Controlling the direction of kinesin-driven microtubule movements along microlithographic tracks.

Motor proteins are able to move protein filaments in vitro. However, useful work cannot be extracted from the existing in vitro systems because filament motions are in random directions on two-dimensional surfaces. We succeeded in restricting kinesin-driven movements of microtubules along linear tracks by using micrometer-scaled grooves lithographically fabricated on glass surfaces. We also accomplished the extraction of unidirectional movement from the bidirectional movements along the linear tracks by adding arrowhead patterns on the tracks. These "rectifiers" enabled us to construct microminiturized circulators in which populations of microtubules rotated in one direction, and to actively transport microtubules between two pools connected by arrowheaded tracks in the fields of micrometer scales.

Phalloidin affects the myosin-dependent sliding velocities of actin filaments in a bound-divalent cation dependent manner.

We examined sliding velocities in vitro of four types of actin filaments, that is, filaments with Ca2+ or Mg2+ bound at the high affinity metal binding site, each with rhodamine phalloidin bound with a high or low stoichiometry. When surfaces coated with a high density of heavy meromyosin (HMM) were used, high stoichiometric concentrations of rhodamine phalloidin reduced sliding velocities of only Ca2+-actin filaments, by 40%. As the HMM density on surfaces was reduced, continuous movement of actin filaments became dependent on the presence of methylcellulose and sliding velocities of all four types became progressively slower. Interestingly, Ca2+-actin filaments with a high stoichiometric concentration of rhodamine phalloidin were the fastest among the four types of filaments on sparse HMM surfaces. In contrast, phalloidin did not affect steady state ATPase activities of HMM in the presence of Ca2+- or Mg2+-actin filaments. We speculate that the reversal of the order of sliding velocities among the four types of actin filaments between high and low densities of HMM relates with different axial elasticity of the actin filaments, so that stiffer filaments move slower on dense HMM surfaces, but faster on sparse surfaces, than elastic ones.

Genetic approaches to dissect the mechanisms of two distinct pathways of cell cycle-coupled cytokinesis in Dictyostelium.

Dictyostelium discoideum is a unique experimental organism which allows genetic analysis of the mechanism of cytokinesis of the animal type, and a number of mutations which affect cytokinesis in one way or other have been identified. Myosin II filaments accumulate in the equatorial region, and myosin II-null cells cannot divide in suspension, indicating that active, myosin II-dependent constriction of the cleavage furrow contributes to bisection of the cell. We refer to this method of cytokinesis as cytokinesis A. On substrates, however, myosin II-null cells divide efficiently in a cell cycle-coupled manner. This adhesion-dependent but myosin II-independent division method, which we termed cytokinesis B, is carried out by a pathway that is genetically distinct from that of cytokinesis A. Morphological analyses suggested that cytokinesis B is driven by radial traction forces generated along polar peripheries, which indirectly cause furrow ingression. Identification of two redundant pathways have allowed us to search genes involved in either pathway by mutagenizing cells which are already defective in one of the pathways. This approach enabled us to identify a number of novel cytokinesis-related genes, as well as to reclassify known genes as cytokinesis-related.

Functional characterization of vertebrate nonmuscle myosin IIB isoforms using Dictyostelium chimeric myosin II.

The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(B2), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the B2-inserted form of loop 2 from human MHC-IIB(B2). The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin. In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain.

How well can an amoeba climb?

We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of Dictyostelium discoideum were "stalled" or no longer able to "climb up." The "apparent weight" of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly, however, the cell stalled not only in low-density media as expected but also in media with densities greater than the cell density where the buoyant force should push the amoeba upward. We find that the leading pseudopod is bent under centrifugal force in all stalled amoebae, suggesting that this pseudopod is very dense indeed. This finding also suggests that directional cell locomotion against resistive forces requires a turgid forward-pointing pseudopod, most likely sustained by cortical actomyosin II.

Myosin II-independent cytokinesis in Dictyostelium: its mechanism and implications.

Similar to higher animal cells, ameba cells of the cellular slime mold Dictyostelium discoideum form contractile rings containing filaments of myosin II during mitosis, and it is generally believed that contraction of these rings bisects the cells both on substrates and in suspension. In suspension, mutant cells lacking the single myosin II heavy chain gene cannot carry out cytokinesis, become large and multinucleate, and eventually lyze, supporting the idea that myosin II plays critical roles in cytokinesis. These mutant cells are however viable on substrates. Detailed analyses of these mutant cells on substrates revealed that, in addition to "classic" cytokinesis which depends on myosin II ("cytokinesis A"), Dictyostelium has two distinct, novel methods of cytokinesis, 1) attachment-assisted mitotic cleavage employed by myosin II null cells on substrates ("cytokinesis B"), and 2) cytofission, a cell cycle-independent division of adherent cells ("cytokinesis C"). Cytokinesis A, B, and C lose their function and demand fewer protein factors in this order. Cytokinesis B is of particular importance for future studies. Similar to cytokinesis A, cytokinesis B involves formation of a cleavage furrow in the equatorial region, and it may be a primitive but basic mechanism of efficiently bisecting a cell in a cell cycle-coupled manner. Analysis of large, multinucleate myosin II null cells suggested that interactions between astral microtubules and cortices positively induce polar protrusive activities in telophase. A model is proposed to explain how such polar activities drive cytokinesis B, and how cytokinesis B is coordinated with cytokinesis A in wild type cells.

Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium.

The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological, and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis.

Involvement of tail domains in regulation of Dictyostelium myosin II.

The actin-dependent ATPase activity of Dictyostelium myosin II filaments is regulated by phosphorylation of the regulatory light chain. Four deletion mutant myosins which lack different parts of subfragment 2 (S2) showed phosphorylation-independent elevations in their activities. Phosphorylation-independent elevation in the activity was also achieved by a double point mutation to replace conserved Glu932 and Glu933 in S2 with Lys. These results suggested that inhibitory interactions involving the head and S2 are required for efficient regulation. Regulation of wild-type myosin was not affected by copolymerization with a S2 deletion mutant myosin in the same filaments. Furthermore, the activity linearly correlated with the fraction of phosphorylated molecules in wild-type filaments. These latter two results suggest that the inhibitory head-tail interactions are primarily intramolecular.

Disturbed communication between actin- and nucleotide-binding sites in a myosin II with truncated 50/20-kDa junction.

The kinetic and functional consequences of deleting nine residues from an actin-binding surface loop (loop 2) were examined to investigate the role of this region in myosin function. The nucleotide binding properties of myosin were not altered by the deletion. However, the deletion affected actin binding and the communication between the actin- and nucleotide-binding sites. The affinity of M765NL for actin (644 nM) was approximately 100-fold lower than that of wild-type construct M765 (5.8 nM). Despite this reduction in affinity, actin binding weakened the affinity of ADP for the motor to a similar extent for both mutant and wild-type constructs. The addition of 0.5 microM actin decreased ADP affinity from 0.6 to 34 microM for M765NL and from 1.6 to 39 microM for M765. In contrast, communication between the actin- and nucleotide-binding sites appears disturbed in regard to phosphate release: thus, basal ATPase activity for M765NL (0.19 s-1) was 3-fold larger than for M765 (0.06 s-1), and the stimulation of ATPase activity by actin was 5-fold lower for M765NL. These results indicate different paths of communication between the actin- and nucleotide-binding sites, in regard to ADP and Pi release, and they confirm that loop 2 is involved in high affinity actin binding.

Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium.

Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: Mydelta824-941, Mydelta943-1464, Mydelta943-1194 and Mydelta1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for Mydelta943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggest that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow.

Cooperativity between two heads of dictyostelium myosin II in in vitro motility and ATP hydrolysis.

To elucidate the significance of the two-headed structure of myosin II, we have engineered and characterized recombinant single-headed myosin II. A tail segment of a myosin II heavy chain fused with a His-tag was expressed in wild-type Dictyostelium cells. Single-headed myosin, which consists of a full length myosin heavy chain and a tagged tail, was isolated on the basis of the affinities for Nickel agarose and actin. Actin sliding velocity by the single-headed myosin was about half of the two-headed, whereas the minimum density of the heads to support continuous movement was twofold higher. Actin-activated MgATPase activity of the single-headed myosin in solution in the presence of 24 microM actin was less than half of the two headed. This decrease is primarily because of fourfold-elevated Kapp for actin and secondary to 40% lower Vmax. These results suggest that the two heads of a Dictyostelium myosin II molecule act cooperatively on an actin filament. We propose a mechanism by which two heads move actin efficiently based on the cooperativity.

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation.

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyDeltaC1225 and MyDeltaC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.

Transport of myosin II to the equatorial region without its own motor activity in mitotic Dictyostelium cells.

Fluorescently labeled myosin moved and accumulated circumferentially in the equatorial region of dividing Dictyostelium cells within a time course of 4 min, followed by contraction of the contractile ring. To investigate the mechanism of this transport process, we have expressed three mutant myosins that cannot hydrolyze ATP in myosin null cells. Immunofluorescence staining showed that these mutant myosins were also correctly transported to the equatorial region, although no contraction followed. The rates of transport, measured using green fluorescent protein-fused myosins, were indistinguishable between wild-type and mutant myosins. These observations demonstrate that myosin is passively transported toward the equatorial region and incorporated into the forming contractile ring without its own motor activity.

Myosin II can be localized to the cleavage furrow and to the posterior region of Dictyostelium amoebae without control by phosphorylation of myosin heavy and light chains.

To elucidate the role of phosphorylation in regulation of intracellular distribution of myosin II, we have characterized mutant Dictyostelium cells expressing myosin II that could not be regulated by the phosphorylation on the mapped heavy chain sites, the light chain site, or both sites. Immunofluorescence microscopy demonstrated that all three mutant myosin IIs were localized in the furrow region of dividing cells and in the tail region of migrating cells, similar to wild-type cells. Thus, regulation by phosphorylation is not required to direct myosin II toward the furrow region and the tail region in Dictyostelium. However, myosins that were deficient in heavy chain phosphorylation were distributed only in the cortical region of interphase cells, whereas some myosin IIs were present throughout the endoplasm in wild-type cells. Video microscopy showed that the rate of cell migration was significantly lower in cells that were deficient in heavy chain phosphorylation- than in light chain phosphorylation-deficient cells, myosin null cells and wild-type cells. Chemotactic behavior of cells that were deficient in heavy chain phosphorylation was also retarded. These results suggest that loss of regulation by heavy chain phosphorylation results in excessive myosin in the cortex, which leads to retarded motility.

The neck region of the myosin motor domain acts as a lever arm to generate movement.

The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.

Role of highly conserved lysine 130 of myosin motor domain. In vivo and in vitro characterization of site specifically mutated myosin.

We have created a mutant Dictyostelium myosin II heavy chain gene in which a highly conserved lysine residue (Lys-130) is changed to leucine. Lys-130 is a residue that is known to be trimethylated in skeletal muscle myosin and had been thought to play an integral role in the interaction of myosin with ATP during the actomyosin chemomechanical cycle. We report here the first in vivo and in vitro characterization of an engineered missense mutation in the motor domain of myosin. Expression of the K130L myosin in a Dictyostelium strain that lacks the myosin II heavy chain gene is sufficient to restore the ability of that cell line to undergo cytokinesis and multicellular development, processes that require functional myosin. The K130L myosin purified from these cells displays maximal actin-activated ATPase activities and promotes maximal sliding velocities of actin filaments in an in vitro motility assay that are comparable with those of wild-type myosin. These results demonstrate that this lysine residue is not required for the enzymatic or motile activities of myosin. However, the mutant protein exhibits a 4-fold increase in Km for ATP over wild-type myosin, indicating that this residue participates in the interaction of myosin with its nucleotide substrate.

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.

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.