoskeleton in motile cells after stimulation with a chemoattractant requires a signal-transduction chain that creates a very specific environment at distinct regions beneath the plasma membrane. Dictyostelium hisactophilin, a unique actin-binding protein, is a submembranous pH sensor that signals slight changes of the H+ concentration to actin by inducing actin polymerization and binding to microfilaments only at pH values below seven. It has a relative molecular mass of 13.5K and its most unusual feature is the presence of 31 histidine residues among its total of 118 amino acids. The transduction of an external signal from the plasma membrane to the cytoskeleton is poorly understood. Here we report the protein's structure in solution determined by nuclear magnetic resonance spectroscopy. The nuclear Overhauser effect intensities of the three-dimensional nuclear Overhauser spectra were used directly in the calculations. The overall folding of histactophilin is similar to that of interleukin-1 beta and fibroblast growth factor, but the primary amino-acid sequence of hisactophilin is unrelated to these two proteins.

Amino Acid Sequence↗

PubMed1992

Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.

Severin is a Ca(2+)-activated actin-binding protein that nucleates actin assembly and severs and caps the fast growing ends of actin filaments. It consists of three highly conserved domains. To investigate the domain structure of severin, we constructed genetically the N-terminal domain 1, the middle domain 2, and the tandem domains 2 + 3. Their interaction with actin, Ca2+, and lipids was characterized. Domain 1 contains the F-actin capping and a Ca(2+)-binding site [Eichinger, L., Noegel, A. A., & Schleicher, M. (1991) J. Cell Biol. 112, 665-676]. Binding of domain 2 to actin filaments was Ca(2+)-dependent and saturated at a 1:1 molar ratio. In the presence of Ca2+, about 1.5 mol of domains 2 + 3 bound per mole of F-actin subunit. Scatchard analysis gave a Kd of 18 microM for the interaction of domain 2 with F-actin subunits and a Kd of 1.6 microM for domains 2 + 3. Low-shear viscometry, electron microscopy, and low-speed sedimentation assays showed that domains 2 + 3 induced bundling of actin filaments. The influence of PIP2 micelles on the different activities of severin was assayed using native severin and N- and C-terminally truncated fragments. Severin contains at least two PIP2-binding sites since the activities of the two nonoverlapping severin fragments domain 1 and domains 2 + 3 were inhibited by PIP2. The specificity of severin-phospholipid interaction was investigated by studying the regulation of native severin by PIP2 and other pure or mixed phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1992

Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins.

We generated by gene disruption Dictyostelium cells that lacked both the F-actin cross-linking proteins, alpha-actinin and gelation factor. Several major cell functions, such as growth, chemotaxis, phagocytosis, and pinocytosis, were apparently unaltered. However, in all double mutants, development was greatly impaired. After formation of aggregates, cells were very rarely able to form fruiting bodies. This ability was rescued when mutant and wild-type strains were mixed in a ratio of 70 to 30. The developmental program in the mutant was not arrested, since the expression pattern of early and late genes remained unchanged. Development of the mutant was rendered normal when a functional alpha-actinin gene was introduced and expressed, showing the morphogenetic defect to be due to the absence of the two F-actin cross-linking proteins. These findings suggest the existence of a functional network allowing mutual complementation of certain actin-binding proteins.

Actin Cytoskeleton↗

PubMed1992

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.

Actin Cytoskeleton↗

PubMed1992

Dynamics of the Dictyostelium cytoskeleton during chemotaxis.

Movement and chemotaxis are fundamental processes of cells and tissues and are based on the dynamics of the cytoskeleton. The cellular slime mold Dictyostelium discoideum is an excellent model system with which to study the molecular components and the key reactions that are required for a coordinated locomotion of single cells or a cell mass during development. The D. discoideum cytoskeleton relies mainly on the equilibrium between monomeric and filamentous actin and, like other nonmuscle cells, contains a large number of actin-binding proteins that either decrease or increase the rigidity of the microfilament system. The proteins themselves are regulated by phosphorylation, Ca2+, phospholipids, and/or pH and thus are targets for the intracellular changes that occur upon stimulation of a cell with chemoattractant. In a synopsis of the data published during the past years, the properties of numerous cytoskeletal components and the biochemical reactions of the signal transduction chain are combined here in a schematic model that attempts to explain how the directed movement of a cell could be coordinated at the molecular level.

Actins↗

PubMed1991

F-actin capping by cap32/34 requires heterodimeric conformation and can be inhibited with PIP2.

The heterodimeric F-actin capping protein cap32/34 from Dictyostelium discoideum is a typical member of a widely distributed family of cytoskeletal proteins. To analyze its regulation and structure/function relationships we cloned and expressed the subunits separately in Escherichia coli using the ATG-expression vector pT7-7. Studies on the viscosity of F-actin solutions and the kinetics of actin polymerization in the presence of single subunits or the reconstituted protein showed that capping of F-actin absolutely requires the heterodimeric conformation. This activity can be inhibited by phosphatidyl bisphosphate (PIP2), an important component in signal transduction. The regulation of cap32/34 by PIP2 suggests an involvement of this protein in the re-organization of the actin cytoskeleton upon stimulation of D. discoideum cells with chemoattractant.

Actins↗

PubMed1991

Dictyostelium annexin VII (synexin). cDNA sequence and isolation of a gene disruption mutant.

By cloning the cDNA coding for the membrane associated actin-binding protein p24, we identified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum. Using a cDNA probe corresponding to this motif, we isolated cDNA clones coding for a protein of the annexin family. On the basis of a long NH2-terminal sequence encompassing the Gly/Tyr/Pro/Gln motifs, the Dictyostelium annexin was identified as a homolog of vertebrate annexin VII (synexin). The mRNA coding for the Dictyostelium annexin VII has a size of 1.6 kilobases and is present during all developmental stages. Annexin VII is coded for by a single gene in Dictyostelium. A mutant deficient in annexin VII was isolated using a vector which carried the amino-terminal third of the Dictyostelium annexin VII cDNA followed by a viral epitope specific for a monoclonal antibody and a stop codon. Using this approach, homologous recombination in the annexin VII gene led to an expression of the viral epitope under the control of the endogenous annexin VII promoter. Lack of annexin VII is not a lethal event for D. discoideum, and the cells are able to undergo development on agar plates.

Amino Acid Sequence↗