Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.     

Direct visualization of contractile proteins in pertubular cells of the guinea-pig testis using antibodies against highly purified actin and myosin.

Antibodies against actin and myosin from smooth muscle, which may react with contractile elements from both muscular and muscle-like cells, were applied to fresh frozen sections of adult guinea-pig testis. Sections stained with an antibody against pectoralis (striated) muscle myosin or with non-immune globulin were used for controls. Peritubular cells of the lamina propria surrounding seminiferous tubulus contained large amounts of actin and myosin as judged by the intensity of immunofluorescence. Sertoli cells did not stain with the antibodies. Our results support the concept of peritubular cells being the critical force for the contractility of seminiferous tubules.     

Direct visualization of contractile proteins in peritubular cells of the guinea-pig testis using antibodies against highly purified actin and myosin

Summary Antibodies against actin and myosin from smooth muscle, which may react with contractile elements from both muscular and muscle-like cells, were applied to fresh frozen sections of adult guinea-pig testis. Sections stained with an antibody against pectoralis (striated) muscle myosin or with non-immune globulin were used for controls. Peritubular cells of the lamina propria surrounding seminiferous tubulus contained large amounts of actin and myosin as judged by the intensity of immunofluorescence. Sertoli cells did not stain with the antibodies. Our results support the concept of peritubular cells being the critical force for the contractility of seminiferous tubules.     

Myosin switching during amoebo-plasmodial differentiation of slime mold, Physarum polycephalum

We reported previously that myosins from amoebal and plasmodial stages in the life cycle of Physarum polycephalum differ in the primary structure of heavy chains and phosphorylatable 18,000 Mr light chains, while Ca-binding 14,000 Mr light chains are common to both myosins (Kohama & Takano-Ohmuro, Proc Jpn acad 60B (1984) 431; Kohama et al., J biol chem 260 (1986) 8022). We have carried out immunofluorescence microscopical studies upon differentiating cultures of amoebic colonies, which show apogamic amoebo-plasmodial differentiation as follows: Typical amoebae differentiate into mono-nucleate intermediate cells with swollen nuclei and then into two or multi-nucleate young plasmodia (Anderson et al., Protoplasma 89 (1976) 29. Antibodies against plasmodial myosin heavy chain (PMHC) and 18,000 Mr plasmodial myosin light chain (PMLC18) stained intermediate cells and young plasmodia, but not typical amoebae. On the other hand, antibody against amoebal myosin heavy chain (AMHC) stained typical amoebae and intermediate cells--but not young plasmodia. Thus staining was detected using antibodies against both PMHC and AMHC in intermediate cells. Intermediate cells were also stained by antibody against another plasmodium-specific cytoskeletal protein, viz., high molecular weight actin-binding protein (HMWP). We propose that synthesis of myosin subunits switches immediately from amoebal to plasmodial type in mono-nucleate cells with swollen nuclei. This myosin switching is associated with the initiation of HMWP synthesis.     

Myosin IIb activity and phosphorylation status determines dendritic spine and post-synaptic density morphology

Dendritic spines in hippocampal neurons mature from a filopodia-like precursor into a mushroom-shape with an enlarged post-synaptic density (PSD) and serve as the primary post-synaptic location of the excitatory neurotransmission that underlies learning and memory. Using myosin II regulatory mutants, inhibitors, and knockdowns, we show that non-muscle myosin IIB (MIIB) activity determines where spines form and whether they persist as filopodia-like spine precursors or mature into a mushroom-shape. MIIB also determines PSD size, morphology, and placement in the spine. Local inactivation of MIIB leads to the formation of filopodia-like spine protrusions from the dendritic shaft. However, di-phosphorylation of the regulatory light chain on residues Thr18 and Ser19 by Rho kinase is required for spine maturation. Inhibition of MIIB activity or a mono-phosphomimetic mutant of RLC similarly prevented maturation even in the presence of NMDA receptor activation. Expression of an actin cross-linking, non-contractile mutant, MIIB R709C, showed that maturation into a mushroom-shape requires contractile activity. Loss of MIIB also leads to an elongated PSD morphology that is no longer restricted to the spine tip; whereas increased MIIB activity, specifically through RLC-T18, S19 di-phosphorylation, increases PSD area. These observations support a model whereby myosin II inactivation forms filopodia-like protrusions that only mature once NMDA receptor activation increases RLC di-phosphorylation to stimulate MIIB contractility, resulting in mushroom-shaped spines with an enlarged PSD.     

The expression of CD15 in dissociated cultured rat dorsal root ganglion cells

Summary This study describes the presence of CD15 in dorsal root ganglia neurons in five experimental conditions: chemically defined medium and the same medium with added nerve growth factor, retinoic acid or antibodies against insulin or tyrosine phosphate. Positive astrocyte controls were used to differentiate the monoclonal antibodies that did not react with CD15. Those monoclonal antibodies which detected CD15 in this positive control were also used to study CD15 positivity in dorsal root ganglion cells. This study shows: (i) masking of the CD15 antibody, which influences the detection capacity of the monoclonal antibodies used; (ii) that CD15 discerns two subpopulations of DRG neurons: a CD15-positive and a CD15-negative population; (iii) that CD15 expression is not involved in the outgrowth of protrusions or the wrapping by non-neuronal cells of DRG neurons.     

Identification of the surfactant protein A receptor 210 as the unconventional myosin 18A

Mass spectrometric characterization of the surfactant protein A (SP-A) receptor 210 (SP-R210) led to the identification of myosin (Myo) XVIIIA and nonmuscle myosin IIA. Antibodies generated against the unique C-terminal tail of MyoXVIIIA revealed that MyoXVIIIA, MyoIIA, and SP-R210 have overlapping tissue distribution, all being highly expressed in myeloid cells, bone marrow, spleen, lymph nodes, and lung. Western blot analysis of COS-1 cells stably transfected with either MyoXVIIIA or MyoIIA indicated that SP-R210 antibodies recognize MyoXVIIIA. Furthermore, MyoXVIIIA but not MyoIIA localized to the surface of COS-1 cells, and most importantly, expression of MyoXVIIIA in COS-1 cells conferred SP-A binding. Western analysis of recombinant MyoXVIIIA domains expressed in bacteria mapped the epitopes of previously derived SP-R210 antibodies to the neck region of MyoXVIIIA. Antibodies raised against the neck domain of MyoXVIIIA blocked the binding of SP-A to macrophages. Together, these findings indicate that MyoXVIIIA constitutes a novel receptor for SP-A.     

Two distinct mechanisms for regulation of nonmuscle myosin assembly via the heavy chain: phosphorylation for MIIB and mts 1 binding for MIIA

In search of the regulation mechanisms for isoform specific myosin assembly, we have used the COOH-terminal fragments of nonmuscle myosin isoforms MIIA and MIIB (MIIA(F46) and MIIB(alpha)(F47)) as a model system. Phosphorylation by protein kinase C (PK C) or casein kinase II (CK II) within or near the nonhelical tail-end domain inhibits assembly of MIIB(alpha)(F47) [Murakami, N., et al. (1998) Biochemistry 37, 1989]. In the study presented here, we mutated the kinase sites to analyze the inhibition mechanisms of MIIB assembly by phosphorylation. Replacement of the CK II or PK C sites with Asp (MIIB(alpha)(F47)-CK-5D or -PK-4D) strongly inhibited the filament assembly, with or without Mg(2+), by significantly increasing the critical concentrations for assembly. Without Mg(2+), MIIB(alpha)(F47)-CK-5D or -PK-4D inhibited the assembly of wild-type (wt) MIIB(alpha)(F47) by either mixing as homofragments or forming heterofragments. With 2.5 mM Mg(2+), MIIB(alpha)(F47)-wt promoted assembly of MIIB(alpha)(F47)-CK-5D and -PK-4D in homofragment mixtures, but not by forming heterofragments. MIIA(F46) coassembled with MIIB(alpha)(F47)-wt and -CK-5D and altered their assembly patterns. In contrast, assembly of MIIB(alpha)(F47)-PK-4D was unchanged by MIIA(F46). A metastasis-associated protein, mts 1, bound in a Ca(2+)-dependent manner to MIIA(F46), but not appreciably to MIIB(alpha)(F47). At 0.15 M NaCl, mts 1-Ca(2+) not only inhibited MIIA(F46) assembly but also disassembled the MIIA(F46) filaments. Mts 1, however, did not affect the assembly of MIIB(alpha)(F47) in MIIA(F46) and MIIB(alpha)(F47) mixtures, indicating that mts 1 is an inhibitor specific to MIIA assembly. Our results suggest strongly that assembly of MIIA and MIIB is regulated by distinct mechanisms via tail-end domains: phosphorylation of MIIB and mts 1 binding to MIIA. These mechanisms may also function to form MIIA or MIIB homofilaments by selectively inhibiting MIIB or MIIA assembly.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles.

Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles

Summary Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.     

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.     

Analysis of glia cell differentiation in the developing chick peripheral nervous system: sensory and sympathetic satellite cells express different cell surface antigens.

To identify and analyse precursor cells of neuronal and glial cell lineages during the early development of the chick peripheral nervous system, monoclonal antibodies were raised against a population of undifferentiated cells of E6 dorsal root ganglia (DRG). Non-neuronal cells of E6 DRG express surface antigens that are recognized by four monoclonal antibodies, G1, G2, GLI 1 and GLI 2. The proportion of non-neuronal cells in DRG that express the GLI 1 antigen is very high during ganglion formation (80% at E4) and decreases during later development (15% at E14). GLI 2 antigen is expressed only on a minority of the cells at E6 and increases with development. The G1 and G2 antigens are expressed on about 60-80% of the cells between E6 and E14. All cells that express the established glia marker O4 are also positive for the new antigens. In addition, it was demonstrated that GLI 1-positive cells from early DRG, which are devoid of O4 antigen, could be induced in vitro to express the O4 antigen. Thus, the antigen-positive cells are considered as glial cells or glial precursor cells. Surprisingly, the antigen expression by satellite cells of peripheral ganglia is dependent on the type of ganglion: antigens G1, G2 and GLI 1 were not detectable on glial cells of lumbosacral sympathetic ganglia and GLI 2 was expressed only by a small subpopulation. These results demonstrate an early immunological difference between satellite cells of sensory DRG and sympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunofluorescence comparisons of anti-actin specificity

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.     

Simultaneous localization of myosin and tubulin in human tissue culture cells by double antibody staining

We have studied the distribution of myosin and tubulin molecules inside the same tissue culture cells by using two antibodies labeled with contrasting fluorochromes. Antimyosin raised against human platelet myosin was labeled with rhodamine. Antitubulin raised against sea urchin vinblastine-induced tubulin crystals was labeled with fluorescein. The two antibodies stained entirely different structures inside the same flat interphase cell: antimyosin bound to stress fibers and antitubulin bound to thin, wavy fibers thought to be individual microtubules. Compact interphase cells stained diffusely with both antibodies. From prophase through early anaphase both antibodies stained the mitotic spindle, although the fluorescence contrast between the spindle and the cytoplasm was much higher with antitubulin than with antimyosin. From anaphase through telophase, strong antimyosin staining occurred in the cleavage furrow, while antitubulin stained the region between the separated chromosomes. This study established the feasibility of high-resolution fluorescent antibody localization of pairs of motility proteins in the cytoplasm of single cells, an approach which will make it possible to map out the sites of the various contractile protein interactions in situ.     

Subcellular compartmentalization of myosin isoforms in embryonic chick heart ventricle myocytes during cytokinesis

Embryonic chick heart ventricle myocytes retain the ability to alternate between proliferation and functional differentiation. A cytoplasmic isoform of myosin is present in cleavage furrows of various nonmuscle cells during cytokinesis, whereas one or more of the cardiac myosin isoforms are localized in sarcomeres of beating cardiomyocytes. Antibodies were employed to reveal the subcellular localizations of cytoplasmic and cardiac myosin isoforms in embryonic chick ventricle cardiomyocytes during cytokinesis. Monoclonal anticytoplasmic myosin antibodies were prepared against myosin purified from brains of 1-day-posthatched chickens and shown to react with chick brain myosin heavy chain by Western blots and/or ELISA tests. One monoclonal antibrain myosin antibody also cross-reacted with chick cardiac myosin but not with skeletal or smooth muscle myosins. Two antichick cardiac myosin monoclonal antibodies and one antichick skeletal myosin polyclonal antibody that cross-reacts with cardiac myosin were employed to identify cardiac sarcomeric myosin. Cells were isolated from day 8 embryonic chick heart ventricles, enriched for myocytes, grown in vitro for 3 days, and then examined by immunofluorescence techniques. Monoclonal antibodies against cytoplasmic myosin preferentially localized in the cleavage furrows of both cardiofibroblasts and cardiomyocytes in all stages of cytokinesis. In contrast, antibodies that recognize cardiac myosin were distributed throughout cardiomyocytes during early stages of cytokinesis, but became progressively excluded from the furrow area during middle and late stages of cytokinesis. These data suggest that in cells that contain both cytoplasmic and sarcomeric myosin isoforms, only cytoplasmic myosin isoforms are mobilized to from the contractile ring for cytokinesis.     

The size of the fibre populations in rabbit skeletal muscles as revealed by indirect immunofluorescence with anti-myosin sera

Summary Antibodies against myosin of the fast long. dorsi and the slow soleus muscle of rabbits were induced in guinea pigs. With the aid of a new technique, the gel-electrophoresis-derived-enzyme-linked-immunosorbent assay (GEDELISA) it could be shown that they are directed against the heavy and the light chains of fast (M. long. dorsi) and slow (M. soleus) myosin. In the indirect immunofluorescence test each antiserum only stained one population of fibres in five different muscles tested. The single fibres were observed to react only with one of the two types of antisera. The following percentage of fibres showed a positive reaction with the anti-fast myosin serum: M. long. dorsi, 95%; M. psoas maior, 95%, M. psoas minor, 92%; M. tibialis ant., 90%; M. soleus, 15%.Abbreviations AB antibodies - ETPase adenosintriphosphatase - Anti-LdM antiserum against LdM - Anti-SoM antiserum against SoM - BSA bovine serum albumin - CPf contaminating protein in LdM - CPs contaminating protein in SoM - EDTA ethylene diamine tetra-acetic acid - ELISA enzyme-linked immunosorbent assay - FITC fluoresceinisothiocyanate - FM last myosin showing 3LC in PAGE - GEDELISA gel electrophoresis-derived enzyme-linked immunosorbent assay - HC heavy chains of myosin - LC light chains of myosin - LdM myosin preparation of longissimus dorsi muscle - MCF microcomplement fixation - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline (140 mM NaCl, 20 mM potassium-phosphate, pH 7.4) - SDS sodiumdodecylsulfate - SM slow myosin, showing two LC in PAGE - SoM myosin preparation of soleus muscle     

Biochemical, Immunochemical and Immunohistochemical Identification of Myosin Heavy Chains in Cultured Cells of Catharanthus roseus

In the pollen tubes of the lily Lilium longiflorum, myosin,composed of 170-kDa heavy chains is responsible for the intracellulartransport of organelles [Yokota and Shimmen (1994) Protoplasma177: 153]. Polypeptides of 170 kDa with similar antigenicityto this pollen-tube myosin have also been found in other angiospermcells [Yokota et al. (1995) Protoplasma 185: 178]. To clarifythe role of this type of myosin in cytoplasmic streaming, weprepared partially purified myosin fraction from cultured cellsof Catharanthus roseus by co-precipitation with F-actin. Ina motility assay in vitro with this fraction, rhodamine-phalloidin-labeledF-actin moved with an average velocity of 10.7 µm s^-1.This sliding velocity was similar to that of the cytoplasmicstreaming observed in intact cultured cells. Antibodies raisedagainst the 170-kDa heavy chain of pollen-tube myosin recognizedonly a single polypeptide of 170 kDa in this partially purifiedfraction. The same polypeptide was also identified by theseantibodies in a crude extract of proteins from cultured cells.The myosin-specific fluorescence was concentrated around thenuclei and was associated with particles of various sizes. Duallocalization using antibodies against myosin and against actinrevealed that these particles were preferentially co-localizedwith actin filaments. On the other hand, no component of thecrude extract or of the partially purified myosin fraction cross-reactedwith antibodies against heavy chains of myosin II from animalcells. These results suggest that the 170-kDa polypeptide is the myosinheavy chain and that this myosin generates the motive forcefor cytoplasmic streaming in cultured cells of Catharanthusroseus. (Received March 28, 1995; Accepted September 14, 1995)     

Mechanochemical proteins, cell motility and cell-cell contacts: the localization of mechanochemical proteins inside cultured cells at the edge of an in vitro "wound".

We have examined the distribution of several mechanochemical proteins inside rat A10 cells in monolayer culture, both in sparse cultures and at the edges of in vitro "wounds" in confluent cultures. The proteins examined were actin, myosin, tropomyosin, alpha-actinin, filamin, and tubulin. In each experiment, a pair of these proteins (one of which was usually actin) were examined simultaneously by double fluorescence staining methods. Actin was specificially stained by double fluorescence staining methods. Actin was specifically stained by a method based on heavy meromyosin binding, while the other proteins were specifically stained by indirect immunofluorescence procedures. The most important of the various results described was obtained with cells moving out from the edge of an in vitro wound. Within the flat leading lamella of such a cell, there was an extended region in which myosin was severely depleted or absent compared to the proximal regions of the same cells. By contrast, the other proteins were abundantly present throughout the leading lamella, except for tropomyosin, which was somewhat depleted but not as extensively as myosin. In Nomarski optics, there was no detectable morphological differentiation between the region depleted of myosin and the more proximal portion of the same lamella. While the depletion of myosin from the motile regions of cells does not rule out the involvement of some form of an actomyosin sliding filament mechanism, it suggests that other molecular mechanisms for generating motility be seriously considered.     

Myosin types in cultured muscle cells

Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins.