scholarly journals CONSERVED INTERACTIONS IN CARDIAC SYNTHETIC THICK FILAMENTS DIFFERENTLY AFFECT MYOSIN SUPER-RELAXED STATE IN HEALTHY, DISEASE AND MAVACAMTEN-TREATED MODELS

2020 ◽  
Author(s):  
Sampath K. Gollapudi ◽  
Suman Nag
Keyword(s):  
Clinical Stage ◽  
Thick Filament ◽  
Functional Studies ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Molecule Inhibitor ◽  
Vitro Model ◽  
Low Ionic Strength ◽  
Electrostatic Stability

ABSTRACTA hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their use for functional studies has been very limited. In this report, we show that the ultra-low ATP-consuming super-relaxed (SRX) state of myosin is electrostatically more stable in STFs as compared with shorter myosin sub-fragments that lack the distal tail required for thick filament assembly. However, this electrostatic stability of the SRX state is weakened by phosphorylation of myosin light chains or the hypertrophic cardiomyopathy-causing myosin R403Q mutation. We also show that ADP binding to myosin depopulates the SRX population in STFs made of wild-type (WT) myosin, but not in S1, HMM, or STFs made of mutant R403Q myosin. Collectively, these findings emphasize that a critical network of inter- and intra-molecular interactions that underlie the SRX state of myosin are mostly preserved in STFs, establishing it as a native-like tool to interrogate myosin regulation. Next, using STFs, we show that a clinical-stage small molecule inhibitor, mavacamten, is more effective in promoting the myosin SRX state in STFs than in S1 or HMM and that it is equally potent in STFs made of atrial-WT, ventricular-WT, and mutant-R403Q myosin. Also, we found that mavacamten-bound heads are not permanently protected in the SRX state but can be recruited in response to physiological perturbations, thus providing new insights into its inhibitory mechanism.

scholarly journals Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, disease, and mavacamten-treated cardiac systems

2020 ◽  
pp. jbc.RA120.016506 ◽  
Author(s):  
Sampath K Gollapudi ◽  
Ming Yu ◽  
Qing-Fen Gan ◽  
Suman Nag
Keyword(s):  
Clinical Stage ◽  
Thick Filament ◽  
Functional Evaluation ◽  
Thick Filaments ◽  
Myosin Subfragment ◽  
Molecule Inhibitor ◽  
Myosin Subfragment 1 ◽  
Vitro Model ◽  
Low Ionic Strength

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in-vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their functional evaluation has been limited.  In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin sub-fragments, heavy meromyosin (HMM) and myosin subfragment-1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and increased ADP:ATP ratio destabilize the SRX population. Furthermore, wild-type myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small molecule inhibitor, mavacamten, showed that it is not only more effective in increasing myosin SRX population in STFs than in S1 or HMM ,  but it also increases myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations   such as elevated ADP concentration weakens the mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited   into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, disease, and therapeutic conditions.

scholarly journals A Structural Model for Phosphorylation Control of Dictyostelium Myosin II Thick Filament Assembly

1999 ◽  
Vol 147 (5) ◽  
pp. 1039-1048 ◽  
Author(s):  
Wenchuan Liang ◽  
Hans M. Warrick ◽  
James A. Spudich
Keyword(s):  
Structural Model ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Thick Filament ◽  
Tail Region ◽  
Filament Assembly ◽  
Dictyostelium Myosin ◽  
Thick Filaments ◽  
Coil Structure

Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3×Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3×Asp myosin II fails to rescue myosin II–null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II–null cells containing 3×Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled–coil structure.

Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle

1994 ◽  
Vol 107 (5) ◽  
pp. 1115-1129 ◽  
Author(s):  
C. Ferguson ◽  
A. Lakey ◽  
A. Hutchings ◽  
G.W. Butcher ◽  
K.R. Leonard ◽  
...  
Keyword(s):  
Insect Flight ◽  
Regular Structure ◽  
Thick Filament ◽  
Cytoskeletal Proteins ◽  
Stretch Activation ◽  
Insect Muscle ◽  
Thick Filaments ◽  
Flight Muscles ◽  
Leg Muscle ◽  
Low Ionic Strength

Asynchronous insect flight muscles produce oscillatory contractions and can contract at high frequency because they are activated by stretch as well as by Ca2+. Stretch activation depends on the high stiffness of the fibres and the regular structure of the filament lattice. Cytoskeletal proteins may be important in stabilising the lattice. Two proteins, zeelin 1 (35 kDa) and zeelin 2 (23 kDa), have been isolated from the cytoskeletal fraction of Lethocerus flight muscle. Both zeelins have multiple isoforms of the same molecular mass and different charge. Zeelin 1 forms micelles and zeelin 2 forms filaments when renatured in low ionic strength solutions. Filaments of zeelin 2 are ribbons 10 nm wide and 3 nm thick. The position of zeelins in fibres from Lethocerus flight and leg muscle was determined by immunofluorescence and immunoelectron microscopy. Zeelin 1 is found in flight and leg fibres and zeelin 2 only in flight fibres. In flight myofibrils, both zeelins are in discrete regions of the A-band in each half sarcomere. Zeelin 1 is across the whole A-band in leg myofibrils. Zeelins are not in the Z-disc, as was thought previously, but migrate to the Z-disc in glycerinated fibres. Zeelins are associated with thick filaments and analysis of oblique sections showed that zeelin 1 is closer to the filament shaft than zeelin 2. The antibody labelling pattern is consistent with zeelin molecules associated with myosin near the end of the rod region. Alternatively, the position of zeelins may be determined by other A-band proteins. There are about 2.0 to 2.5 moles of myosin per mole of each zeelin. The function of these cytoskeletal proteins may be to maintain the ordered structure of the thick filament.

scholarly journals Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans.

1996 ◽  
Vol 135 (2) ◽  
pp. 371-382 ◽  
Author(s):  
P E Hoppe ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Striated Muscle ◽  
Coiled Coil ◽  
Thick Filament ◽  
Surface Hydrophobicity ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Characteristic Variation ◽  
Muscle Myosin

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

The Perfused Human Liver Wedge Biopsy: A New In Vitro Model for Morphological and Functional Studies

Hepatology ◽  
2007 ◽  
Vol 2 (4) ◽  
pp. 426S-432S ◽  
Author(s):  
Susan J. Burwen ◽  
Albert L. Jones ◽  
Ira S. Goldman ◽  
Lawrence W. Way ◽  
Sussan Dejbakhsh
Keyword(s):  
Human Liver ◽  
In Vitro Model ◽  
Functional Studies ◽  
Vitro Model ◽  
Wedge Biopsy

scholarly journals VASP is a processive actin polymerase that requires monomeric actin for barbed end association

2010 ◽  
Vol 191 (3) ◽  
pp. 571-584 ◽  
Author(s):  
Scott D. Hansen ◽  
R. Dyche Mullins
Keyword(s):  
Ionic Strength ◽  
Total Internal Reflection ◽  
Molecular Mechanisms ◽  
Binding Modes ◽  
Cellular Functions ◽  
Actin Networks ◽  
Filament Assembly ◽  
Binding Event ◽  
Low Ionic Strength

Ena/VASP proteins regulate the actin cytoskeleton during cell migration and morphogenesis and promote assembly of both filopodial and lamellipodial actin networks. To understand the molecular mechanisms underlying their cellular functions we used total internal reflection fluorescence microscopy to visualize VASP tetramers interacting with static and growing actin filaments in vitro. We observed multiple filament binding modes: (1) static side binding, (2) side binding with one-dimensional diffusion, and (3) processive barbed end tracking. Actin monomers antagonize side binding but promote high affinity (Kd = 9 nM) barbed end attachment. In low ionic strength buffers, VASP tetramers are weakly processive (Koff = 0.69 s−1) polymerases that deliver multiple actin monomers per barbed end–binding event and effectively antagonize filament capping. In higher ionic strength buffers, VASP requires profilin for effective polymerase and anti-capping activity. Based on our observations, we propose a mechanism that accounts for all three binding modes and provides a model for how VASP promotes actin filament assembly.

The self-assembly of synthetic filaments of myosin isolated from Chaos carolinensis and Amoeba proteus

1977 ◽  
Vol 25 (1) ◽  
pp. 387-402
Author(s):  
J.S. Condeelis
Keyword(s):  
Ionic Strength ◽  
Self Assembly ◽  
Divalent Cations ◽  
Central Zone ◽  
Thick Filament ◽  
Amoeba Proteus ◽  
High Ionic Strength ◽  
Thick Filaments ◽  
Myosin Filaments ◽  
Low Ionic Strength

Synthetic myosin thick filaments were formed from preparations of electrophoretically homogeneous myosin isolated from Chaos carolinensis and Amoeba proteus when dialysed to physiological ionic strength and pH. Myosin dialysed directly against low ionic strength buffers formed native-like thick filaments in the presence and absence of exogenous divalent cations. The average dimensions of the synthetic filaments grown under these conditions were 455 nm long and 16 nm wide with a distinct bare central zone 174 nm long. Myosin predialysed against EGTA-EDTA solutions at high ionic strength and then dialysed to low ionic strength formed native-like filaments only in the presence of 1mM Mg2+. 1 mM Ca2+ could not be substituted for Mg2+ under these conditions to achieve native-like filaments. Filaments grown from predialysed myosin in the absence of Mg2+ resembled EGTA-dissociated myosin filaments observed in EGTA-treated cytoplasm and were highly branched, poorly formed filaments lacking a distinct bare central zone. The average dimensions of the filaments grown from predialysed myosin in the absence of Mg2+ were 328 nm long, 13 nm wide with a bare central zone 111 nm long. Under the conditions tested, myosin isolated from these amoebae did not demonstrate a divalent cation requirement for thick filament formation. The results obtained with myosin isolated from the 2 organisms were identical.

scholarly journals The carboxyl-terminal isoforms of smooth muscle myosin heavy chain determine thick filament assembly properties

2002 ◽  
Vol 156 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Arthur S. Rovner ◽  
Patricia M. Fagnant ◽  
Susan Lowey ◽  
Kathleen M. Trybus
Keyword(s):  
Smooth Muscle ◽  
Thick Filament ◽  
Smooth Muscle Myosin ◽  
Myosin Isoforms ◽  
Heavy Chains ◽  
Filament Assembly ◽  
Filament Structure ◽  
Alternatively Spliced ◽  
Muscle Myosin

The alternatively spliced SM1 and SM2 smooth muscle myosin heavy chains differ at their respective carboxyl termini by 43 versus 9 unique amino acids. To determine whether these tailpieces affect filament assembly, SM1 and SM2 myosins, the rod region of these myosin isoforms, and a rod with no tailpiece (tailless), were expressed in Sf 9 cells. Paracrystals formed from SM1 and SM2 rod fragments showed different modes of molecular packing, indicating that the tailpieces can influence filament structure. The SM2 rod was less able to assemble into stable filaments than either SM1 or the tailless rods. Expressed full-length SM1 and SM2 myosins showed solubility differences comparable to the rods, establishing the validity of the latter as a model for filament assembly. Formation of homodimers of SM1 and SM2 rods was favored over the heterodimer in cells coinfected with both viruses, compared with mixtures of the two heavy chains renatured in vitro. These results demonstrate for the first time that the smooth muscle myosin tailpieces differentially affect filament assembly, and suggest that homogeneous thick filaments containing SM1 or SM2 myosin could serve distinct functions within smooth muscle cells.

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