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M Schleicher

Publications and source records attributed to M Schleicher.

At least 19 recordsLinked to original sources

The genome of the social amoeba Dictyostelium discoideum.

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal-fungal lineage after the plant-animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

ATP-Binding Cassette Transporters↗

Formins and VASPs may co-operate in the formation of filopodia.

Filopodia are finger-like cell protrusions composed of parallel arrays of actin filaments, which elongate through actin polymerization at their tips. These highly dynamic structures seem to be used by many cell types as sensing organs to explore environmental cues and have been implicated in cell motility as well as in cell-substrate adhesion. Formins are highly conserved multidomain proteins that play important roles in the nucleation of actin and the formation of linear actin filaments, yet their role in filopodia formation has remained poorly defined. The Dictyostelium diaphanous-related formin dDia2 is strongly enriched in filopodia tips. Genetic and biochemical analysis revealed that this protein is important for cell migration and cell adhesion, but most importantly for the formation of filopodia. Recently, we have identified the Dictyostelium VASP (vasodilator-stimulated phosphoprotein) orthologue as a binding partner of dDia2 and provide evidence for a co-operative role of both proteins in filopodia formation.

Animals↗

Villin-type headpiece domains show a wide range of F-actin-binding affinities.

The villin-type "headpiece" domain is a modular motif found at the extreme C-terminus of larger "core" domains in over 25 cytoskeletal proteins in plants and animals. Although headpiece is classified as an F-actin-binding domain, it has been suggested that some expressed fusion-proteins containing headpiece may lack F-actin-binding in vivo. To determine the intrinsic F-actin affinity of headpiece domains, we quantified the F-actin affinity of seven headpiece domains and three N-terminal truncations, under identical in vitro conditions. The constructs are folded and adopt the native headpiece structure. However, they show a wide range of affinities that can be grouped into high, low, and nonspecific-binding categories. Computer models of the structure and charged surface potential of these headpiece domains suggest features important for high F-actin affinity. We conclude that not all headpiece domains are intrinsically F-actin-binding motifs, and suggest that the surface charge distribution may be an important element for F-actin recognition.

Actins↗

Characterization of CD36/LIMPII homologues in Dictyostelium discoideum.

The CD36/LIMPII family is ubiquitously expressed in higher eukaryotes and consists of integral membrane proteins that have in part been characterized as cell adhesion receptors, scavenger receptors, or fatty acid transporters. However, no physiological role has been defined so far for the members of this family that localize specifically to vesicular compartments rather than to the cell surface, namely lysosomal integral membrane protein type II (LIMPII) from mammals and LmpA from the amoeba Dictyostelium discoideum. LmpA, the first described CD36/LIMPII homologue from lower eukaryotes, has initially been identified as a suppressor of the profilin-minus phenotype. We report the discovery and initial characterization of two new CD36/LIMPII-related proteins, both of which share several features with LmpA: (i) their size is considerably larger than that of the CD36/LIMPII proteins from higher eukaryotes; (ii) they show the characteristic "hairpin" topology of this protein family; (iii) they are heavily N-glycosylated; and (iv) they localize to vesicular structures of putative endolysosomal origin. However, they show intriguing differences in their developmental regulation and exhibit different sorting signals of the di-leucine or tyrosine-type in their carboxyl-terminal tail domains. These features make them promising candidates as a paradigm for the study of the function and evolution of the as yet poorly understood CD36/LIMPII proteins.

Amino Acid Sequence↗

Dictyostelium discoideum: a genetic model system for the study of professional phagocytes. Profilin, phosphoinositides and the lmp gene family in Dictyostelium.

Profilin is a key regulator of actin polymerization, and plays a pivotal role at the interface of the phosphoinositide signal transduction pathway and the cytoskeleton. Recent evidence suggests the involvement of profilin in the regulation of phagocytosis and macropinocytosis, and the transport along the endosomal pathway. Disruption of profilin leads to a complex phenotype that includes abnormal cytokinesis, a block in development and defects in the endosomal pathway. Macropinocytosis, fluid phase efflux and secretion of lysosomal enzymes were reduced, whereas the rate of phagocytosis was increased as compared to wild-type cells. The lmpA gene, a homolog of the CD36/LIMPII family, was identified as a suppressor for most of the profilin-minus defects. This gene encodes an integral membrane protein, it localizes to lysosomes and macropinosomes, and binds to phosphoinositides. Even though phosphatidylinositol lipids constitute only a small fraction of total lipids in the membranes of eukaryotic cells, they play an important role in vesicle transport, signal transduction and cytoskeletal regulation. Disruption of lmpA in wild-type cells resulted in defects in fluid phase efflux and macropinocytosis, but not in phagocytosis. The discovery and initial characterization of two additional members of the CD36/LIMPII family in Dictyostelium, lmpB and lmpC, that exhibit intriguing differences in developmental regulation and their putative sorting signals, suggests that a set of lysosomal integral membrane proteins contribute to the crosstalk between vesicles and cytoskeletal proteins.

Animals↗

Filamins as integrators of cell mechanics and signalling.

Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Cell Membrane↗

Mapping the functional surface of domain 2 in the gelsolin superfamily.

The crystal structure of the F-actin binding domain 2 of severin, the gelsolin homologue from Dictyostelium discoideum, has been determined by multiple isomorphous replacement and refined to 1.75 A resolution. The structure reveals an alpha-helix-beta-sheet sandwich similar to the domains of gelsolin and villin, and contains two cation-binding sites, as observed in other domain 1 and domain 2 homologues. Comparison of the structures of several gelsolin family domains has identified residues that may mediate F-actin binding in gelsolin domain 2 homologues. To assess the involvement of these residues in F-actin binding, three mutants of human gelsolin domain 2 were assayed for F-actin binding activity and thermodynamic stability. Two of the mutants, RRV168AAA and RLK210AAA, demonstrated a lowered affinity for F-actin, indicating a role for those residues in filament binding. Using both structural and biochemical data, we have constructed a model of the gelsolin domain 1-domain 2-F-actin complex. This model highlights a number of interactions that may serve as positive and negative determinants of filament end- and side-binding.

Actins↗

Clinical utility of the modified barium swallow.

The purpose of this investigation was to evaluate the immediate and clinically relevant information gained from the modified barium swallow study and to determine the impact of the procedure on patient management. A database containing a nonrandom sample of 608 swallowing studies was reviewed. Results showed that only 10.4% of the studies were classified as normal examinations and aspiration occurred in 32.4%. However, swallowing abnormality without aspiration was recorded in 57.2% of the studies. Five additional outcome variables were assessed: referrals made to other specialties, effectiveness of applied compensatory strategies, treatment recommendations, mode of intake change, and diet grade change. Nearly 83% of the 608 studies showed change in at least one of the variables: needed referral to a specialist was identified on 26.3%; compensatory strategies that improved swallow physiology were identified on 48.4%; swallowing therapy was recommended on 37.2%; changes in mode of intake occurred on 31.4%; and diet texture changes were recommended on 43.8%. The low percentage of normal studies coupled with the high percentage of change in measurable variables indicate high clinical utility for the modified barium swallow study. The misguided tendency to refer to the modified barium study only as a tool for identifying aspiration and the appropriate utilization of the examination for identification of underlying abnormality in swallowing physiology are explained.

Aged↗

Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.

The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii. Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)-tagged legionellae rarely co-localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum. This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild-type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.

Acanthamoeba↗

Dissection of functional domains by expression of point-mutated profilins in Dictyostelium mutants.

Profilin is a ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site-directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline-binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited a profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79, 303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline-binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities.

Animals↗

Cloning and characterization of beta-COP from Dictyostelium discoideum.

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum by polymerase chain reaction using degenerate primers derived from rat beta-COP. The complete cDNA clone has a size of 2.8 kb and codes for a protein with a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between the highly conserved N- and C-terminal domains. Dictyostelium beta-COP is encoded by a single gene, which is transcribed at moderate levels into two RNAs that are present throughout development. To localize the protein, full-length beta-COP was fused to GFP and expressed in Dictyostelium cells. The fusion protein was detected on vesicles distributed all over the cells and was strongly enriched in the perinuclear region. Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this compartment was identified as the Golgi apparatus. Beta-COP distribution in Dictyostelium was not brefeldin A sensitive being most likely due to the presence of a brefeldin A resistance gene. However, upon DMSO treatment we observed a reversible disassembly of the Golgi apparatus. In mammalian cells DMSO treatment had a similar effect on beta-COP distribution.

3T3 Cells↗

Inactivation of lmpA, encoding a LIMPII-related endosomal protein, suppresses the internalization and endosomal trafficking defects in profilin-null mutants.

Profilin is a key phosphoinositide and actin-binding protein connecting and coordinating changes in signal transduction pathways with alterations in the actin cytoskeleton. Using biochemical assays and microscopic approaches, we demonstrate that profilin-null cells are defective in macropinocytosis, fluid phase efflux, and secretion of lysosomal enzymes but are unexpectedly more efficient in phagocytosis than wild-type cells. Disruption of the lmpA gene encoding a protein (DdLIMP) belonging to the CD36/LIMPII family suppressed, to different degrees, most of the profilin-minus defects, including the increase in F-actin, but did not rescue the secretion defect. Immunofluorescence microscopy indicated that DdLIMP, which is also capable of binding phosphoinositides, was associated with macropinosomes but was not detected in the plasma membrane. Also, inactivation of the lmpA gene in wild-type strains resulted in defects in macropinocytosis and fluid phase efflux but not in phagocytosis. These results suggest an important role for profilin in regulating the internalization of fluid and particles and the movement of material along the endosomal pathway; they also demonstrate a functional interaction between profilin and DdLIMP that may connect phosphoinositide-based signaling through the actin cytoskeleton with endolysosomal membrane trafficking events.

Biological Transport↗

The actin cytoskeleton of Dictyostelium: a story told by mutants.

Actin-binding proteins are effectors of cell signalling and coordinators of cellular behaviour. Research on the Dictyostelium actin cytoskeleton has focused both on the elucidation of the function of bona fide actin-binding proteins as well as on proteins involved in signalling to the cytoskeleton. A major part of this work is concerned with the analysis of Dictyostelium mutants. The results derived from these investigations have added to our understanding of the role of the actin cytoskeleton in growth and development. Furthermore, the studies have identified several cellular and developmental stages that are particularly sensitive to an unbalanced cytoskeleton. In addition, use of GFP fusion proteins is revealing the spatial and temporal dynamics of interactions between actin-associated proteins and the cytoskeleton.

Actins↗

Dictyostelium as model system for studies of the actin cytoskeleton by molecular genetics.

The actin cytoskeleton is an essential structure for most movements at the cellular and intracellular level. Whereas for contraction a muscle cell requires a rather static organisation of cytoskeletal proteins, cell motility of amoeboid cells relies on a tremendously dynamic turnover of filamentous networks in a matter of seconds and at distinct regions inside the cell. The best model system for studying cell motility is Dictyostelium discoideum. The cells live as single amoebae but can also start a developmental program that leads to multicellular stages and differentiation into simple types of tissues. Thus, cell motility can be studied on single cells and on cells in a tissue-like aggregate. The ability to combine protein purification and biochemistry with fairly easy molecular genetics is a unique feature for investigation of the cytoskeleton and cell motility. The actin cytoskeleton in Dictyostelium harbours essentially all classes of actin-binding proteins that have been found throughout eukaryotes. By conventional mutagenesis, gene disruption, antisense approaches, or gene replacements many genes that code for cytoskeletal proteins have been disrupted, and altered phenotypes in transformants that lacked one or more of those cytoskeletal proteins allowed solid conclusions about their in vivo function. In addition, tagging the proteins or selected domains with green fluorescent protein allows the monitoring of protein redistribution during cell movement. Gene tagging by restriction enzyme mediated integration of vectors and the ongoing international genome and cDNA sequencing projects offer the chance to understand the dynamics of the cytoskeleton by identification and functional characterisation of all proteins involved.

Actins↗

Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).

The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.

Animals↗

The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.

Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.

Adenosine Monophosphate↗

Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue.

Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin.

Amino Acid Sequence↗