scholarly journals Muscle actin filaments bind pituitary secretory granules in vitro.

1977 ◽  
Vol 73 (1) ◽  
pp. 78-87 ◽  
Author(s):  
R E Ostlund ◽  
J T Leung ◽  
D M Kipnis
Keyword(s):  
Skeletal Muscle ◽  
Metal Ions ◽  
Secretory Granule ◽  
Actin Filaments ◽  
Anterior Pituitary ◽  
Secretory Granules ◽  
Muscle Actin ◽  
Viscous Solutions ◽  
Styrene Butadiene

Hog anterior pituitary secretory granules sediment at 3,000 g. When rat or rabbit skeletal muscle actin filaments are present with the granules, the sedimentation decreases markedly. Depolymerized actin or viscous solutions of Ficoll and collagen have no effect on granule sedimentation. With this assay, actin filaments bind secretory granules (consisting of the proteinaceous core plus limiting membrane), secretory granule membranes, mitochondria, artificial lecithin liposomes, and styrene-butadiene microspheres, but have little or no interaction with membrane-free secretory granule cores and albumin microspheres. A secretory granule-actin complex sedimentable between 3,000 g and 25,000 g can be isolated. Metal ions, nucleotides, salts, dithiothreitol, or pretreatment of the granules with trypsin do not destroy the binding, which appears to be a lipophilic interaction.

scholarly journals ATP-dependent movement of myosin in vitro: characterization of a quantitative assay.

1984 ◽  
Vol 99 (5) ◽  
pp. 1867-1871 ◽  
Author(s):  
M P Sheetz ◽  
R Chasan ◽  
J A Spudich
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Quantitative Assay ◽  
Vitro Assay ◽  
Skeletal Muscle Myosin ◽  
In Vitro Characterization ◽  
Actin Cables ◽  
Muscle Myosin

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement of myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 micron/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (approximately 20 micrograms myosin/2.5 X 10(9) beads of 1 micron in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations less than 70 mM, at ATP concentrations greater than 0.1 mM, and at Mg2+ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 90 kJ/mol below 22 degrees C and 40 kJ/mol above 22 degrees C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.

scholarly journals Cyclophosphamide treatment evoked side effect on skeletal muscle actin, monitored by DSC

2021 ◽  
Author(s):  
Péter Farkas ◽  
Dávid Szatmári ◽  
Franciska Könczöl ◽  
Dénes Lőrinczy
Keyword(s):  
Skeletal Muscle ◽  
Side Effect ◽  
Main Peak ◽  
Muscle Actin ◽  
Scanning Calorimetry ◽  
Drug Induced ◽  
Cyclophosphamide Treatment ◽  
Intact Muscle ◽  
Binding Cleft

AbstractSeveral kind of drugs—used in cancer treatments—such as cyclophosphamide (CP) can also trigger a disease classified as toxic polyneuropathy. Polyneuropathy is a simultaneous malfunction of several peripheral nerves, typical side effect of a cancer therapy. In our previous study, we used CP treated in vitro animal model (Guinea pig) with a comparable dosage and time handling of human protocol to show evidences of this drug-induced effects. We could show a dose-dependent difference between in Tm and ΔHcal of untreated and treated samples assigned to their intact muscle and nerve, blood plasma and red blood cells. In our current study we analyze this side effect on skeletal muscle actin (prepared from m. psoas of rabbit) by DSC (differential scanning calorimetry), to follow the possible consequence of drug treatment on the “activator” of muscle contraction. We have demonstrated that run of DSC curves, Tms together with the ΔHcal exhibit clear CP effect. In case of Ca2+ G actin it is manifested in a well separated second high denaturing temperature as a consequence of CP binding into the cleft. This way the nucleotide binding cleft with subdomains 1 and 3 becomes less flexible, indicating clear sensitivity to CP treatment. In F-actin samples, the main peak represents the thermal denaturation of subdomains 1 and 3, and the increased calorimetric enthalpy administrating Ca2+ as well as CP refers to a more rigid structure. These alterations can be the molecular background in the malfunction of muscle in case of polyneuropathy after CP treatment.

Polymorphism of Actin Paracrystals

1981 ◽  
Vol 39 ◽  
pp. 420-421
Author(s):  
W.E. Fowler ◽  
U. Aebi
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Muscle Cells ◽  
New Method ◽  
Structural Studies ◽  
Muscle Actin ◽  
Structural Detail ◽  
Naturally Occurring ◽  
Synthetic Filament

In the muscle sarcomere and in certain specialized non-muscle cells actin filaments are organized in bundles or paracrystalline arrays. Structural studies of these naturally occurring filament arrays have been limited to about 3nm resolution, mainly due to inherent disorder of the specimen and/or difficulties with the preparation of these arrays for EM. Skeletal muscle G-actin can be induced to form synthetic filament paracrystals upon addition of non-physiological concentrations of Mg++ (e.g. 50mM) . The structural resolution obtained with these synthetic paracrystals has been of the same order (about 3nm) as that encountered with the naturally occurring filament arrays. Using a new method of induction, we have obtained synthetic paracrystals with two non-muscle actins which reveal structural detail to almost 2nm resolution (Figs. 1,2,3). While the same types of paracrystals were observed with Physarum and Acanthamoeba actin, skeletal muscle actin displayed a different polymorphism under identical conditions.

scholarly journals Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals Talin at myotendinous junctions.

1986 ◽  
Vol 103 (4) ◽  
pp. 1465-1472 ◽  
Author(s):  
J G Tidball ◽  
T O'Halloran ◽  
K Burridge
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Skeletal Muscles ◽  
Immunofluorescence Microscopy ◽  
Protein Separations ◽  
Force Transmission ◽  
Electron Microscopic ◽  
Focal Contacts ◽  
Myotendinous Junctions

Junctions formed by skeletal muscles where they adhere to tendons, called myotendinous junctions, are sites of tight adhesion and where forces generated by the cell are placed on the substratum. In this regard, myotendinous junctions and focal contacts of fibroblasts in vitro are analogues. Talin is a protein located at focal contacts that may be involved in force transmission from actin filaments to the plasma membrane. This study investigates whether talin is also found at myotendinous junctions. Protein separations on SDS polyacrylamide gels and immunolabeling procedures show that talin is present in skeletal muscle. Immunofluorescence microscopy using anti-talin indicates that talin is found concentrated at myotendinous junctions and in lesser amounts in periodic bands over nonjunctional regions. Electron microscopic immunolabeling shows talin is a component of the digitlike processes of muscle cells that extend into tendons at myotendinous junctions. These findings indicate that there may be similarities in the molecular composition of focal contacts and myotendinous junctions in addition to functional analogies.

scholarly journals Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

2021 ◽  
Vol 22 (8) ◽  
pp. 4036
Author(s):  
Małgorzata Śliwinska ◽  
Katarzyna Robaszkiewicz ◽  
Piotr Wasąg ◽  
Joanna Moraczewska
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thin Filament ◽  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Troponin Complex ◽  
Increased Sensitivity

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca2+-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca2+, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca2+]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca2+]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca2+-dependent regulation of actin–myosin interactions.

Putative myosin heavy and light chains in Tetrahymena: co-localization to the basal body-cage complex and association of the heavy chain with skeletal muscle actin filaments in vitro

1995 ◽  
Vol 108 (3) ◽  
pp. 869-881
Author(s):  
J.A. Garces ◽  
J.G. Hoey ◽  
R.H. Gavin
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Basal Body ◽  
Actin Filaments ◽  
Protein Fraction ◽  
Immunofluorescence Microscopy ◽  
Myosin Ii ◽  
Light Chains ◽  
Muscle Actin ◽  
Cage Complex

The basal body cage is a fibrillar chamber which surrounds each basal body in the ciliate cytoskeleton. The function of this chamber is unknown. In Tetrahymena, the cage contains actin filaments which connect the cage to triplet microtubules. In this study, we have examined the cage for the presence of myosin. Skeletal muscle myosin-II heavy and light chains were used to affinity-purify anti-MHC and anti-MLC antibodies, respectively, from an antiserum raised against Tetrahymena oral apparatus proteins. On western immunoblots of ATP-solubilized Tetrahymena proteins, the anti-MHC antibody detected a putative myosin heavy (180 kDa) chain, and the anti-MLC antibody detected a putative myosin light (18 kDa) chain. The anti-MHC antibody specifically labeled the AI zone of sarcomeres. In cosedimentation assays with an ATP-solubilized protein fraction, the 180 kDa polypeptide associated with skeletal muscle actin filaments in an ATP-dependent manner. The sedimented actin filaments appeared to be organized into bundles. Immunodepletion of the 180 kDa rendered the ATP-solubilized protein fraction ineffective in bundling actin filaments in a cosedimentation assay. ATP-solubilized Tetrahymena proteins, which included the 180 kDa polypeptide, exhibited F-actin-stimulated, Mg2+ ATPase activity and K+, EDTA ATPase activity which are characteristic of myosin ATPases. Immunodepletion of the 180 kDa polypeptide reduced the F-actin, Mg2+ ATPase activity of the ATP-solubilized protein fraction by more than 80%. Based on these various observations, we conclude that the 180 kDa polypeptide is a putative myosin heavy chain, probably a myosin-II and that the 18 kDa polypeptide is probably a myosin-II light chain. We have used the affinity-purified, anti-myosin antibodies with immunofluorescence microscopy and immunogold electron microscopy to map the location of the putative myosin heavy and light chains in Tetrahymena. Immunofluorescence microscopy showed that the anti-myosin antibodies localized to Tetrahymena somatic and oral region basal bodies. At the ultrastructural level, the anti-myosin antibodies localized to filaments in the basal body-cage complex. The labeling patterns with both anti-myosin antibodies were identical to the labeling pattern observed with an anti-actin antibody reported in a previous study. The co-localization of myosin and actin argue for a motility system within the basal body-cage complex.

THE EFFECT OF THYROTROPHIC RELEASING FACTOR ON DIFFERENT HYPOTHALAMIC AREAS AND THE ANTERIOR PITUITARY GLAND: A BIOCHEMICAL AND ULTRASTRUCTURAL STUDY

1973 ◽  
Vol 56 (1) ◽  
pp. 99-109 ◽  
Author(s):  
A. MOGUILEVSKY ◽  
SILVIA CUERDO-ROCHA ◽  
JANINE CHRISTOT ◽  
D. ZAMBRANO
Keyword(s):  
Pituitary Gland ◽  
Anterior Pituitary ◽  
Regulatory Mechanism ◽  
Secretory Granules ◽  
Anterior Pituitary Gland ◽  
Morphological Identification ◽  
Ultrastructural Studies ◽  
Medial Hypothalamus ◽  
The Central Nervous System

SUMMARY A study on the effect of synthetic thyrotrophic releasing factor (TRF) on different areas of the central nervous system and on the anterior pituitary gland was performed in a system in vitro. These observations were combined with ultrastructural studies of the anterior pituitary gland after intracarotid TRF administration. TRF specifically decreased the incorporation in vitro of labelled amino acids into proteins of the medial hypothalamus, whereas the anterior pituitary gland showed enhanced protein synthesis. At the electron microscope level, only thyrotrophs showed enhanced signs of activation after 5, 15 and 30 min of TRF administration, which consisted in hypertrophy of the rough endoplasmic reticulum and Golgi complexes associated with a marked increase in the number of secretory granules undergoing extrusion into the perivascular and extracellular spaces. These observations indicate that the medial hypothalamus may be related to regulatory mechanism(s) of TRF secretion. They provide further morphological identification of the thyrotrophs and corroborate that TRF acts on these cells by promoting release and synthesis of thyrotrophin.

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