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.

Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle.

The muscle myosins and hexomeric proteins consisting of two heavy chains and two pairs of light chains, the latter called essential (ELC) and regulatory (RLC). The light chains stabilize the long alpha helical neck of the myosin head. Their function in striated muscle, however, is only partially understood. We report here the identification of distinct missense mutations in a skeletal/ventricular ELC and RLC, each of which are associated with a rare variant of cardiac hypertrophy as well as abnormal skeletal muscle. We show that myosin containing the mutant ELC has abnormal function, map the mutant residues on the three-dimensional structure of myosin and suggest that the mutations disrupt the stretch activation response of the cardiac papillary muscles.

Calmodulin dissociation regulates brush border myosin I (110-kD-calmodulin) mechanochemical activity in vitro.

110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin-dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium-inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium-sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments.

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

Localization and topography of antigenic domains within the heavy chain of smooth muscle myosin.

We have produced and characterized monoclonal antibodies that label antigenic determinants distributed among three distinct, nonoverlapping peptide domains of the 200-kD heavy chain of avian smooth muscle myosin. Mice were immunized with a partially phosphorylated chymotryptic digest of adult turkey gizzard myosin. Hybridoma antibody specificities were determined by solid-phase indirect radioimmunoassay and immunoreplica techniques. Electron microscopy of rotary-shadowed samples was used to directly visualize the topography of individual [antibody.antigen] complexes. Antibody TGM-1 bound to a 50-kD peptide of subfragment-1 (S-1) previously found to be associated with actin binding and was localized by immunoelectron microscopy to the distal aspect of the myosin head. However, there was no antibody-dependent inhibition of the actin-activated heavy meromyosin ATPase, nor was antibody TGM-1 binding to actin-S-1 complexes inhibited. Antibody TGM-2 detected an epitope of the subfragment-2 (S-2) domain of heavy meromyosin but not the S-2 domain of intact myosin or rod, consistent with recognition of a site exposed by chymotryptic cleavage of the S-2:light meromyosin junction. Localization of TGM-2 to the carboxy-terminus of S-2 was substantiated by immunoelectron microscopy. Antibody TGM-3 recognized an epitope found in the light meromyosin portion of myosin. All three antibodies were specific for avian smooth muscle myosin. Of particular interest is that antibody TGM-1, unlike TGM-3, bound poorly to homogenates of 19-d embryonic smooth muscles. This indicates the expression of different myosin heavy chain epitopes during smooth muscle development.

Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities.

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A was 2.6-fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions.

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption.

Polarity and velocity of sliding filaments: control of direction by actin and of speed by myosin.

Myosin filaments, which are responsible for a large repertoire of motile activities in muscle and nonmuscle cells, can translocate actin filaments both toward and away from their central bare zone. This bidirectional movement suggests that there is enough flexibility in the head portion of the tightly packed myosin molecules in the native myosin filaments to move actin filaments not only in the expected direction, but also in the direction opposite to that predicted by the regular structure of muscle--away from the center of the myosin filament.

Skeletal muscle expression and abnormal function of beta-myosin in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is an important inherited disease. The phenotype has been linked, in some kindreds, to the beta-myosin heavy chain (beta-MHC) gene. Missense and silent mutations in the beta-MHC gene were used as markers to demonstrate the expression of mutant and normal cardiac beta-MHC gene message in skeletal muscle of hypertrophic cardiomyopathy patients. Mutant beta-myosin, also shown to be present in skeletal muscle by Western blot analysis, translocated actin filaments slower than normal controls in an in vitro motility assay. Thus, single amino acid changes in beta-myosin result in abnormal actomyosin interactions, confirming the primary role of missense mutations in beta-MHC gene in the etiology of hypertrophic cardiomyopathy.

Interferometric Scattering Microscopy for the Study of Molecular Motors.

Our understanding of molecular motor function has been greatly improved by the development of imaging modalities, which enable real-time observation of their motion at the single-molecule level. Here, we describe the use of a new method, interferometric scattering microscopy, for the investigation of motor protein dynamics by attaching and tracking the motion of metallic nanoparticle labels as small as 20nm diameter. Using myosin-5, kinesin-1, and dynein as examples, we describe the basic assays, labeling strategies, and principles of data analysis. Our approach is relevant not only for motor protein dynamics but also provides a general tool for single-particle tracking with high spatiotemporal precision, which overcomes the limitations of single-molecule fluorescence methods.

Myosins: a diverse superfamily.

Myosins constitute a large superfamily of actin-dependent molecular motors. Phylogenetic analysis currently places myosins into 15 classes. The conventional myosins which form filaments in muscle and non-muscle cells form class II. There has been extensive characterization of these myosins and much is known about their function. With the exception of class I and class V myosins, little is known about the structure, enzymatic properties, intracellular localization and physiology of most unconventional myosin classes. This review will focus on myosins from class IV, VI, VII, VIII, X, XI, XII, XIII, XIV and XV. In addition, the function of myosin II in non-muscle cells will also be discussed.

Catalytic fragment of protein kinase C exhibits altered substrate specificity toward smooth muscle myosin light chain.

Smooth muscle myosin light chain (LC) can be phosphorylated by myosin light chain kinase (MLCK) at Ser19 and Thr18 and by protein kinase C (PKC) at Thr9 and Ser1 or Ser2 under the in vitro assay conditions. Conversion of PKC to the spontaneously active protein kinase M (PKM) by proteolysis resulted in a change in the substrate specificity of the kinase. PKM phosphorylated both sets of sites in LC recognized by MLCK and PKC as analyzed by peptide mapping analysis. The PKM-catalyzed phosphorylation of these sites was not greatly affected by a MLCK inhibitor, ML-9, nor by the activators of MLCK, Ca2+ and calmodulin.

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton.

Myosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser.

A cytotoxicity assay utilizing a fluorescent dye that determines accurate surviving fractions of cells.

A cytotoxicity assay has been developed based on the measurement of the proliferative activity of surviving cells as quantified by a cell-incorporated fluorescent dye, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The BCECF proliferative assay is fast (the results are obtained within 3-4 days depending on the cell line), accurate, not labor-intensive, does not require the use of radioisotopes or toxic compounds, and is amenable to automation. The BCECF proliferative assay was compared with two other indirect cytotoxicity tests, a trypan blue exclusion test and a BCECF viability test. Neither of these two latter assays reflected in any way the killing of cells by ricin. In contrast, using the BCECF proliferation assay, an optimal period of cell culturing after exposure to a toxin could be found so that the cytotoxicity values produced agreed with the surviving fractions of cells measured in a direct cytotoxicity assay. Under non-optimal conditions, the assay reflected the cell kill only qualitatively. Although it is common practice to conduct indirect cytotoxicity tests without validating them with a direct assay, our experiments demonstrate that the values obtained in such non-optimized indirect cytotoxicity tests may not parallel the cell kill and may, therefore, be meaningless.

Effects of calcium on vascular smooth muscle contraction.

Calcium initiates smooth muscle contraction by binding to calmodulin and activating the enzyme myosin light chain kinase. The activated form of myosin light chain kinase phosphorylates myosin on the 20,000-dalton light chain and contractile activity ensues. Calcium may also enhance smooth muscle contractile activity by binding directly to myosin, the main component of the thick filament. Recent studies raise the possibility that the calcium-calmodulin complex may also modulate smooth muscle contractile activity by removing the inhibition imposed by caldesmon, a protein that is bound to the thin (i.e., actin-containing) filaments of smooth muscle. In vitro studies have demonstrated that the calcium-activated, phospholipid-dependent kinase, protein kinase C, can phosphorylate smooth muscle myosin at a different site than does myosin light chain kinase and down-regulate its actin-activated magnesium adenosine triphosphatase activity. This raises the possibility that protein kinase C phosphorylation of myosin may play a role in modulating vascular contractile activity in vivo.

Unconventional myosins and the genetics of hearing loss.

Mutations of the unconventional myosins genes encoding myosin VI, myosin VIIA and myosin XV cause hearing loss and thus these motor proteins perform fundamental functions in the auditory system. A null mutation in myosin VI in the congenitally deaf Snell's waltzer mice (Myo6(sv)) results in fusion of stereocilia and subsequent progressive loss of hair cells, beginning soon after birth, thus reinforcing the vital role of cytoskeletal proteins in inner ear hair cells. To date, there are no human families segregating hereditary hearing loss that show linkage to MYO6 on chromosome 6q13. The discovery that the mouse shaker1 (Myo7(ash1)) locus encodes myosin VIIA led immediately to the identification of mutations in this gene in Usher syndrome type 1B; subsequently, mutations in this gene were also found associated with recessive and dominant nonsyndromic hearing loss (DFNB2 and DFNA11). Stereocilla of sh1 mice are severely disorganized, and eventually degenerate as well. Myosin VIIA has been implicated in membrane trafficking and/or endocytosis in the inner ear. Mutant alleles of a third unconventional myosin, myosin XV, are associated with nonsyndromic, recessive, congenital deafness DFNB3 on human chromosome 17p11.2 and deafness in shaker2 (Myo15(sh2)) mice. In outer and inner hair cells, myosin XV protein is detectable in the cell body and stereocilia. Hair cells are present in homozygous sh2 mutant mice, but the stereocilia are approximately 1/10 of the normal length. This review focuses on what we know about the molecular genetics and biochemistry of myosins VI, VIIA and XV as relates to hereditary hearing loss. Am. J. Med. Genet. (Semin. Med. Genet.) 89:147-157, 1999. Published 2000 Wiley-Liss, Inc.

In vitro studies of determinants of smooth muscle mechanics.

Smooth muscle contraction is dependent upon phosphorylation of the 20,000 Da light chain subunits of myosin. Whereas the kinetics of the hydrolysis of MgATP by smooth muscle myosin suggest a simple phosphorylation-dependent "on-off" mechanism, the contractile response in smooth muscle tissue is complex. Experiments to unravel this complexity have been performed in vitro using a combination of motility assays and kinetic techniques. Some insight into this complexity is obtained, but the mechanism and the regulation of smooth muscle contraction is still not completely known.