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.

scholarly journals UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

2004 ◽  
Vol 167 (2) ◽  
pp. 315-325 ◽  
Author(s):  
Matthew Lord ◽  
Thomas D. Pollard
Keyword(s):  
Atpase Activity ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Gliding Motility ◽  
Light Chains ◽  
Temperature Sensitive ◽  
Contractile Ring ◽  
In Vitro Motility

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis.

Localization of actin in the Tetrahymena basal body-cage complex

1992 ◽  
Vol 103 (3) ◽  
pp. 629-641 ◽  
Author(s):  
J.G. Hoey ◽  
R.H. Gavin
Keyword(s):  
Basal Body ◽  
Specific Binding ◽  
Ultrastructural Level ◽  
Basal Bodies ◽  
Muscle Actin ◽  
Cage Complex ◽  
Contractile System ◽  
Chicken Muscle ◽  
Filament Bundles

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify anti-chicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahymena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.

scholarly journals Regulatory Light Chains Fine-Tune Skeletal Muscle Myosin-II Function

2021 ◽  
Vol 120 (3) ◽  
pp. 344a
Author(s):  
Arnab Nayak ◽  
Tianbang Wang ◽  
Peter Franz ◽  
Walter Steffen ◽  
Igor Chizhov ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myosin Ii ◽  
Light Chains ◽  
Skeletal Muscle Myosin ◽  
Regulatory Light Chains ◽  
Muscle Myosin ◽  
Fine Tune

scholarly journals Tropomyosin and Myosin-II Cellular Levels Promote Actomyosin Ring Assembly in Fission Yeast

2010 ◽  
Vol 21 (6) ◽  
pp. 989-1000 ◽  
Author(s):  
Benjamin C. Stark ◽  
Thomas E. Sladewski ◽  
Luther W. Pollard ◽  
Matthew Lord
Keyword(s):  
Motor Activity ◽  
Atpase Activity ◽  
Fission Yeast ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Bound State ◽  
Contractile Ring ◽  
Ring Assembly

Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.

The influence of caldesmon on ATPase activity of the skeletal muscle actomyosin and bundling of actin filaments

1985 ◽  
Vol 842 (1) ◽  
pp. 70-75 ◽  
Author(s):  
Renata Da̧browska ◽  
Andrzej Goch ◽  
Barbara Gała̧zkiewicz ◽  
Hanna Osińska
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Actin Filaments

scholarly journals Myosin isozymes in normal and cross-reinnervated cat skeletal muscle fibers.

1983 ◽  
Vol 97 (3) ◽  
pp. 756-771 ◽  
Author(s):  
G F Gauthier ◽  
R E Burke ◽  
S Lowey ◽  
A W Hobbs
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Muscle Fibers ◽  
Motor Units ◽  
Myosin Atpase ◽  
Light Chains ◽  
Slow Myosin ◽  
Fast Myosin ◽  
Physiological Properties ◽  
Contraction Times

Immunocytochemical characteristics of myosin have been demonstrated directly in normal and cross-reinnervated skeletal muscle fibers whose physiological properties have been defined. Fibers belonging to individual motor units were identified by the glycogen-depletion method, which permits correlation of cytochemical and physiological data on the same fibers. The normal flexor digitorum longus (FDL) of the cat is composed primarily of fast-twitch motor units having muscle fibers with high myosin ATPase activity. These fibers reacted with antibodies specific for the two light chains characteristic of fast myosin, but not with antibodies against slow myosin. Two categories of fast fibers, corresponding to two physiological motor unit types (FF and FR), differed in their immunochemical response, from which it can be concluded that their myosins are distinctive. The soleus (SOL) consists almost entirely of slow-twitch motor units having muscle fibers with low myosin ATPase activity. These fibers reacted with antibodies against slow myosin, but not with antibodies specific for fast myosin. When the FDL muscle was cross-reinnervated by the SOL nerve, twitch contraction times were slowed about twofold, and motor units resembled SOL units in a number of physiological properties. The corresponding muscle fibers had low ATPase activity, and they reacted with antibodies against slow myosin only. The myosin of individual cross-reinnervated FDL muscle units was therefore transformed, apparently completely, to a slow type. In contrast, cross-reinnervation of the SOL muscle by FDL motoneurons did not effect a complete converse transformation. Although cross-reinnervated SOL motor units had faster than normal twitch contraction times (about twofold), other physiological properties characteristic of type S motor units were unchanged. Despite the change in contraction times, cross-reinnervated SOL muscle fibers exhibited no change in ATPase activity. They also continued to react with antibodies against slow myosin, but in contrast to the normal SOL, they now showed a positive response to an antibody specific for one of the light chains of fast myosin. The myosins of both fast and slow muscles were thus converted by cross-reinnervation, but in the SOL, the newly synthesized myosin was not equivalent to that normally present in either the FDL or SOL. This suggests that, in the SOL, alteration of the nerve supply and the associated dynamic activity pattern are not sufficient to completely respecify the type of myosin expressed.

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.

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