Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness.

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.

Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.

Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations.

RATIONALE: Subcellular Ca2+ indicators have yet to be developed for the myofilament where disease mutation or small molecules may alter contractility through myofilament Ca2+ sensitivity. Here, we develop and characterize genetically encoded Ca2+ indicators restricted to the myofilament to directly visualize Ca2+ changes in the sarcomere. OBJECTIVE: To produce and validate myofilament-restricted Ca2+ imaging probes in an adenoviral transduction adult cardiomyocyte model using drugs that alter myofilament function (MYK-461, omecamtiv mecarbil, and levosimendan) or following cotransduction of 2 established hypertrophic cardiomyopathy disease-causing mutants (cTnT [Troponin T] R92Q and cTnI [Troponin I] R145G) that alter myofilament Ca2+ handling. METHODS AND RESULTS: When expressed in adult ventricular cardiomyocytes RGECO-TnT (Troponin T)/TnI (Troponin I) sensors localize correctly to the sarcomere without contractile impairment. Both sensors report cyclical changes in fluorescence in paced cardiomyocytes with reduced Ca2+ on and increased Ca2+ off rates compared with unconjugated RGECO. RGECO-TnT/TnI revealed changes to localized Ca2+ handling conferred by MYK-461 and levosimendan, including an increase in Ca2+ binding rates with both levosimendan and MYK-461 not detected by an unrestricted protein sensor. Coadenoviral transduction of RGECO-TnT/TnI with hypertrophic cardiomyopathy causing thin filament mutants showed that the mutations increase myofilament [Ca2+] in systole, lengthen time to peak systolic [Ca2+], and delay [Ca2+] release. This contrasts with the effect of the same mutations on cytoplasmic Ca2+, when measured using unrestricted RGECO where changes to peak systolic Ca2+ are inconsistent between the 2 mutations. These data contrast with previous findings using chemical dyes that show no alteration of [Ca2+] transient amplitude or time to peak Ca2+. CONCLUSIONS: RGECO-TnT/TnI are functionally equivalent. They visualize Ca2+ within the myofilament and reveal unrecognized aspects of small molecule and disease-associated mutations in living cells.

SarcTrack.

RATIONALE: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. OBJECTIVE: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. METHODS AND RESULTS: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. CONCLUSIONS: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

Heart failure drug changes the mechanoenzymology of the cardiac myosin powerstroke.

Omecamtiv mecarbil (OM), a putative heart failure therapeutic, increases cardiac contractility. We hypothesize that it does this by changing the structural kinetics of the myosin powerstroke. We tested this directly by performing transient time-resolved FRET on a ventricular cardiac myosin biosensor. Our results demonstrate that OM stabilizes myosin's prepowerstroke structural state, supporting previous measurements showing that the drug shifts the equilibrium constant for myosin-catalyzed ATP hydrolysis toward the posthydrolysis biochemical state. OM slowed the actin-induced powerstroke, despite a twofold increase in the rate constant for actin-activated phosphate release, the biochemical step in myosin's ATPase cycle associated with force generation and the conversion of chemical energy into mechanical work. We conclude that OM alters the energetics of cardiac myosin's mechanical cycle, causing the powerstroke to occur after myosin weakly binds to actin and releases phosphate. We discuss the physiological implications for these changes.

ZO-1 protein is required for hydrogen peroxide to increase MDCK cell paracellular permeability in an ERK 1/2-dependent manner.

Hydrogen peroxide (H2O2) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with H2O2 activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of H2O2 to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of H2O2 to increase paracellular permeability. H2O2 treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. H2O2 treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, H2O2 increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to H2O2 was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. H2O2 treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that H2O2 increases MDCK cell paracellular permeability through activation of ERK 1/2. This H2O2 action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

Acute exposure to progesterone attenuates cardiac contraction by modifying myofilament calcium sensitivity in the female mouse heart.

Acute application of progesterone attenuates cardiac contraction, although the underlying mechanisms are unclear. We investigated whether progesterone modified contraction in isolated ventricular myocytes and identified the Ca2+ handling mechanisms involved in female C57BL/6 mice (6-9 mo; sodium pentobarbital anesthesia). Cells were field-stimulated (4 Hz; 37°C) and exposed to progesterone (0.001-10.0 µM) or vehicle (35 min). Ca2+ transients (fura-2) and cell shortening were recorded simultaneously. Maximal concentrations of progesterone inhibited peak contraction by 71.4% (IC50 = 160 ± 50 nM; n = 12) and slowed relaxation by 75.4%. By contrast, progesterone had no effect on amplitudes or time courses of underlying Ca2+ transients. Progesterone (1 µM) also abbreviated action potential duration. When the duration of depolarization was controlled by voltage-clamp, progesterone attenuated contraction and slowed relaxation but did not affect Ca2+ currents, Ca2+ transients, sarcoplasmic reticulum (SR) content, or fractional release of SR Ca2+ Actomyosin MgATPase activity was assayed in myofilaments from hearts perfused with progesterone (1 µM) or vehicle (35 min). While maximal responses to Ca2+ were not affected by progesterone, myofilament Ca2+ sensitivity was reduced (EC50 = 0.94 ± 0.01 µM for control, n = 7 vs. 1.13 ± 0.05 µM for progesterone, n = 6; P < 0.05) and progesterone increased phosphorylation of myosin binding protein C. The effects on contraction were inhibited by lonaprisan (progesterone receptor antagonist) and levosimendan (Ca2+ sensitizer). Unlike results in females, progesterone had no effect on contraction or myofilament Ca2+ sensitivity in age-matched male mice. These data indicate that progesterone reduces myofilament Ca2+ sensitivity in female hearts, which may exacerbate manifestations of cardiovascular disease late in pregnancy when progesterone levels are high. NEW & NOTEWORTHY: We investigated myocardial effects of acute application of progesterone. In females, but not males, progesterone attenuates and slows cardiomyocyte contraction with no effect on calcium transients. Progesterone also reduces myofilament calcium sensitivity in female hearts. This may adversely affect heart function, especially when serum progesterone levels are high in pregnancy.Listen to this article's corresponding podcast at https://ajpheart.podbean.com/e/acute-progesterone-modifies-cardiac-contraction/.

Cortical Actin Alteration at the Matrix-Side Cytoplasm in Lung Adenocarcinoma Cells and Its Significance in Invasion.

OBJECTIVES: Cortical actin is a thin layer of filamentous (F-)actin that lies beneath the plasma membrane, and its role in pathophysiology remains unclear. We investigated the subcellular localization of cortical actin by the histopathological and experimental studies of lung adenocarcinomas. MATERIALS AND METHODS: The subcellular localization of cortical actin was studied in surgically resected lung adenocarcinomas tissues and in 3-dimensionally cultured lung adenocarcinoma A549 cells. RESULTS: In normal type II alveolar cells and the bronchiolar epithelium, cortical actin was localized to the apical-side cytoplasm. In invasive adenocarcinoma cells, cortical actin was frequently localized to the matrix side. The degree of cortical actin localized to the matrix side was associated with the loss of basement membrane and a poor prognosis. In A549 cell spheroids cultured in a type I collagen and basement membrane extract Matrigel™ mixed gel, cortical F-actin was localized to the matrix side with phosphorylated myosin light chain. Super-resolution and electron microscopy results suggest that compact wrinkling of the plasma membrane by myosin-mediated F-actin contraction is an explanation for cortical actin accumulation at the matrix side. The myosin II inhibitor blebbistatin suppressed the 3-dimensional collective migration of A549 cells induced by constitutively active Cdc42 and MT1-MMP. CONCLUSION: Cortical actin accumulation at the matrix-side cytoplasm of cancer cells occurs in invasive lung adenocarcinomas and it possibly participates in the migration of cancer cells through myosin-mediated contraction.

Long-term physiological T3 supplementation in hypertensive heart disease in rats.

Animal studies suggest that hypertension leads to cardiac tissue hypothyroidism, a condition that can by itself lead to heart failure. We have previously shown that short-term thyroid hormone treatment in Spontaneously Hypertensive Heart Failure (SHHF) rats near heart failure is beneficial. This study tested the hypothesis that therapeutic, long-term T3 treatment in SHHF rats can prevent or attenuate cardiac dysfunction. Female SHHF rats were treated orally with a physiological T3 dose (0.04 µg/ml) from 12 to 24 mo of age. Age-matched female SHHF and Wistar-Kyoto rats served as hypertensive and normotensive controls, respectively. SHHF rats had reduced serum free thyroid hormone levels and cardiac tissue T3 levels, LV dysfunction, and elevated LV collagen content compared with normotensive controls. Restoration of serum and cardiac tissue thyroid hormone levels in T3-treated rats was associated with no change in heart rate, but strong trends for improvement in LV systolic function and collagen levels. For instance, end-systolic diameter, fractional shortening, systolic wall stress, and LV collagen levels were no longer significantly different from controls. In conclusion, longstanding hypertension in rats led to chronic low serum and cardiac tissue thyroid hormone levels. Long-term treatment with low-dose T3 was safe. While cardiac dysfunction could not be completely prevented in the absence of antihypertensive treatment, T3 may offer additional benefits as an adjunct therapy with possible improvement in diastolic function.

Myosin X regulates netrin receptors and functions in axonal path-finding.

Netrins regulate axon path-finding during development, but the underlying mechanisms are not well understood. Here, we provide evidence for the involvement of the unconventional myosin X (Myo X) in netrin-1 function. We find that Myo X interacts with the netrin receptor deleted in colorectal cancer (DCC) and neogenin, a DCC-related protein. Expression of Myo X redistributes DCC to the cell periphery or to the tips of neurites, whereas its silencing prevents DCC distribution in neurites. Moreover, expression of DCC, but not neogenin, stimulates Myo X-mediated formation and elongation of filopodia, suggesting that Myo X function may be differentially regulated by DCC and neogenin. The involvement of Myo X in netrin-1 function was further supported by the effects of inhibiting Myo X function in neurons. Cortical explants derived from mouse embryos expressing a motor-less Myo X exhibit reduced neurite outgrowth in response to netrin-1 and chick commissural neurons expressing the motor-less Myo X, or in which Myo X is silenced using microRNA (miRNA), show impaired axon projection in vivo. Taken together, these results identify a novel role for Myo X in regulating netrin-1 function.

AMP-activated protein kinase phosphorylates cardiac troponin I and alters contractility of murine ventricular myocytes.

RATIONALE: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. OBJECTIVE: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. METHODS AND RESULTS: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca(2+) sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca(2+) transient and prolonged relaxation despite shortening the time constant of Ca(2+) transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca(2+) sensitivity. CONCLUSIONS: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Fixed endothelial cells exhibit physiologically relevant nanomechanics of the cortical actin web.

It has been unknown whether cells retain their mechanical properties after fixation. Therefore, this study was designed to compare the stiffness properties of the cell cortex (the 50-100 nm thick zone below the plasma membrane) before and after fixation. Atomic force microscopy was used to acquire force indentation curves from which the nanomechanical cell properties were derived. Cells were pretreated with different concentrations of actin destabilizing agent cytochalasin D, which results in a gradual softening of the cell cortex. Then cells were studied 'alive' or 'fixed'. We show that the cortical stiffness of fixed endothelial cells still reports functional properties of the actin web qualitatively comparable to those of living cells. Myosin motor protein activity, tested by blebbistatin inhibition, can only be detected, in terms of cortical mechanics, in living but not in fixed cells. We conclude that fixation interferes with motor proteins while maintaining a functional cortical actin web. Thus, fixation of cells opens up the prospect of differentially studying the actions of cellular myosin and actin.

[Role of myosins in depression of sensitivity of Helix neurons to acetylcholine in a cellular analog of habituation].

We investigated the involvement of cytoskeleton motor proteins, myosins, in the molecular mechanism of sensitivity depression to acetylcholine in Helix command neurons of defensive behavior in a cellular analog of habituation. There were analyzed the effects of several drugs disturbing myosin function: ML-7 and MLCK-IP-18--blockers of myosin light chain kinase, blebbistatin--an inhibitor of non-muscle myosin II, Y-27632--inhibitor of kinases ROCK-I and ROCK-II (activate mainly non-muscle myosin II) on the depression of acetylcholine-induced inward current. It was found that ML-7 and MLCK-IP- 18 weakened current depression; blebbistatin and Y-27632 did not change the depression. The results of experimental inhibitory analysis and mathematical modeling of the effects of inhibitors on the number of membrane-bound cholinergic receptors allow to suggest the involvement ofmyosins (excluding non-muscle myosin II) in the transports of acetylcholine receptors (endo- and exocytosis) that are responsible for sensitivity changes in neuron somatic membrane to acetylcholine in a cellular analog of habituation.

Contrast enhancement in the phase plate transmission electron microscopy using an objective lens with a long focal length.

A new optical condition using an objective lens (OL) of a long focal length (objective mini lens: OM) was tested to enhance image contrast in phase plate transmission electron microscopy (P-TEM). A phase plate was set on the selected area aperture plane where diffraction patterns were formed under the optical condition using the OM. A phase shift by the phase plate was added to the electron waves to visualize phase objects. The application of the OM to the P-TEM should provide higher phase contrast than that obtained by the OL for the phase objects. One of the reasons for the contrast enhancement is that high-angle scattering electron waves which would give the background intensity were not used for image formation due to the large spherical aberration. Another reason is that the cut-on frequency above which the phase shift was added by the phase plate could be smaller using the OL with a long focal length. Experimental results and model calculations showed the contrast enhancement of the biological specimens using the OM.

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain.

We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

Peptidomic analysis of hippocampal tissue for explore leptin neuroprotective effect on the preterm ischemia-hypoxia brain damage model rats.

The most common injury of preterm infants is periventricular leukomalacia (PVL) and to date there is still no safe and effective treatment. In our previous studies, leptin has been found to have neuroprotective effects on the preterm ischemia-hypoxia brain damage model rats in animal behavior. To gain insight into the neuroprotective mechanisms of leptin on preterm brain damage model rats, we constructed a comparative peptidomic profiling of hippocampal tissue between leptin-treated after model and preterm ischemia-hypoxia brain damage model rats using a stable isobaric labeling strategy involving tandem mass tag reagents, followed by nano liquid chromatography tandem mass spectrometry. We identified and quantified 4164 peptides, 238 of which were differential expressed in hippocampal tissue in the two groups. A total of 150 peptides were up regulated and 88 peptides were down regulated. These peptides were imported into the Ingenuity Pathway Analysis (IPA) and identified putative roles in nervous system development, function and diseases. We concluded that the preterm ischemia-hypoxia brain damage model with leptin treatment induced peptides changes in hippocampus, and these peptides, especially for the peptides associated "microtubule-associated protein 1b (MAP1b), Elastin (Eln), Piccolo presynaptic cytomatrix protein (Pclo), Zinc finger homeobox 3(Zfhx3), Alpha-kinase 3(Alpk3) and Myosin XVA(Myo15a) ", could be candidate bio-active peptides and participate in neuroprotection of leptin. These may advance our current understanding of the mechanism of leptin's neuroprotective effect on preterm brain damage and may be involved in the etiology of preterm brain damage. Meanwhile, we found that repression of ILK signaling pathway plays a significant role in neuroprotection of leptin. A better understanding of the role of ILK signaling pathway in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious drug for therapy against human nervous system disorders.

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.

Myosin is involved in postmitotic cell spreading.

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time-lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.

Characterization of secophalloidin-induced force loss in cardiac myofibrils.

Secophalloidin (SPH) is known to cause in cardiac myofibrils force without Ca(2+) (half-maximal effect approximately 2 mM) followed by irreversible loss of Ca(2+)-activated force. At maximal Ca(2+) activation, SPH increases force (half-maximal effect < 0.1 mM). We found that SPH at low concentration (0.5 mM) did not cause either force activation or force loss at pCa 8.7, but both of these effects did occur when force was activated by Ca(2+). The force loss was prevented when SPH was applied during rigor or in the presence of 2,3-butanedione monoxime (85 mM). Furthermore, studying muscle in which the force was previously reduced by SPH (up to 50%) did not reveal significant changes in Ca(2+) sensitivity and cooperativity of Ca(2+) activation or qualitative alterations in SPH-induced changes in Ca(2+)-activated contraction. Data suggest that the force loss is mediated by cycling cross-bridges, and might reflect a reduction in force generated by individual cross-bridges.

Quantitative analysis of auxin-regulated proteins from basal part of leaf sheaths in rice by two-dimensional difference gel electrophoresis.

To identify the effects of auxin on rice root formation, proteins induced by exogenous addition of auxin to rice seedlings were analyzed by a proteomic approach. Root formation by rice seedlings was promoted by 0.45microM 2,4-dichlorophenoxyacetic acid (2,4-D) and repressed by 60microM p-chlorophenoxyisobutyric acid (PCIB). Proteins extracted from the basal part of leaf sheaths of rice seedlings treated with 2,4-D or PCIB for 48h were labeled with Cy3 and Cy5, and separated by two-dimensional polyacrylamide gel electrophoresis. Out of nine proteins up-regulated by 2,4-D and down-regulated by PCIB, five proteins showing significant difference in abundance were used for expression analysis at the transcript abundance level. Transcript abundance of the mitochondrial complex I subunit slightly increased with 2,4-D treatment and were repressed by PCIB. The transcript abundance of EF-1beta', myosin heavy chain and mitochondrial [Mn]SOD increased with 2,4-D treatment but did not decrease with PCIB. The transcript abundance of aldehyde dehydrogenase was not effected by 2,4-D or PCIB. These results indicate that mitochondrial complex I subunit is part of the downstream signal cascade of PCIB, whereas myosin heavy chain, mitochondrial [Mn]SOD and EF-1beta' are involved in the 2,4-D signal cascade but are probably upstream of PCIB.