Biological roles of actin-binding proteins in Dictyostelium discoideum examined using genetic techniques (original) (raw)
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PLoS ONE, 2008
Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes. Citation: Joseph JM, Fey P, Ramalingam N, Liu XI, Rohlfs M, et al. (
A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor
The Journal of Cell Biology, 1990
Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation
Journal of Cell Biology, 1983
We have studied the abundance, relative gelation activity, and distribution of the 95,000-dalton actin-binding protein in Dictyostelium discoideum amoebae. The 95,000-dalton protein was a prominent polypeptide as assessed using quantitative densitometry and radioimmunoassay. We estimated that this protein comprised ~1.2% of the protein in a soluble extract of amoebae. The molar ratio of the dimeric 95,000-dalton protein to actin in the soluble extract was 1:30. The apparent viscosities of actin mixtures with either the purified 95,000-dalton protein or the soluble extract were measured by falling ball viscometry in an attempt to assess the contribution of the 95,000-dalton protein to gelation of the soluble extract. The gelation of the soluble extract was significantly less than that expected from the contribution of the 95,000-dalton protein alone. Consequently, we questioned the validity of quantitative analyses of the contributions of specific actin-binding proteins to the gelation of cell extracts. The apparent distribution of the 95,000-dalton protein was observed in chemically fixed and extracted cells by immunofluorescence microscopy and compared with the distribution of cytoplasm and organelles visible using light microscopy. The 95,000-dalton protein was dispersed throughout the cytoplasm of fixed cells, was apparently excluded from prominent organelles, and displayed brightest fluorescence in regions of hyaline cytoplasm. These regions of hyaline cytoplasm that exhibited the brightest fluorescence were observed in the cortical region of rounded cells and in pseudopods of polarized cells. Thus, cell shape and polarity may also have influenced the apparent distribution of the 95,000-dalton protein observed by immunofluorescence microscopy. Study of the distribution of fluorescein-labeled ovalbumin injected into living cells supported the interpretation that the thickness of the cell and the distribution of organelles contributed to the apparent distribution of the 95,000dalton protein observed in fixed cells using immunofluorescence microscopy. We suggest that the 95,000-dalton protein contributes to modulation of the consistency and contractility of the cytoplasm of D. discoideum amoebae, since it could cross-link actin filaments in vitro in a reversible process that was regulated by changes in the concentration of calcium and of protons, and since it was present in large quantity in the cytoplasm of these cells.
Journal of Cell Science
The contribution of three actin cross-linking proteins, α α-actinin (α αA), gelation factor (ABP-120), and the 34 kDa actin-bundling protein to cellular functions has been studied in three single mutant α αA − − , 120 − − , and 34 − − ) and three double mutant (α αA − − /120 − − , 34 − − /α αA − − , 34 − − /120 − − ) strains of Dictyostelium generated by homologous recombination. Strains α αA − − /120 − − and 34 − − /α αA − − exhibited a reduced rate of pinocytosis, grew to lower saturation densities, and produced small cells in shaking cultures. All strains grew normally in bacterial suspensions and on agar plates with a bacterial lawn. Slow growth under conditions of reduced temperature and increased osmolarity was observed in single mutants 34 − − and α αA − − , respectively, as well as in some of the double mutant strains. Motility, chemotaxis, and development were largely unaltered in 34 − − /α αA − − and 34 − − /120 − − cells. However, 34 − − /α αA − − cells showed enhanced aggregation when starved in suspension. Moreover, morphogenesis was impaired in both double mutant strains and fruiting bodies of aberrant morphology were observed. These defects were reverted by re-expression of one of the lacking crosslinking proteins. The additive and synthetic phenotypes of these mutations indicate that actin cross-linking proteins serve both unique and overlapping functions in the actin cytoskeleton.
Journal of Cell Science, 1999
The contribution of three actin cross-linking proteins, α α-actinin (α αA), gelation factor (ABP-120), and the 34 kDa actin-bundling protein to cellular functions has been studied in three single mutant α αA − − , 120 − − , and 34 − − ) and three double mutant (α αA − − /120 − − , 34 − − /α αA − − , 34 − − /120 − − ) strains of Dictyostelium generated by homologous recombination. Strains α αA − − /120 − − and 34 − − /α αA − − exhibited a reduced rate of pinocytosis, grew to lower saturation densities, and produced small cells in shaking cultures. All strains grew normally in bacterial suspensions and on agar plates with a bacterial lawn. Slow growth under conditions of reduced temperature and increased osmolarity was observed in single mutants 34 − − and α αA − − , respectively, as well as in some of the double mutant strains. Motility, chemotaxis, and development were largely unaltered in 34 − − /α αA − − and 34 − − /120 − − cells. However, 34 − − /α αA − − cells showed enhanced aggregation when starved in suspension. Moreover, morphogenesis was impaired in both double mutant strains and fruiting bodies of aberrant morphology were observed. These defects were reverted by re-expression of one of the lacking crosslinking proteins. The additive and synthetic phenotypes of these mutations indicate that actin cross-linking proteins serve both unique and overlapping functions in the actin cytoskeleton.
Severe developmental defects in Dictyostelium null mutants for actin-binding proteins
Mechanisms of Development, 2000
The actin cytoskeleton is implicated in many cellular processes, such as cell adhesion, locomotion, contraction and cytokinesis, which are central to any development. The extent of polymerization, cross-linking, and bundling of actin is regulated by several actin-binding proteins. Knock-out mutations in these proteins have revealed in many cases only subtle, if any, defects in development, suggesting that the actin system is redundant, with multiple proteins sharing overlapping functions. The apparent redundancy may, however, re¯ect limitations of available laboratory assays in assessing the developmental role of a given protein. By using a novel assay, which reproduces conditions closer to the natural ones, we have re-examined the effects of disruption of many actin-binding proteins, and show here that deletion of a-actinin, interaptin, synexin, 34-kDa actin-bundling protein, and gelation factor affect to varying degrees the ef®ciency of Dictyostelium cells to complete development and form viable spores. No phenotypic defects were found in hisactophilin or comitin null mutants. q
Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum
Journal of Cell Biology, 1986
The binding of native, 125I-Bolton-Hunterlabeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. In the presence of gelsolin, the amount of actin bound at saturation to three different membrane preparations was 80, 120, and 200 ~g/mg of membrane protein. The respective concentrations of actin at half-saturation were 8, 12, and 18 ~g/ml. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape.
The Journal of Cell Biology, 1989
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, HGl132, 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 HGl132 is recessive and has been mapped to linkage group VII.