A Novel Positive Selection for Identifying Cold-Sensitive Myosin II Mutants in Dictyostelium

We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.     

Functional expression of a chimeric myosin-containing motor domain of Chara myosin and neck and tail domains of Dictyostelium myosin II

We succeeded in expressing a chimeric myosin that comprises the motor domain of characean algal myosin, (the fastest known motor protein), and the neck and tail domains of Dictyostelium myosin II. Although the chimeric myosin showed an ATPase activity comparable to that of muscle myosin (15 times higher than that of the wild-type Dictyostelium myosin II), the motile activity of the chimera was only 1.3 times higher than that of the wild-type. However, this is the first chimeric myosin that showed motile activity faster than at least one of the parent myosins. It was suggested, therefore, that the motor domain of Chara myosin has the potential for performing fast sliding movement.     

Roles of the hydrophobic triplet in the motor domain of myosin in the interaction between myosin and actin

Myosin is a molecular motor and a member of a protein family comprising at least 18 classes. There is an about 1,000-fold difference in the in vitro sliding velocity between the fastest myosin and the slowest one. Previous studies revealed that the hydrophobic triplet in the motor domain (Val534, Phe535, and Pro536 in Dictyostelium myosin) is important for the strong binding of myosin to actin. We studied the role of the triplet in the sliding motion of myosin by means of site directed mutagenesis because the sliding velocity is determined by the time that myosin interacts with actin strongly. We produced mutant Dictyostelium myosins and subfragment-1s that have the triplet sequences of various classes of myosin with different sliding velocities. The V(max) and K(actin) values of the actin-activated ATPase for all these mutant subfragment-1s were lower than those of the wild-type Dictyostelium myosin. The mutant myosins exhibited much lower sliding velocities than the wild type. The time that the mutant subfragment-1s are in the strongly bound state did not correlate well with the sliding velocity. Our results suggested that (i) the hydrophobic triplet alone does not determine the sliding velocity of myosin, (ii) the size of the amino acid side chain in the triplet is crucial for the ATPase activity and the motility of myosin, and (iii) the hydrophobic triplet is important not only for strong binding to actin but also for the structural change of the myosin motor domain during the power stroke.     

Rat myr 4 defines a novel subclass of myosin I: identification,distribution, localization,and mapping of calmodulin-binding sites with differential calcium sensitivity

We report the identification and characterization of myr 4 (myosin from rat), the first mammalian myosin I that is not closely related to brush border myosin I. Myr 4 contains a myosin head (motor) domain, a regulatory domain with light chain binding sites and a tail domain. Sequence analysis of myosin I head (motor) domains suggested that myr 4 defines a novel subclass of myosin I''s. This subclass is clearly different from the vertebrate brush border myosin I subclass (which includes myr 1) and the myosin I subclass(es) identified from Acanthamoeba castellanii and Dictyostelium discoideum. In accordance with this notion, a detailed sequence analysis of all myosin I tail domains revealed that the myr 4 tail is unique, except for a newly identified myosin I tail homology motif detected in all myosin I tail sequences. The Ca(2+)-binding protein calmodulin was demonstrated to be associated with myr 4. Calmodulin binding activity of myr 4 was mapped by gel overlay assays to the two consecutive light chain binding motifs (IQ motifs) present in the regulatory domain. These two binding sites differed in their Ca2+ requirements for optimal calmodulin binding. The NH2-terminal IQ motif bound calmodulin in the absence of free Ca2+, whereas the COOH-terminal IQ motif bound calmodulin in the presence of free Ca2+. A further Ca(2+)-dependent calmodulin binding site was mapped to amino acids 776-874 in the myr 4 tail domain. These results demonstrate a differential Ca2+ sensitivity for calmodulin binding by IQ motifs, and they suggest that myr 4 activity might be regulated by Ca2+/calmodulin. Myr 4 was demonstrated to be expressed in many cell lines and rat tissues with the highest level of expression in adult brain tissue. Its expression was developmentally regulated during rat brain ontogeny, rising 2-3 wk postnatally, and being maximal in adult brain. Immunofluorescence localization demonstrated that myr 4 is expressed in subpopulations of neurons. In these neurons, prominent punctate staining was detected in cell bodies and apical dendrites. A punctate staining that did not obviously colocalize with the bulk of F- actin was also observed in C6 rat glioma cells. The observed punctate staining for myr 4 is reminiscent of a membranous localization.     

Expression of Dictyostelium myosin tail segments in Escherichia coli: domains required for assembly and phosphorylation

The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.     

Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins

A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.     

PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis.

We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain. PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain. paka null cells are defective in completing cytokinesis in suspension. PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells. paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly. PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling. However, PAKa does not phosphorylate myosin II. We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.     

Stretching actin filaments within cells enhances their affinity for the myosin II motor domain

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.     

Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.     

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.     

Use of the myosin motor domain as large-affinity tag for the expression and purification of proteins in Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum is increasingly be used for the overexpression of proteins. Dictyostelium is amenable to classical and molecular genetic approaches and can easily be grown in large quantities. It contains a variety of chaperones and folding enzymes, and is able to perform all kinds of post-translational protein modifications. Here, new expression vectors are presented that have been designed for the production of proteins in large quantities for biochemical and structural studies. The expression cassettes of the most successful vectors are based on a tandem affinity purification tag consisting of an octahistidine tag followed by the myosin motor domain tag. The myosin motor domain not only strongly enhances the production of fused proteins but is also used for a fast affinity purification step through its ATP-dependent binding to actin. The applicability of the new system has been demonstrated for the expression and purification of subunits of the dynein-dynactin motor protein complex from different species.     

The structural basis of blebbistatin inhibition and specificity for myosin II

Molecular motors play a central role in cytoskeletal-mediated cellular processes and thus present an excellent target for cellular control by pharmacological agents. Yet very few such compounds have been found. We report here the structure of blebbistatin, which inhibits specific myosin isoforms, bound to the motor domain of Dictyostelium discoideum myosin II. This reveals the structural basis for its specificity and provides insight into the development of new agents.     

Developmentally expressed myosin heavy-chain kinase possesses a diacylglycerol kinase domain.

In Dictyostelium, an ordered actin and myosin assembly-disassembly process is necessary for proper development, differentiation, and motility (Yumura S, Fukui F, 1985, Nature 314(6007): 194-196; Ravid S, Spudich JA, 1989, J Biol Chem 264(25): 15144-15150), and phosphorylation of myosin heavy chains has been implicated in the myosin assembly-disassembly process (Egelhoff TT, Lee RJ, Spudich JA, 1993, Cell 75(2):363-371). The developmentally expressed 84-kDa myosin heavy-chain kinase (MHCK) from Dictyostelium (Ravid S, Spudich JA, 1992, Proc Natl Acad Sci USA 89(13):5877-5881) is known to be a member of the protein kinase C (PKC) family. We have observed a rather striking homology between the large central domain of MHCK and the catalytic domain of diacylglycerol kinase (DGK), indicating that MHCK is in fact a gene fusion between a DGK and a PKC, possessing two separate kinase domains. The combined diacylglycerol kinase/myosin heavy-chain kinase (DGK/MHCK) may therefore have dual functionality, possessing the ability to phosphorylate both protein and lipid. We present a hypothesis that DGK/MHCK can antagonize both actin and myosin assembly, as well as other cellular processes, by coordinated down regulation of signaling via myosin heavy-chain kinase activity and diacylglycerol kinase activity.     

Functional characterisation of Dictyostelium myosin II with conserved tryptophanyl residue 501 mutated to tyrosine.

We created a Dictyostelium discoideum myosin II mutant in which the highly conserved residue Trp-501 was replaced by a tyrosine residue. The mutant myosin alone, when expressed in a Dictyostelium strain lacking the functional myosin II heavy chain gene, supported cytokinesis and multicellular development, processes which require a functional myosin in Dictyostelium. Additionally, we expressed the W501 Y mutant in the soluble myosin head fragment M761-2R (W501Y-2R) to characterise the kinetic properties of the mutant myosin motor domain. The affinity of the mutant myosin for actin was approximately 6-fold decreased, but other kinetic properties of the protein were changed less than 2-fold by the W501Y mutation. Based on spectroscopic studies and structural considerations, Trp-501, corresponding to Trp-510 in chicken fast skeletal muscle myosin, has been proposed to be the primary ATP-sensitive tryptophanyl residue. Our results confirm these conclusions. While the wild-type construct displayed a 10% fluorescence increase, addition of ATP to W501Y-2R was not followed by an increase in tryptophan fluorescence emission.     

Mutation in the SH1 helix reduces the activation energy of the ATP-induced conformational transition of myosin

The SH1 helix is a joint that links the converter subdomain to the rest of the myosin motor domain. Recently, we showed that a mutation within the SH1 helix in Dictyostelium myosin II (R689H) reduced the elasticity and thermal stability of the protein. To reveal the involvement of the SH1 helix in ATP-dependent conformational changes of the motor domain, we have investigated the effects of the R689H mutation on the conformational changes of the converter, using a GFP-based fluorescence resonance energy transfer method. Although the mutation does not seem to strongly affect conformations, we found that it significantly reduced the activation energy required for the ATP-induced conformational transition corresponding to the recovery stroke. Given the effects of the mutation on the mechanical properties of myosin, we propose that the SH1 helix plays an important role in the mechanochemical energy conversion underlying the conformational change of the myosin motor domain.     

Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II.

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.     

Drosophila crinkled, mutations of which disrupt morphogenesis and cause lethality, encodes fly myosin VIIA

Myosin VIIs provide motor function for a wide range of eukaryotic processes. We demonstrate that mutations in crinkled (ck) disrupt the Drosophila myosin VIIA heavy chain. The ck/myoVIIA protein is present at a low level throughout fly development and at the same level in heads, thoraxes, and abdomens. Severe ck alleles, likely to be molecular nulls, die as embryos or larvae, but all allelic combinations tested thus far yield a small fraction of adult "escapers" that are weak and infertile. Scanning electron microscopy shows that escapers have defects in bristles and hairs, indicating that this motor protein plays a role in the structure of the actin cytoskeleton. We generate a homology model for the structure of the ck/myosin VIIA head that indicates myosin VIIAs, like myosin IIs, have a spectrin-like, SH3 subdomain fronting their N terminus. In addition, we establish that the two myosin VIIA FERM repeats share high sequence similarity with only the first two subdomains of the three-lobed structure that is typical of canonical FERM domains. Nevertheless, the approximately 100 and approximately 75 amino acids that follow the first two lobes of the first and second FERM domains are highly conserved among myosin VIIs, suggesting that they compose a conserved myosin tail homology 7 (MyTH7) domain that may be an integral part of the FERM domain or may function independently of it. Together, our data suggest a key role for ck/myoVIIA in the formation of cellular projections and other actin-based functions required for viability.     

Functional characterization of the N-terminal region of myosin-2

All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release approximately 40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.