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J A Spudich

Publications and source records attributed to J A Spudich.

At least 73 records · Page 4Linked to original sources
PubMed1994

Enzymatic activities correlate with chimaeric substitutions at the actin-binding face of myosin.

Myosins are a functionally divergent group of mechanochemical enzymes involved in various motile activities in cells. Despite a high degree of conservation in the amino-acid sequence of the 130K motor domain (head region) of the molecule, there are large differences in the enzymatic and motile activities (Tables 1 and 2) of myosins from diverse species and cell types. However, the degree of conservation is not uniform throughout the head sequence; therefore, one reasonable hypothesis is that the functional differences between myosins derive from the poorly conserved areas. The most prominent divergent region occurs at the 50K/20K junction, a region of the molecule sensitive to proteolytic digestion and a binding site for actin. We have now constructed chimaeras of this region of myosin by substituting the 9-amino-acid Dictyostelium junction region with those from myosins from other species and find that the actin-activated ATPase correlates well with the activity of the myosin from which the junction region was derived. Our results suggest that this region, likely to be part of the myosin head that interacts directly with actin, is important in determining the enzymatic activity of myosin.

Actins↗
PubMed1994

Single myosin molecule mechanics: piconewton forces and nanometre steps.

A new in vitro assay using a feedback enhanced laser trap system allows direct measurement of force and displacement that results from the interaction of a single myosin molecule with a single suspended actin filament. Discrete stepwise movements averaging 11 nm were seen under conditions of low load, and single force transients averaging 3-4 pN were measured under isometric conditions. The magnitudes of the single forces and displacements are consistent with predictions of the conventional swinging-crossbridge model of muscle contraction.

Actins↗
PubMed1994

Functional analysis of a cardiac myosin rod in Dictyostelium discoideum.

Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament-forming, or light meromyosin (LMM) domain of the myosin molecule in cytokinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1-11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677-688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat beta cardiac LMM was transfected into both wild-type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild-type and Dd mhc null cells. Although null cells expressing chimeric mhc at approximately 10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility.

Actins↗
PubMed1994

Stage-specific requirement for myosin II during Dictyostelium development.

Dictyostelium cells that lack a functional myosin II heavy chain are motile and are capable of aggregation, but fail to undergo further multicellular development. We have used a Dictyostelium mutant expressing a cold-sensitive myosin heavy chain to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature-shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between those stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. Calcofluor staining and reporter gene fusions reveal that these structures contain defective spores and a miniature stalk.

Animals↗
PubMed1994

Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain.

Conventional myosin ('myosin II') is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail that is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail.

Animals↗
PubMed1993

A functional recombinant myosin II lacking a regulatory light chain-binding site.

Myosin II, which converts the energy of adenosine triphosphate hydrolysis into the movement of actin filaments, is a hexamer of two heavy chains, two essential light chains, and two regulatory light chains (RLCs). Dictyostelium myosin II is known to be regulated in vitro by phosphorylation of the RLC. Cells in which the wild-type myosin II heavy chain was replaced with a recombinant form that lacks the binding site for RLC carried out cytokinesis and almost normal development, processes known to be dependent on functional myosin II. Characterization of the purified recombinant protein suggests that a complex of RLC and the RLC binding site of the heavy chain plays an inhibitory role for adenosine triphosphatase activity and a structural role for the movement of myosin along actin.

Actins↗
PubMed1993

Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo.

Three threonine residues in the tail region of Dictyostelium myosin II heavy chain have been implicated previously in control of myosin filament formation. Here we report the in vitro and in vivo consequences of converting these sites to alanine residues, which eliminates phosphorylation at these positions, or to aspartate residues, which mimics the negative charge state of the phosphorylated molecule. Alanine substitution allows in vitro assembly and in vivo contractile activity, although this myosin shows substantial over-assembly in vivo. Aspartate substitution eliminates filament assembly in vitro and renders the myosin unable to drive any tested contractile event in vivo. These results demonstrate that heavy chain phosphorylation plays a key modulatory role in controlling myosin function in vivo.

Actin Cytoskeleton↗
PubMed1993

Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1.

Elucidation of the molecular contacts between actin and myosin is central to understanding the force-generating process in muscle and other cells. Actin, a highly conserved globular protein found in all eukaryotes, polymerizes into filaments (F-actin) for most of its biological functions. Myosins, which are more diverse in sequence, share a conserved globular head of about 900 amino acids in length (subfragment-1 or S1) at the N-terminal end of the molecule. S1 contains all the elements necessary for mechano-chemical force transduction in vitro. Here we report an atomic model for the actomyosin complex produced by combining the atomic X-ray structure of F-actin and chicken myosin S1 with a three-dimensional reconstruction from electron micrographs of frozen-hydrated F-actin decorated with recombinant Dictyostelium myosin S1. The accuracy of the reconstruction shows the position of actin and myosin molecules unambiguously.

Actins↗
PubMed1993

Molecular evolution of the myosin family: relationships derived from comparisons of amino acid sequences.

To examine the evolutionary relationships between members of the myosin family, we have used two different phylogenetic methods, distance matrix and maximum parsimony, to analyze all available myosin head sequences. We find that there are at least three equally divergent classes of myosin, demonstrating that the current classification of myosin into only two classes needs to be reexamined. In the myosin II class, smooth muscle myosin is more closely related to nonmuscle myosin than to striated muscle myosin, implying that smooth muscle and skeletal muscle myosins were independently derived from nonmuscle myosin and suggesting that similarities between these types of muscle are the result of convergent evolution. The grouping of head sequences produced by phylogenetic analysis is consistent with classifications based on enzymology and structural localization and is generally consistent with grouping based on common tail structure elements. This result demonstrates that specific head sequences are tightly coupled to specific tail sequences throughout evolution and challenges the idea that myosin heads are freely interchangeable units whose unique function is determined only by the tail structure to which it is attached.

Animals↗
PubMed1993

Force on single actin filaments in a motility assay measured with an optical trap.

We have used an optical trap to measure or exert a force on single actin filaments via the attachment of polystyrene beads which were coated with NEM-modified HMM. In the simplest experiment, beads were attached to rhodamine phalloidin labelled actin filaments and observed to move on an HMM coated surface in the presence of ATP. Moving beads were steered into the vicinity of the trap using a PZT operated microscope stage. The minimum force needed to stop a moving bead was measured by lowering the trap strength until the bead resumed movement. By aligning the optical trap with the centre of a quadrant detector placed in an image plane of the microscope, it was possible to measure the force exerted on a filament by measuring the displacement of the bead position from the centre of the trap. In each of these experiments, the trap was calibrated by applying a Stokes force to a bead in free solution. The characteristics of the trap were studied, and the displacement of the bead from the centre of the trap was shown to be directly proportional to the applied force over a large part of the total range of the trap. The compliance of the trap could be substantially reduced by the use of feedback control to deflect the laser beam via an acousto-optic modulator. The advantages and limitations of this technique will be discussed.

Actin Cytoskeleton↗
PubMed1993

The unconventional myosin encoded by the myoA gene plays a role in Dictyostelium motility.

The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.

Animals↗
PubMed1992

Membrane-bound Dictyostelium myosin heavy chain kinase: a developmentally regulated substrate-specific member of the protein kinase C family.

A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought.

Amino Acid Sequence↗
PubMed1992

Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.

The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because it is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act1 mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been significant conservation of actin function, specifically the ability to be moved by interaction with myosin.

Actins↗
PubMed1992

A Dictyostelium myosin II lacking a proximal 58-kDa portion of the tail is functional in vitro and in vivo.

We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.

Actin Cytoskeleton↗
PubMed1991

Characterization and bacterial expression of the Dictyostelium myosin light chain kinase cDNA. Identification of an autoinhibitory domain.

A full-length cDNA corresponding to the Dictyostelium myosin light chain kinase gene has been isolated and characterized. Sequence analysis of the cDNA confirms conserved protein kinase subdomains and reveals that the Dictyostelium sequence is highly homologous to those of calcium/calmodulin-dependent protein kinases, including myosin light chain kinases from higher eukaryotes. Despite the high homologies to calcium/calmodulin-dependent protein kinases, there is no recognizable calmodulin-binding domain within the Dictyostelium sequence. However, the Dictyostelium myosin light chain kinase possesses a putative auto-inhibitory domain near its carboxyl terminus. To further characterize this domain, the full-length enzyme as well as a truncated form lacking this domain were expressed in bacterial cells and purified. The full-length enzyme expressed in bacteria exhibits essentially the same biochemical characteristics as the enzyme isolated from Dictyostelium. The truncated form however exhibits a Vmax that is approximately ten times greater than that of the native enzyme. In addition, unlike the native kinase and the full-length kinase expressed in bacteria, the truncated enzyme does not undergo autophosphorylation. These results suggest that the Dictyostelium enzyme, like myosin light chain kinases from higher eukaryotes, is regulated by an autoinhibitory domain but that the specific molecular signals necessary for activation of the Dictyostelium enzyme are entirely distinct.

Amino Acid Sequence↗
PubMed1991

Quantized velocities at low myosin densities in an in vitro motility assay.

An in vitro motility assay has been developed in which single actin filaments move on one or a few heavy meromyosin (HMM) molecules. This movement is slower than when many HMM molecules are involved, in contrast to analogous experiments with microtubules and kinesin. Frequency analysis shows that sliding speeds distribute around integral multiples of a unitary velocity. This discreteness may be due to differences in the numbers of HMM molecules interacting with each actin filament, where the unitary velocity reflects the activity of one HMM molecule. The value of the unitary velocity predicts a step size of 5-20 nm per ATP, which is consistent with the conventional swinging crossbridge model for myosin function.

Adenosine Triphosphate↗