Regulation by molluscan myosins

Molluscan myosins are regulated molecules that control muscle contraction by the selective binding of calcium. The essential and the regulatory light chains are regulatory subunits. Scallop myosin is the favorite material for studying the interactions of the light chains with the myosin heavy chain since the regulatory light chains can be reversibly removed from it and its essential light chains can be exchanged. Mutational and structural studies show that the essential light chain binds calcium provided that the Ca-binding loop is stabilized by specific interactions with the regulatory light chain and the heavy chain. The regulatory light chains are inhibitory subunits. Regulation requires the presence of both myosin heads and an intact headrod junction. Heavy meromyosin is regulated and shows cooperative features of activation while subfragment-1 is non-cooperative. The myosin heavy chains of the functionally different phasic striated and the smooth catch muscle myosins are products of a single gene, the isoforms arise from alternative splicing. The differences between residues of the isoforms are clustered at surface loop-1 of the heavy chain and account for the different ATPase activity of the two muscle types. Catch muscles contain two regulatory light chain isoforms, one phosphorylatable by gizzard myosin light chain kinase. Phosphorylation of the light chain does not alter ATPase activity. We could not find evidence that light chain phosphorylation is responsible for the catch state.     

The observation of quasi-molecular ions from a protein neurotoxin (MW 7821) using 127I-plasma desorption mass spectroscopy

Fast heavy ions, i.e. 90 MeV ¹²⁷I from the Uppsala tandem accelerator have been used to desorb and ionize molecules from a cobra venom neurotoxin. The protein is built up by 71 amino acid residues in a single polypeptide chain, tightly cross-linked by 5 disulfide bridges. The molecular weight as confirmed by protein sequence analysis is 7821. The ions were mass analyzed by the time-of-flight technique. This is to our knowledge the largest protein for which it has been possible to detect quasimolecular ions by a mass spectrometric technique.     

Conformationally altered aortic myosin light chains

Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidende: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation.     

Myosin S2 is not required for effects of myosin binding protein-C on motility

The unique myosin binding protein-c "motif" near the N-terminus of myosin binding protein-C (MyBP-C) binds myosin S2. Previous studies demonstrated that recombinant proteins containing the motif and flanking regions (e.g., C1C2) affect thin filament movement in motility assays using heavy meromyosin (S1 plus S2) as the molecular motor. To determine if S2 is required for these effects we investigated whether C1C2 affects motility in assays using only myosin S1 as the motor protein. Results demonstrate that effects of C1C2 are comparable in both systems and suggest that the MyBP-C motif affects motility through direct interactions with actin and/or myosin S1.     

Refined model of the 10S conformation of smooth muscle myosin by cryo-electron microscopy 3D image reconstruction

The actin-activated ATPase activity of smooth muscle myosin and heavy meromyosin (smHMM) is regulated by phosphorylation of the regulatory light chain (RLC). Complete regulation requires two intact myosin heads because single-headed myosin subfragments are always active. 2D crystalline arrays of the 10S form of intact myosin, which has a dephosphorylated RLC, were produced on a positively charged lipid monolayer and imaged in 3D at 2.0 nm resolution by cryo-electron microscopy of frozen, hydrated specimens. An atomic model of smooth muscle myosin was constructed from the X-ray structures of the smooth muscle myosin motor domain and essential light chain and a homology model of the RLC was produced based on the skeletal muscle S1 structure. The initial model of the 10S myosin, based on the previous reconstruction of smHMM, was subjected to real space refinement to obtain a quantitative fit to the density. The smHMM was likewise refined and both refined models reveal the same asymmetric interaction between the upper 50 kDa domain of the "blocked" head and parts of the catalytic, converter domains and the essential light chain of the "free" head observed previously. This observation suggests that this interaction is not simply due to crystallographic packing but is enforced by elements of the myosin heads. The 10S reconstruction shows additional alpha-helical coiled-coil not seen in the earlier smHMM reconstruction, but the location of one segment of S2 is the same in both.     

Three-dimensional reconstruction of thin filaments decorated with a Ca2+-regulated myosin

Three-dimensional reconstructions of “barbed” and “blunted” arrowheads (Craig et al., 1980) show that these two forms arise from arrangement of scallop myosin subfragments (S1) that appear about 40 Å longer in the presence of the regulatory light chain than in its absence. A similar difference in apparent length is indicated by images of single myosin subfragments in partially decorated filaments. The extra mass is located at the end of the subfragment furthest from actin, and probably comprises part of the regulatory light chain as well as a segment of the myosin heavy chain. The fact that barbed arrowheads are also formed by myosin subfragments from vertebrate striated and smooth muscles implies that the homologous light chains in these myosins have locations similar to that of the scallop light chain.The scallop light chain probably does not extend into the actin-binding site on the myosin head, and is therefore unlikely to interfere physically with binding. Rather, regulation of actin-myosin interaction by light chains may involve Ca²⁺-dependent changes in the structure of a region near the head-tail junction of myosin.The reconstructions suggest locations for actin and tropomyosin relative to myosin that are similar to those proposed by Taylor & Amos (1981) and are consistent with a revised steric blocking model for regulation by tropomyosin. The identification of actin from these reconstructions is supported by images of partially decorated filaments that display the polarity of the actin helix relative to that of bound myosin subfragments.     

Alternative splicing of smooth muscle myosin heavy chains and its functional consequences

The aim of our study was to determine the relation between alternatively spliced myosin heavy chain (MHC) isoforms and the contractility of smooth muscle. The relative amount of MHC with an alternatively spliced insert in the 5′ (amino terminal) domain was determined on the protein level using a peptide-directed antibody (a25K/50K) raised against the inserted sequence (QGPSFAY). Smooth muscle MHC isoforms of both bladder and myometrium but not nonmuscle MHC reacted with a25/50K. Using a quantitative Western-blot approach the amount of 5′-inserted MHC in rat bladder was detected to be about eightfold higher than in normal rat myometrium. The amount of heavy chain with insert was found to be decreased by about 50% in the myometrium of pregnant rats. Although bladder contained significantly more 5′-inserted MHC than myometrium, apparent maximal shortening velocities (Vmax) were comparable, being 0.138 ± 0.012 and 0.114 ± 0.023 muscle length per second of skinned bladder and normal myometrium fibers, respectively. Phosphorylation of myosin light chain 20 induced by maximal Ca²⁺/calmodulin activation was the same in bladder and myometrial fibers. These results suggest that the amount of 5′-inserted MHC is not necessarily associated with contractile properties of smooth muscle. © 1996 Wiley-Liss, Inc.     

人心肌肌球蛋白轻链1与重链和肌动蛋白的结合

在测得中国人心肌肌球蛋白轻链 1cDNA的核苷酸序列 ,并获得一株单克隆抗体 (HCMLC1 8)的基础上 ,用PCR方法 ,以中国人心肌肌球蛋白轻链 1的cDNA为模板 ,分别获得中国人心肌肌球蛋白轻链 1的各为 98个氨基酸的N端和C端片段cDNA的克隆并进行了表达。同时进行了其表达产物和大鼠心肌肌球蛋白重链和人心肌肌动蛋白以及单克隆抗体结合的研究 ,发现三者均和轻链 1的N端相结合 ,结合位点各不相同。这些结合位点可能均位于轻链 1的分子表面 ,而且如果轻链 1在实验状态下先与肌动蛋白结合 ,则有可能影响轻链与重链间的彼此结合。肌动蛋白在体外能以不同位点结合肌球蛋白重链和轻链 ,可能在肌肉收缩过程中具有重要的生理意义     

调节轻链在平滑肌肌球蛋白分子上的定位

 应用凝胶电泳覆盖技术和放射自显影法研究了32~P-标记的平滑肌肌球蛋白调节轻链在肌球蛋白分子上的定位。实验结果表明调节轻链(LC_(20))可重新结合于平滑肌肌球蛋白重链(200kD),重酶解肌球蛋白(130kD)及其62kD和26kD肽段上。这提示调节轻链的结合点位于平滑肌肌球蛋白亚段-1羧基端的26kD肽段上。     

Serial deletion constructs of human cardiac myosin heavy chain genes generated by PCR amplification

Summary Serial deletion constructs derived from the 5-flanking regions of the human cardiac - and -myosin heavy chain genes were generated by polymerase chain reaction (PCR) amplifications. Generation of different length chimeric constructs were based on the complete sequence of the human cardiac myosin heavy chain genes [1, 2]. The primers were synthesized with HindIII and BamH₁ sites and were linked to any designed nucleotide of the 5 flanking sequence of the myosin heavy chain gene(s). Following the PCR amplification and the site-directed mutagenesis, the PCR products were verified by DNA sequencing and subsequently ligated to the chloramphenical acetyltransferase (pBLCAT₃) reporter gene which was restricted with Hind III and BamH₁. Neonatal rat cardiocytes were used to assay the promotor activity (i.e. CAT activity) of different lengths of the chimeric constructs of the gene.     

Structural and functional changes of myosin during development: comparison with adult fast, slow and cardiac myosin.

ATPase (Ca²⁺ and K⁺ activated) activity of myosin prepared from muscles of 3–4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO₄ polyacrylamide gel electrophoresis, of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5′-dithio-2-dinitrobenzoate treatment of LC₂ except that the relative amount of LC₃ is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains.     

Alternative splicing and cycling kinetics of myosin change during hypertrophy of human smooth muscle cells

We investigated in vivo expression of myosin heavy chain (MHC) isoforms, 17 kDa myosin light chain (MLC₁₇), and phosphorylation of the 20 kDa MLC (MLC₂₀) as well as mechanical performance of chemically skinned fibers of normal and hypertrophied smooth muscle (SM) of human myometrium. According to their immunological reactivity, we identified three MHC isoenzymes in the human myometrium: two SM-MHC (SM1 with 204 kDa and SM2 with 200 kDa), and one non-muscle specific MHC (NM with 196 kDa). No cross-reactivity was detected with an antibody raised against a peptide corresponding to a seven amino acid insert at the 25K/50K junction of the myosin head (a-25K/50K) in both normal and hypertrophied myometrium. In contrast, SM-MHC of human myomatous tissue strongly reacted with a-25K/50K. Expression of SM1/SM2/NM (%) in normal myometrium was 31.7/34.7/33.6 and 35.1/40.9/24 in hypertrophied myometrium. The increased SM2 and decreased NM expression in the hypertrophied state was statistically significant (P < 0.05). MHC isoform distribution in myomatous tissue was similar to normal myometrium (35.3/35.3/29.4). In vivo expression of MLC_17a increased from 25.5% in normal to 44.2% in hypertrophied (P < 0.001) myometrium. Phosphorylation levels of MLC₂₀ upon maximal Ca²⁰-calmodulin activation of skinned myometrial fibers were the same in normal and hypertrophied myometrial fibers. Maximal force of isometric contraction of skinned fibers (pCa 4.5, slack-length) was 2.85 mN/mm² and 5.6 mN/mm² in the normal and hypertrophied state, respectively (P < 0.001). Apparent maximal shortening velocity (Vmax_app, extrapolated from the force-velocity relation) of myometrium rose from 0.13 muscle length s ¹ (ML/s) in normal to 0.24 ML/s in hypertrophied fibers (P < 0.001). J. Cell. Biochem, 64:171–181. © 1997 Wiley-Liss, Inc.     

The B2 alternatively spliced isoform of nonmuscle myosin II-B lacks actin-activated MgATPase activity and in vitro motility

We report the initial biochemical characterization of an alternatively spliced isoform of nonmuscle heavy meromyosin (HMM) II-B2 and compare it with HMM II-B0, the nonspliced isoform. HMM II-B2 is the HMM derivative of an alternatively spliced isoform of endogenous nonmuscle myosin (NM) II-B, which has 21-amino acids inserted into loop 2, near the actin-binding region. NM II-B2 is expressed in the Purkinje cells of the cerebellum as well as in other neuronal cells [X. Ma, S. Kawamoto, J. Uribe, R.S. Adelstein, Function of the neuron-specific alternatively spliced isoforms of nonmuscle myosin II-B during mouse brain development, Mol. Biol. Cell 15 (2006) 2138-2149]. In contrast to any of the previously described isoforms of NM II (II-A, II-B0, II-B1, II-C0 and II-C1) or to smooth muscle myosin, the actin-activated MgATPase activity of HMM II-B2 is not significantly increased from a low, basal level by phosphorylation of the 20 kDa myosin light chain (MLC-20). Moreover, although HMM II-B2 can bind to actin in the absence of ATP and is released in its presence, it cannot propel actin in the sliding actin filament assay following MLC-20 phosphorylation. Unlike HMM II-B2, the actin-activated MgATPase activity of a chimeric HMM with the 21-amino acid II-B2 sequence inserted into the homologous location in the heavy chain of HMM II-C is increased following MLC-20 phosphorylation. This indicates that the effect of the II-B2 insert is myosin heavy chain specific.     

The functional cooperation of MAP1A heavy chain and light chain 2 in the binding of microtubules

Microtubule-associated protein 1A (MAP1A) is a high-molecular-weight protein that is comprised of a heavy chain and a light chain (LC2) and is widely distributed along the microtubules in both mature neurons and glial cells. To illustrate the interaction among the MAP1A heavy chain, light chain, and microtubule, we prepared DNA constructs with Myc-, EGFP-, or DsRed-tags for full-length MAP1A DNA expressing whole MAP1A protein, two domains of MAP1A heavy chain, and light chain. Distribution patterns of various MAP1A domains as well as their interactions with microtubules were monitored in a non-neuronal COS7 and a neuronal Neuro2A cells. Our data revealed that a complete MAP1A protein, which contains both heavy chain and LC2, could be colocalized with microtubule networks not only in Neuro2A cells but also in transfected COS7 cells. Filamentous structures failed to be visualized along microtubules in COS7 cells transfected with MAP1A heavy chain or LC2 alone. Whereas, after introducing MAP1A heavy chain with LC2 into COS7 cells, both heavy chain and LC2 could be colocalized with microtubules. From our functional analysis, both MAP1A and its LC2 could protect microtubules against the challenge of nacodazol. Data collected from yeast two-hybrid assays of various MAP1A domains confirmed that the interaction of LC2 and NH2-terminal of MAP1A heavy chain is important for microtubule binding. From our analysis of MAP1A functional domains, we suggest that interactions between MAP1A heavy chain and LC2 are critical for the binding of microtubules.     

Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

Interaction of pollen F-actin with rabbit muscle myosin and its subfragments (HMM,S1)

Summary Actin purified from maize pollen grains can be polymerized into F-actin which increased the ATPase activities of proteolytic fragments (HMM, S₁) of rabbit muscle myosin. The values of K_app is 232 M for HMM and 290 M for S₁, which are six- and seven-fold higher than those of rabbit muscle F-actin under the same conditions. Pollen actin and rabbit muscle myosin form hybrid actomyosin showing increase in viscosity and turbidity of solution. Viscosity and turbidity of the actomyosin dropped and then increased again with addition of ATP. Polymerized pollen actin can be decorated in vitro with both rabbit muscle HMM and S₁ to form an arrowhead-shaped structure like that observed in living plant cells. The results show that pollen actin is similar to muscle actin at a qualitative level. But there are differences between them at a quantitative level.Abbreviations HMM heavy meromyosin - S₁ myosin subfragment 1 - ATP adenosine-5-triphosphate     

The effect of angiotensin II on myosin heavy chain expression in cultured myocardial cells

Summary Angiotensin II (AII), the principal mediator of the renin-angiotensin system, is an important regulator of vascular and cardiac homeostasis. AII has also been shown to be a regulator of cardiac hypertrophy and of the corresponding changes in amount and composition of certain tissue proteins. We examined the trophic effects of AII on cultured myocytes derived from neonatal rat ventricles and followed, by Northern blot analysis and polyacrylamide gel electrophoresis, the expression of α- and β-myosin heavy chain iso-mRNAs and isoproteins. Our findings show that a single administration of AII is sufficient to induce a trophic response in cultured beating myocytes and to enhance the expression of β-myosin heavy chain iso-mRNA and isoprotein, having no effect on α-myosin heavy chain. Induction of α-myosin heavy chain expression by thyroid hormone before AII was administered showed that AII could not potentiate a shift from α- to β-myosin heavy chain predominance. We suggest that the potency of AII to regulate the expression of myosin heavy chain isogenes is restricted to the β isoform and is overridden by thyroid hormone.     

A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows

Approaches with high spatial and temporal resolution are required to understand the regulation of nonmuscle myosin II in vivo. Using fluorescence resonance energy transfer we have produced a novel biosensor allowing simultaneous determination of myosin light chain kinase (MLCK) localization and its [Ca2+]4/calmodulin-binding state in living cells. We observe transient recruitment of diffuse MLCK to stress fibers and its in situ activation before contraction. MLCK is highly active in the lamella of migrating cells, but not at the retracting tail. This unexpected result highlights a potential role for MLCK-mediated myosin contractility in the lamella as a driving force for migration. During cytokinesis, MLCK was enriched at the spindle equator during late metaphase, and was maximally activated just before cleavage furrow constriction. As furrow contraction was completed, active MLCK was redistributed to the poles of the daughter cells. These results show MLCK is a myosin regulator in the lamella and contractile ring, and pinpoints sites where myosin function may be mediated by other kinases.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

Orientational changes of crossbridges during single turnover of ATP

Muscle contraction results from rotation of actin-bound myosin crossbridges. Crossbridges consist of the globular N-terminal catalytic domain and the alpha-helical C-terminal regulatory domain containing the essential and regulatory light chains. The essential light chain exists in two isoforms, of which the larger one has a 41-amino acid extension piece added at the N-terminus. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. We measured the kinetics of the swing associated with the turnover of a single molecule of ATP. Muscle was labeled at the regulatory domain by replacing native essential or regulatory light chain with fluorescent adducts. The rotations were measured by the anisotropy of fluorescence originating from approximately 400 crossbridges residing in a small volume defined by a confocal aperture of a microscope. The crossbridges were synchronized by rapid photogeneration of a stoichiometric amount of ATP. The rotations reflected dissociation from thin filaments followed by a slow reattachment. The dissociation was the same for each light chain (halftime approximately 120 ms) but the rate of reattachment depended on the type of light chain. The halftimes were 920 +/- 50 ms and 660 +/- 100 ms for isoforms 1 and 3 of the essential light chain, respectively. The reason that the lifetimes were so long was creation of a small amount of ATP, enough only for a single turnover of crossbridges. A model was constructed that quantified this effect. After accounting for the slowdown, the halftimes of dissociation and attachment were 34 and 200 ms, respectively.