Label-free, all-optical detection, imaging, and tracking of a single protein.

Optical detection of individual proteins requires fluorescent labeling. Cavity and plasmonic methodologies enhance single molecule signatures in the absence of any labels but have struggled to demonstrate routine and quantitative single protein detection. Here, we used interferometric scattering microscopy not only to detect but also to image and nanometrically track the motion of single myosin 5a heavy meromyosin molecules without the use of labels or any nanoscopic amplification. Together with the simple experimental arrangement, an intrinsic independence from strong electronic transition dipoles and a detection limit of <60 kDa, our approach paves the way toward nonresonant, label-free sensing and imaging of nanoscopic objects down to the single protein level.

The Qdot-labeled actin super-resolution motility assay measures low-duty cycle muscle myosin step size.

Myosin powers contraction in heart and skeletal muscle and is a leading target for mutations implicated in inheritable muscle diseases. During contraction, myosin transduces ATP free energy into the work of muscle shortening against resisting force. Muscle shortening involves relative sliding of myosin and actin filaments. Skeletal actin filaments were fluorescently labeled with a streptavidin conjugate quantum dot (Qdot) binding biotin-phalloidin on actin. Single Qdots were imaged in time with total internal reflection fluorescence microscopy and then spatially localized to 1-3 nm using a super-resolution algorithm as they translated with actin over a surface coated with skeletal heavy meromyosin (sHMM) or full-length ß-cardiac myosin (MYH7). The average Qdot-actin velocity matches measurements with rhodamine-phalloidin-labeled actin. The sHMM Qdot-actin velocity histogram contains low-velocity events corresponding to actin translation in quantized steps of ~5 nm. The MYH7 velocity histogram has quantized steps at 3 and 8 nm in addition to 5 nm and larger compliance compared to that of sHMM depending on the MYH7 surface concentration. Low-duty cycle skeletal and cardiac myosin present challenges for a single-molecule assay because actomyosin dissociates quickly and the freely moving element diffuses away. The in vitro motility assay has modestly more actomyosin interactions, and methylcellulose inhibited diffusion to sustain the complex while preserving a subset of encounters that do not overlap in time on a single actin filament. A single myosin step is isolated in time and space and then characterized using super-resolution. The approach provides a quick, quantitative, and inexpensive step size measurement for low-duty cycle muscle myosin.

Direct observation of oriented behavior of actin filaments interacting with desmin intermediate filaments.

BACKGROUND: Associations between actin filaments (AFs) and intermediate filaments (IFs) are frequently observed in living cells. The crosstalk between these cytoskeletal components underpins cellular organization and dynamics; however, the molecular basis of filamentous interactions is not fully understood. Here, we describe the mode of interaction between AFs and desmin IFs (DIFs) in a reconstituted in vitro system. METHODS: AFs (rabbit skeletal muscle) and DIFs (chicken gizzard) were labeled with fluorescent dyes. DIFs were immobilized on a heavy meromyosin (HMM)-coated collodion surface. HMM-driven AFs with ATP hydrolysis was assessed in the presence of DIFs. Images of single filaments were obtained using fluorescence microscopy. Vector changes in the trajectories of single AFs were calculated from microscopy images. RESULTS: AF speed transiently decreased upon contact with DIF. The difference between the incoming and outgoing angles of a moving AF broadened upon contact with a DIF. A smaller incoming angle tended to result in a smaller outgoing angle in a nematic manner. The percentage of moving AFs decreased with an increasing DIF density, but the speed of the moving AFs was similar to that in the no-desmin control. An abundance of DIFs tended to exclude AFs from the HMM-coated surfaces. CONCLUSIONS: DIFs agitate the movement of AFs with the orientation. DIFs can bind to HMMs and weaken actin-myosin interactions. GENERAL SIGNIFICANCE: The study indicates that apart from the binding strength, the accumulation of weak interactions characteristic of filamentous structures may affect the dynamic organization of cell architecture.

Characterization of metal and trace element contents of particulate matter (PM10) emitted by vehicles running on Brazilian fuels-hydrated ethanol and gasoline with 22% of anhydrous ethanol.

Emission of fine particles by mobile sources has been a matter of great concern due to its potential risk both to human health and the environment. Although there is no evidence that one sole component may be responsible for the adverse health outcomes, it is postulated that the metal particle content is one of the most important factors, mainly in relation to oxidative stress. Data concerning the amount and type of metal particles emitted by automotive vehicles using Brazilian fuels are limited. The aim of this study was to identify inhalable particles (PM(10)) and their trace metal content in two light-duty vehicles where one was fueled with ethanol while the other was fueled with gasoline mixed with 22% of anhydrous ethanol (gasohol); these engines were tested on a chassis dynamometer. The elementary composition of the samples was evaluated by the particle-induced x-ray emission technique. The experiment showed that total emission factors ranged from 2.5 to 11.8 mg/km in the gasohol vehicle, and from 1.2 to 3 mg/km in the ethanol vehicle. The majority of particles emitted were in the fine fraction (PM(2.5)), in which Al, Si, Ca, and Fe corresponded to 80% of the total weight. PM(10) emissions from the ethanol vehicle were about threefold lower than those of gasohol. The elevated amount of fine particulate matter is an aggravating factor, considering that these particles, and consequently associated metals, readily penetrate deeply into the respiratory tract, producing damage to lungs and other tissues.

The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes.

The topographical relationship between stress fiber-like structures (SFLS) and nascent myofibrils was examined in cultured chick cardiac myocytes by immunofluorescence microscopy. Antibodies against muscle-specific light meromyosin (anti-LMM) and desmin were used to distinguish cardiac myocytes from fibroblastic cells. By various combinations of staining with rhodamine-labeled phalloidin, anti-LMM, and antibodies against chick brain myosin and smooth muscle alpha-actinin, we observed the following relationships between transitory SFLS and nascent and mature myofibrils: (a) more SFLS were present in immature than mature myocytes; (b) in immature myocytes a single fluorescent fiber would stain as a SFLS distally and as a striated myofibril proximally, towards the center of the cell; (c) in regions of a myocyte not yet penetrated by the elongating myofibrils, SFLS were abundant; and (d) in regions of a myocyte with numerous mature myofibrils, SFLS had totally disappeared. Spontaneously contracting striated myofibrils with definitive Z-band regions were present long before anti-desmin localized in the I-Z-band region and long before morphologically recognizable structures periodically link Z-bands to the sarcolemma. These results suggest a transient one-on-one relationship between individual SFLS and newly emerging individual nascent myofibrils. Based on these and other relevant data, a complex, multistage molecular model is presented for myofibrillar assembly and maturation. Lastly, it is of considerable theoretical interest to note that mature cardiac myocytes, like mature skeletal myotubes, lack readily detectable stress fibers.

Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method.

Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.

Differential distribution of subsets of myofibrillar proteins in cardiac nonstriated and striated myofibrils.

Cultured cardiac myocytes were stained with antibodies to sarcomeric alpha-actinin, troponin-I, alpha-actin, myosin heavy chain (MHC), titin, myomesin, C-protein, and vinculin. Attention was focused on the distribution of these proteins with respect to nonstriated myofibrils (NSMFs) and striated myofibrils (SMFs). In NSMFs, alpha-actinin is found as longitudinally aligned, irregular approximately 0.3-microns aggregates. Such aggregates are associated with alpha-actin, troponin-I, and titin. These I-Z-I-like complexes are also found as ectopic patches outside the domain of myofibrils in close apposition to the ventral surface of the cell. MHC is found outside of SMFs in the form of discrete fibrils. The temporal-spatial distribution and accumulation of the MHC-fibrils with respect to the I-Z-I-like complexes varies greatly along the length of the NSMFs. There are numerous instances of I-Z-I-like complexes without associated MHC-fibrils, and also cases of MHC-fibrils located many microns from I-Z-I-like complexes. The transition between the terminal approximately 1.7-microns sarcomere of any given SMF and its distal NSMF-tip is abrupt and is marked by a characteristic narrow alpha-actinin Z-band and vinculin positive adhesion plaque. A titin antibody T20, which localizes to an epitope at the Z-band in SMFs, precisely costains the 0.3-microns alpha-actinin aggregates in ectopic patches and NSMFs. Another titin antibody T1, which in SMFs localizes to an epitope at the A-I junction, typically does not stain ectopic patches and NSMFs. Where detectable, the T1-positive material is adjacent to rather than part of the 0.3-microns alpha-actinin aggregates. Myomesin and C-protein are found only in their characteristic sarcomeric locations (even in just perceptible SMFs). These A-band-associated proteins appear to be absent in ectopic patches and NSMFs.

Purification and characterization of actin from normal and chronic lymphocytic leukemia lymphocytes.

Previous studies from this laboratory have shown actin to be a major protein of human lymphocytes (Stark, R., Liebes, L. F., Nevrla, D., and Silber, R. Biochem. Med., 27: 200-206, 1982). We now report the purification to homogeneity and characterization of actin from blood lymphocytes of normal subjects and patients with chronic lymphocytic leukemia. The recovery of the purified protein was about 20%. The properties of the lymphocyte actins were compared to each other and to those of rabbit skeletal muscle actin. Lymphocyte actin consisted of beta and gamma forms in a 2:1 ratio. The Mr 42,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Normal and leukemic lymphocyte actin had similar polymerization properties as assessed by viscosity measurements at 25 degrees and 4 degrees, and the ultrastructural appearance of the filaments was the same. Similar patterns were observed between normal and chronic lymphocytic leukemia actin tryptic digests analyzed by high-performance liquid chromatography. The Vmax of the actin-activated myosin Mg2+ ATPase activity was compared using rabbit skeletal muscle heavy meromyosin and subfragment 1 preparations. The values obtained with rabbit skeletal muscle and normal lymphocyte actin were identical. The Vmax observed with chronic lymphocytic leukemia lymphocyte actin was 70% of that obtained with normal lymphocyte actin. The amount of actin needed to produce half-maximal activation (Kapparent) of heavy meromyosin and subfragment 1 were, respectively, 26 and 25 microM for normal lymphocytes and 18 and 24 microM for chronic lymphocytic leukemia lymphocytes. The anomalous ATP activation by actin did not reflect differences in B-:T-cell subpopulations between chronic lymphocytic leukemia and normal lymphocytes. The possible significance of the observed differences between the myosin Mg2+ ATPase activation by chronic lymphocytic leukemia and normal lymphocyte actin is discussed.

Periodic features in the amino acid sequence of nematode myosin rod.

Properties of the amino acid sequence of the nematode myosin rod region, deduced from cloned DNA, are analysed. The rod sequence of 1117 residues contains a regular region of 1094 residues, which has features typical of an alpha-helical coiled coil, followed by a short non-helical tailpiece at the carboxyl end. The hydrophobic amino acids show the expected seven-residue pattern a, b, c, d, e, f, g, which is modulated by a longer repeat of 28-residue zones. In addition, there are four one-residue insertions, or skip residues, at the ends of zones, at positions 351, 548, 745 and 970. Myosin is considerably less hydrophobic than tropomyosin or alpha-keratin and the outer surface of the coiled coil is covered by clusters of positive and negatively charged amino acid side-chains. Molecular models suggest that the coiled coil is continuous throughout the rod, with an approximately uniform left-handed twist, except for a few turns of helix near each skip region, where the twist flattens out to accommodate the extra residue. Fourier transforms of the amino acid profiles show strong periodicities based on repeats of seven residues (7/2 and 7/3) and 28 residues (especially 28/3 and 28/9). The positive and negative charges each have strong 28/3-residue periodicities that are out of phase with one another. The negative charges also show a 196/9-residue modulation frequency, which may reflect the presence of a 196-residue structural unit in muscle, approximately 2 X 143 A long. The distribution of charged amino acids suggests that electrostatic forces are dominant in forming the thick filament structure. Models that allow regular patterns of interacting charges are restricted and the simplest types are discussed.

ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure.

To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.

Proteolysis and Flavor Characteristics of Serrano Ham Processed under Different Ripening Temperature Conditions.

UNLABELLED: Physicochemical, proteolysis and sensory characteristics of Serrano hams processed under low, medium and high ripening temperature conditions (RTC), with respective average temperatures of 9.3, 14.3, and 19.1 °C, were determined throughout a 15-mo period. In addition, quantitative relationships among variables were calculated. Medium and high RTC hams showed lower moisture contents and lower levels of low- and high-ionic-strength soluble proteins than low RTC hams. At 15 mo, aldolase was the most abundant low-ionic-strength soluble protein and actin the most abundant high-ionic-strength soluble protein in all hams while creatine kinase was no longer detected and H-meromyosin was detected only in low and medium RTC hams. Levels of all the molecular-weight peptide fractions increased during ripening, with higher factors of increase for the fractions of lower molecular weight. Total free amino acids were at significantly higher concentrations in medium and high RTC hams than in low RTC hams from month 7 onwards. The correlations of flavor preference and flavor intensity with ripening time, thermal integral, total free amino acids and most individual free amino acids were highly significant, while raw-meat taste was negatively correlated with all those variables. From month 5 to month 9 of ripening, development of a high quality flavor evolved more rapidly in medium RTC hams, flavor intensity increased at a faster rate in high RTC hams and raw-meat taste declined more rapidly in medium and high RTC hams. Medium and high RTC may be applied to accelerate the ripening process of Serrano ham without impairing flavor preference. PRACTICAL APPLICATION: Medium and high ripening temperature conditions (RTC) may be applied to Serrano ham in order to enhance the phenomena associated with ripening, without loss of product quality. Moisture loss, degradation of proteins and formation of free amino acids were accelerated in medium and high RTC hams. From month 5 to month 9 of ripening, development of a high quality flavor evolved more rapidly in medium RTC hams, flavor intensity increased at a faster rate in high RTC hams, and raw-meat taste declined more rapidly in medium and high RTC hams.

Curtailing Oxidation-Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment.

UNLABELLED: In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron-redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl2 at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross-linking was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation-initiated cross-linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G') of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation-induced G' by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats. PRACTICAL APPLICATION: The association of myosin S1 subfragment possibly S2 as well with pyrophosphate prevents extensive myosin head­head cross-linking. This alleviates the negative impact of oxidation on the gel formation of myosin, an expected main form of myofibrillar protein due to pyrophosphate-induced actomyosin dissociation. Likewise, oxidative S1­S1 association inhibits the binding of pyrophosphate thereby reducing its gel-weakening effect. The mutual constraining roles of oxidation and pyrophosphate can be a significant factor for the minimization of gelling property losses during the manufacture of comminuted meat products.

Both isoforms of skeletal muscle subfragment 1 (S1A1 and S1A2) can induce actin polymerization with equal speed in the absence of ATP.

The ability of myosin subfragment 1 to induce actin polymerization was reinvestigated using the DNase I inhibition assay, by electron microscopy after negative staining, cosedimentation, measurement of viscosity and the fluorescence increase of pyrenyl-labeled actin. Using these techniques we demonstrate that rabbit skeletal muscle myosin subfragment 1 containing either the alkali light chain 1 (S1A1) or the alkali light chain 2 (S1A2) is able to promote actin polymerization even in the absence of divalent cations or salt. In the presence of ATP the rate of induction of actin polymerization by S1A2 is slower than by A1A1. In contrast, in the absence of free ATP, both subfragment 1 variants exhibit equal ability to induce actin polymerization. Evidence is given that the slower rate of induction of actin polymerization by S1A2 in the presence of free ATP is due to a slower rate of ATP-hydrolysis by S1A2 and thus to a slower rate of ATP depletion. We therefore assume that the formation of rigor type complexes involving the subfragment 1 heavy chain is necessary for the induction of actin polymerization. The ability of subfragment 1 to induce actin polymerization is retarded by a synthetic heavy chain mimetic peptide which inhibits its actin binding or after proteolytic cleavage of the subfragment 1 heavy chain by trypsin.

Human embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene.

A 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.

Heart contractile proteins.

That several proteins of the sarcomere differ from one muscle to the next is well documented, and it is becoming evident that homogeneous muscles, like the heart, are also species specific. 1) Clear-cut evidence is available concerning myosin, and, to date, several types of molecules have been described. a) The myosins of white skeletal, heart, and smooth muscle differ in the activity of their Ca2+ and K+ATPases, as also in the structure of their light subunits. b) The Ca2+ATPases of the various cardiac myosins have been shown to exhibit species differences and correlate with the speed of shortening of the muscle. 2) The structures of tropomyosin, some troponin components, and alpha actinin (but not actin) appear to be unlike in the different types of muscle. 3) These phylogenic modifications may be related to the changes characteristic of the particular muscles under pathological conditions, which are accompanied by substantial increase in protein synthesis.

Histochemical, biochemical, and ultrastructural analyses of single human muscle fibers, with special reference to the C-fiber population.

A muscle biopsy from the vastus lateralis muscle of a strength-trained woman was found to contain an unusual fiber type composition and was analyzed by histochemical, biochemical, and ultrastructural techniques. Special attention was given to the C-fibers, which comprised over 15% of the total fiber number in the biopsy. The mATPase activity of the C-fibers remained stable to varying degrees over the pH range normally used for routine mATPase histochemistry. Although a continuum existed, the C-fibers were histochemically subdivided into three main fiber types: IC, IIC, and IIAC. The IC fibers were histochemically more similar to the Type I, the IIAC were more similar to the Type IIA, and the IIC were darkly stained throughout the pH range. Biochemical analysis revealed that all C-fibers coexpressed myosin heavy chains (MHC) I and IIa in variable ratios. The histochemical staining intensity correlated with the myosin heavy chain composition such that the Type IC fibers contained a greater ratio of MHCI/MHCIIa, the IIAC contained a greater ratio of MHCIIa/MHCI, and the Type IIC contained equal amounts of these two heavy chains. Ultrastructural data of the C-fiber population revealed an oxidative capacity between fiber Types I and IIA and suggested a range of mitochondrial volume percent from highest to lowest such that I greater than IC greater than IIC greater than IIA-C greater than IIA. Under physiological conditions, it appears that the IC fibers represent Type I fibers that additionally express some fast characteristics, whereas the Type IIAC are Type IIA fibers that additionally express some slow characteristics. Fibers expressing a 50:50 mixture of MHCI and MHCIIa (IIC fibers) were rarely found. It is not known whether C-fibers represent a distinct population between the fast- and slow-twitch fibers that is specifically adapted to a particular usage or whether they are transforming fibers in the process of going from fast to slow or slow to fast.

Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin.

The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein (5-IAF). The conjugate is purified by DEAE-cellulose ion exchange chromatography and cycles of polymerization-depolymerization, yielding a relatively homogeneous product with the fluorescein group covalently attached to cystein 373. The fluorescently labeled actin maintains normal polymerizability and activates heavy meromyosin Mg2+ adenosine triphosphatase to the same extent as unlabeled actin. Furthermore, fluoresecent paracrystals are readily detectable in fluroescence microscope upon adding excess Mg2+ or Ni2+ ions. Spectrofluorimetric studies of the bound fluorescein indicate that the peak excitation and emission wavelengths, the shapes of the spectra, and the peak fluorescence intensities are somewhat sensitive to polymerization and heavy meromyosin binding. Possible causes of these spectral changes are analyzed and future applications of this fluorescently labeled actin in vitro as well as in vivo are discussed.

Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide.

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.

Interaction of Lys-61 labeled actin with myosin subfragment-1 and the regulatory proteins.

Actin modified at Lys-61 with fluorescein 5-isothiocyanate (FITC) recovers the ability to polymerize following the binding of phalloidin. The resulting polymer (FITC-P-actin) activates the S1-Mg2+-ATPase activity to the same extent as non-labeled F-actin. However, in the absence of phalloidin, FITC-actin (0.5 mg/ml) neither polymerized nor activated the S1-Mg2+-ATPase activity effectively even when it was preincubated with S1 for 3 h in 0.1 mM ATP, 0.1 mM CaCl2, and 1 mM Tris/HCl (pH 8.0), in contrast to the previous report [Miller, L., Phillips, M., & Reisler, E. (1988) Eur. J. Biochem. 174, 23-29]. The modification of Lys-61 did not impair the ability to bind tropomyosin or tropomyosin-troponin. On the other hand, the fluorescence polarization of FITC-P-actin increased when tropomyosin or troponin-tropomyosin was added. Moreover, the modification of Lys-61 affected the regulation of the actin activation of the S1-Mg2+-ATPase activity by the tropomyosin and troponin complex. In 30 mM KCl, 2.5 mM ATP, and 5 mM MgCl2, tropomyosin alone has been shown to inhibit the actin-activated S1-Mg2+-ATPase. This inhibition did not occur with FITC-P-actin even though tropomyosin was tightly bound. When troponin-tropomyosin was added, the FITC-P-actin activation of S1-Mg2+-ATPase activity was regulated in response to micromolar Ca2+ concentrations. On the other hand, in 30 mM KCl, 2.5 mM ATP, and 2 mM MgCl2, tropomyosin alone did not inhibit the actin-activated S1-Mg2+-ATPase activity with either non-labeled F-actin or FITC-actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemically detected structural difference in the heavy chains of chicken breast muscle myosin isozymes.

Antisera against the isozymes of heavy meromyosin from chicken breast muscle myosin were prepared by immunizing rabbits. Both antisera against heavy meromyosin containing g1 light chain (HMM(g1) and that containing g3 light chain (HMM(g3)) reacted with both heavy meromyosin isozymes and myosin, but not with whole light chain mixture. This indicates that only antibodies against the heavy chain of heavy meromysin were elicited. The antisera were applied successively to two columns, one coupled with subfragment-2 and the other with heterologous subfragment-1 isozymes. When the antiserum against HMM(g3) was absorbed with subfragment-1 containing g1 (S-1(g1)), it did not react with HMM(g1), but reacted with HMM(g3). On the other hand, when the antiserum against HMM(g1) was absorbed with subfragment-1 containing g3 (S-1(g3)), it lost its ability to react with both heavy meromyosin isozymes. This indicates the presence of a specific antibody against the heavy chain of HMM(g3). The head portion of myosin containing g3 may hold an unique antigenic determinant which is not present in the head of a myosin containing g1.