Packing of α-Helical Coiled-Coil Myosin Rods in Vertebrate Muscle Thick Filaments

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.147.5.1039 ◽

1999 ◽

Vol 147 (5) ◽

pp. 1039-1048 ◽

Cited By ~ 30

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.

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UNC-98 links an integrin-associated complex to thick filaments in Caenorhabditis elegans muscle

The Journal of Cell Biology ◽

10.1083/jcb.200608043 ◽

2006 ◽

Vol 175 (6) ◽

pp. 853-859 ◽

Cited By ~ 23

Author(s):

Rachel K. Miller ◽

Hiroshi Qadota ◽

Megan L. Landsverk ◽

Kristina B. Mercer ◽

Henry F. Epstein ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Cell Membrane ◽

Transmembrane Protein ◽

Focal Adhesions ◽

Terminal Portion ◽

Dense Bodies ◽

Thick Filaments ◽

Vertebrate Muscle ◽

Associated Proteins ◽

Lines Of Evidence

Focal adhesions are multiprotein assemblages that link cells to the extracellular matrix. The transmembrane protein, integrin, is a key component of these structures. In vertebrate muscle, focal adhesion–like structures called costameres attach myofibrils at the periphery of muscle cells to the cell membrane. In Caenorhabditis elegans muscle, all the myofibrils are attached to the cell membrane at both dense bodies (Z-disks) and M-lines. Clustered at the base of dense bodies and M-lines, and associated with the cytoplasmic tail of β-integrin, is a complex of many proteins, including UNC-97 (vertebrate PINCH). Previously, we showed that UNC-97 interacts with UNC-98, a 37-kD protein, containing four C2H2 Zn fingers, that localizes to M-lines. We report that UNC-98 also interacts with the C-terminal portion of a myosin heavy chain. Multiple lines of evidence support a model in which UNC-98 links integrin-associated proteins to myosin in thick filaments at M-lines.

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Myosin and paramyosin of Caenorhabditis elegans embryos assemble into nascent structures distinct from thick filaments and multi-filament assemblages

The Journal of Cell Biology ◽

10.1083/jcb.122.4.845 ◽

1993 ◽

Vol 122 (4) ◽

pp. 845-858 ◽

Cited By ~ 42

Author(s):

HF Epstein ◽

DL Casey ◽

I Ortiz

Keyword(s):

Caenorhabditis Elegans ◽

Functional Relationship ◽

Immunofluorescence Microscopy ◽

Myosin Heavy Chains ◽

Wild Type ◽

Linear Structures ◽

Heavy Chains ◽

Thick Filaments ◽

Vertebrate Muscle ◽

Charge Change

The organization of myosin heavy chains (mhc) A and B and paramyosin (pm) which are the major proteins of thick filaments in adult wild-type Caenorhabditis elegans were studied during embryonic development. As a probe of myosin-paramyosin interaction, the unc-15 mutation e73 which produces a glu342lys charge change in pm and leads to the formation of large paracrystalline multi-filament assemblages was compared to wild type. These three proteins colocalized in wild-type embryos from 300 to 550 min of development after first cleavage at 20 degrees C on the basis of immunofluorescence microscopy using specific monoclonal antibodies. Linear structures which were diversely oriented around the muscle cell peripheries appeared at 360 min and became progressively more aligned parallel to the embryonic long axis until distinct myofibrils were formed at 550 min. In the mutant, mhc A and pm were colocalized in the linear structures, but became progressively separated until they showed no spatial overlap at the myofibril stage. These results indicate that the linear structures represent nascent assemblies containing myosin and pm in which the proteins interact differently than in wild-type thick filaments of myofibrils. In e73, these nascent structures were distinct from the multi-filament assemblages. The overlapping of actin and mhc A in the nascent linear structures suggests their possible structural and functional relationship to the "stress fiber-like structures" of cultured vertebrate muscle cells.

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Structure of myosin filaments from relaxed Lethocerus flight muscle by cryo-EM at 6 Å resolution

Science Advances ◽

10.1126/sciadv.1600058 ◽

2016 ◽

Vol 2 (9) ◽

pp. e1600058 ◽

Cited By ~ 38

Author(s):

Zhongjun Hu ◽

Dianne W. Taylor ◽

Michael K. Reedy ◽

Robert J. Edwards ◽

Kenneth A. Taylor

Keyword(s):

Flight Muscle ◽

Three Dimensional ◽

Coiled Coil ◽

Thick Filament ◽

Coiled Coils ◽

Muscle Physiology ◽

Layer Arrangement ◽

Thick Filaments ◽

Unusual Position ◽

Filament Structure

We describe a cryo–electron microscopy three-dimensional image reconstruction of relaxed myosin II–containing thick filaments from the flight muscle of the giant water bug Lethocerus indicus. The relaxed thick filament structure is a key element of muscle physiology because it facilitates the reextension process following contraction. Conversely, the myosin heads must disrupt their relaxed arrangement to drive contraction. Previous models predicted that Lethocerus myosin was unique in having an intermolecular head-head interaction, as opposed to the intramolecular head-head interaction observed in all other species. In contrast to the predicted model, we find an intramolecular head-head interaction, which is similar to that of other thick filaments but oriented in a distinctly different way. The arrangement of myosin’s long α-helical coiled-coil rod domain has been hypothesized as either curved layers or helical subfilaments. Our reconstruction is the first report having sufficient resolution to track the rod α helices in their native environment at resolutions ~5.5 Å, and it shows that the layer arrangement is correct for Lethocerus. Threading separate paths through the forest of myosin coiled coils are four nonmyosin peptides. We suggest that the unusual position of the heads and the rod arrangement separated by nonmyosin peptides are adaptations for mechanical signal transduction whereby applied tension disrupts the myosin heads as a component of stretch activation.

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CryoEM structure of Drosophila flight muscle thick filaments at 7 Å resolution

Life Science Alliance ◽

10.26508/lsa.202000823 ◽

2020 ◽

Vol 3 (8) ◽

pp. e202000823

Author(s):

Nadia Daneshparvar ◽

Dianne W Taylor ◽

Thomas S O’Leary ◽

Hamidreza Rahmani ◽

Fatemeh Abbasiyeganeh ◽

...

Keyword(s):

Striated Muscle ◽

Flight Muscle ◽

Myosin Ii ◽

Coiled Coil ◽

Fruit Fly ◽

Actin Binding ◽

Thick Filaments ◽

Flight Muscles ◽

Lethocerus Indicus ◽

Globular Head

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins. Myosin II’s long α-helical coiled-coil tail forms the dense protein backbone of filaments, whereas its N-terminal globular head containing the catalytic and actin-binding activities extends outward from the backbone. Here, we report the structure of thick filaments of the flight muscle of the fruit fly Drosophila melanogaster at 7 Å resolution. Its myosin tails are arranged in curved molecular crystalline layers identical to flight muscles of the giant water bug Lethocerus indicus. Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; one new density, possibly stretchin-mlck, is found on the backbone outer surface. Surprisingly, the myosin heads are disordered rather than ordered along the filament backbone. Our results show striking myosin tail similarity within flight muscle filaments of two insect orders separated by several hundred million years of evolution.

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Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet

The Journal of Cell Biology ◽

10.1083/jcb.200207153 ◽

2002 ◽

Vol 159 (3) ◽

pp. 431-440 ◽

Cited By ~ 141

Author(s):

Jun Yang ◽

Xiaoqing Liu ◽

Guohua Yue ◽

Michael Adamian ◽

Oleg Bulgakov ◽

...

Keyword(s):

Basal Body ◽

Structural Component ◽

Coiled Coil ◽

Basal Bodies ◽

Tail Domain ◽

Kinesin Light Chain ◽

Thick Filaments ◽

Head Domain ◽

Retinal Photoreceptors

The ciliary rootlet, first recognized over a century ago, is a prominent structure originating from the basal body at the proximal end of a cilium. Despite being the largest cytoskeleton, its structural composition has remained unknown. Here, we report a novel 220-kD protein, designated rootletin, found in the rootlets of ciliated cells. Recombinant rootletin forms detergent-insoluble filaments radiating from the centrioles and resembling rootlets found in vivo. An mAb widely used as a marker for vertebrate rootlets recognizes an epitope in rootletin. Rootletin has a globular head domain and a tail domain consisting of extended coiled-coil structures. Rootletin forms parallel in register homodimers and elongated higher order polymers mediated by the tail domain alone. The head domain may be required for targeting to the basal body and binding to a kinesin light chain. In retinal photoreceptors where rootlets appear particularly robust, rootlets extend from the basal bodies to the synaptic terminals and anchor ER membranes along their length. Our data indicate that rootlets are composed of homopolymeric rootletin protofilaments bundled into variably shaped thick filaments. Thus, rootletin is the long-sought structural component of the ciliary rootlet.

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CryoEM Structure of Drosophila Flight Muscle Thick Filaments at 7Å Resolution

10.1101/2020.06.05.136580 ◽

2020 ◽

Author(s):

Nadia Daneshparvar ◽

Dianne W. Taylor ◽

Thomas S. O’Leary ◽

Hamidreza Rahmani ◽

Fatemeh Abbasi Yeganeh ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Striated Muscle ◽

Flight Muscle ◽

Coiled Coil ◽

Thick Filament ◽

Fruit Fly ◽

Actin Binding ◽

Thick Filaments ◽

Flight Muscles ◽

Lethocerus Indicus

AbstractStriated muscle thick filaments are composed of myosin II and several non-myosin proteins. Myosin II’s long α-helical coiled-coil tail forms the dense protein backbone of filaments while its N-terminal globular head containing the catalytic and actin binding activities extends outward from the backbone. Here we report the structure of thick filaments of the flight muscle of the fruit fly Drosophila melanogaster at 7 Å resolution. Its myosin tails are arranged in curved molecular crystalline layers identical to flight muscles of the giant waterbug Lethocerus indicus. Four non-myosin densities are observed, three of which correspond to ones found in Lethocerus; one new density, possibly stretchin-Mlck, is found on the backbone outer surface. Surprisingly, the myosin heads are disordered rather than ordered along the filament backbone. Our results show striking myosin tail similarity within flight muscle filaments of two insect orders separated by several hundred million years of evolution.Significance StatementMyosin thick filaments are one of striated muscle’s key structures, but also one of its least understood. A key question is how the myosin a-helical coiled-coil tail is arranged in the backbone. At 7Å resolution, sufficient to resolve individual a-helices, the myosin tail arrangement in thick filaments from the flight muscle of the fruit fly Drosophila melanogaster is strikingly similar to the myosin tail arrangement in flight muscles of the giant waterbug Lethocerus indicus. Nearly every other thick filament feature is different. Drosophila and Lethocerus evolved separately >245 million years ago suggesting myosin tail packing into curved molecular crystalline layers forms a highly conserved thick filament building block and different properties are obtained by alterations in non-myosin proteins.

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