Cooperativity between Two Heads of Dictyostelium Myosin II in in Vitro Motility and ATP Hydrolysis

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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Molecular motors: forty years of interdisciplinary research

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0447 ◽

2011 ◽

Vol 22 (21) ◽

pp. 3936-3939 ◽

Cited By ~ 10

Author(s):

James A. Spudich

Keyword(s):

Single Molecule ◽

Molecular Motors ◽

Atp Hydrolysis ◽

Molecular Motor ◽

Single Molecule Level ◽

In Vitro Motility ◽

Nonmuscle Cell ◽

Nonmuscle Cells ◽

City Plan

A mere forty years ago it was unclear what motor molecules exist in cells that could be responsible for the variety of nonmuscle cell movements, including the “saltatory cytoplasmic particle movements” apparent by light microscopy. One wondered whether nonmuscle cells might have a myosin-like molecule, well known to investigators of muscle. Now we know that there are more than a hundred different molecular motors in eukaryotic cells that drive numerous biological processes and organize the cell's dynamic city plan. Furthermore, in vitro motility assays, taken to the single-molecule level using techniques of physics, have allowed detailed characterization of the processes by which motor molecules transduce the chemical energy of ATP hydrolysis into mechanical movement. Molecular motor research is now at an exciting threshold of being able to enter into the realm of clinical applications.

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An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays

Journal of Cell Science ◽

10.1242/jcs.104.3.899 ◽

1993 ◽

Vol 104 (3) ◽

pp. 899-906

Author(s):

R. Chandra ◽

S.A. Endow ◽

E.D. Salmon

Keyword(s):

Heavy Chain ◽

Atp Hydrolysis ◽

Motor Protein ◽

Longitudinal Axis ◽

Opposite Polarity ◽

Truncated Protein ◽

In Vitro Motility ◽

Kinesin Heavy Chain

The nonclaret disjunctional (ncd) protein is a kinesin-related microtubule motor protein that is encoded at the claret locus in Drosophila and is required for proper chromosome distribution in meiosis and early mitosis. The protein contains a region with 41% amino acid sequence identity to kinesin heavy chain, but translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends. The overall structure of ncd also differs from kinesin heavy chain, in that the proposed motor domain is present at the C terminus of the molecule instead of the N terminus, as in kinesin heavy chain. In studies to define the molecular determinants of ncd function, we constructed and expressed a protein with a deletion of the N-terminal 208 amino acids of the non-motor region. Analysis of the truncated protein shows that the protein exhibits microtubule-stimulated Mg(2+)-ATPase activity and binds microtubules in pelleting assays. In contrast to near full-length ncd, the truncated protein does not support directional movement of microtubules in in vitro motility assays. Instead, microtubules show nucleotide-sensitive binding to the truncated protein on glass surfaces and bound microtubules exhibit one-dimensional diffusional movement that is constrained to their longitudinal axis. The diffusional movement reveals a weak binding state of the ncd motor that may represent a mechanochemical intermediate in its ATP hydrolysis cycle. If diffusional movement is a characteristic intrinsic to the claret motor, it is likely to be important in the in vivo function of the protein.

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Myosin-driven fragmentation of actin filaments triggers contraction of a disordered actin network

10.1101/332684 ◽

2018 ◽

Author(s):

Kyohei Matsuda ◽

Takuya Kobayashi ◽

Mitsuhiro Sugawa ◽

Yurika Koiso ◽

Yoko Y. Toyoshima ◽

...

Keyword(s):

Molecular Mechanism ◽

Actin Filaments ◽

Atp Hydrolysis ◽

Myosin Ii ◽

Network Connectivity ◽

Experimental System ◽

Actin Network ◽

Atp Concentration ◽

Time Required

AbstractThe dynamic cytoskeletal network is responsible for cell shape changes and cell division. The actin-based motor protein myosin II drives the remodeling of a highly disordered actin-based network and enables the network to perform mechanical work such as contraction, migration and adhesion. Myosin II forms bipolar filaments that self-associate via their tail domains. Such myosin minifilaments generate both extensile and compressive forces that pull and push actin filaments, depending on the relative position of myosin and actin filaments in the network. However, it remains unclear how the mechanical properties of myosin II that rely on the energy of ATP hydrolysis spontaneously contract the disordered actin network. Here, we used a minimal in vitro reconstituted experimental system consisting of actin, myosin, and a cross-linking protein, to gain insights into the molecular mechanism by which myosin minifilaments organize disordered actin networks into contractile states. We found that contracted cluster size and time required for the onset of network contraction decreased as ATP concentration decreased. Contraction velocity was negatively correlated with ATP concentrations. Reduction of ATP concentration caused fragmentation of actin filaments by myosin minifilament. We also found that gelsolin, a Ca2+-regulated actin filament-severing protein, induced contraction of a mechanically stable network, implying that fragmentations of actin filaments in the network weaken the intra-network connectivity and trigger contraction. Our findings reveal that the disordered actin network contraction can be controlled by fragmentation of actin filaments, highlighting the molecular mechanism underlying the myosin motor-severing activities, other than the sliding tensile and compressive stress in the disordered actin network.

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Studies on the Dynamic Localization of GFP-Myosin During Cytokinesis in Live Cells

Microscopy and Microanalysis ◽

10.1017/s1431927600007534 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 129-130

Author(s):

James Sabry ◽

Sheri Moores ◽

Shannon Ryan ◽

Ji-Hong Zang ◽

James A. Spudich

Keyword(s):

Fluorescent Protein ◽

Molecular Motor ◽

Myosin Ii ◽

Thick Filament ◽

Live Cells ◽

In Vitro Motility ◽

Multimeric Complex ◽

Ring Constriction ◽

Green Fluorescent

Cell division is thought to be powered by the constriction of an actomyosin containing contractile ring found transiently in the cleavage furrow. Conventional myosin II plays a fundamental role in this process of cytokinesis where, in the form of a multimeric complex known as the bipolar thick filament, it is thought to be the molecular motor that generates the force necessary to cause ring constriction.In order to study the dynamics of this protein in the dividing cell, we have made a fusion protein of the green fluorescent protein (GFP) and the amino terminus of the Dictyostelium myosin heavy chain (GFP-myosin), and imaged the location of this protein in dividing Dictyostelium cells were it is the only myosin II present in the cell. The addition of GFP does not compromise the functioning of the myosin motor as evidenced by the fact that purified GFP-myosin has solution ATPase and in vitro motility kinetics similar to that of non-labelled myosin. In addition, GFP-myosin fully complements the myosin null mutation for both development and cytokinesis in suspension suggesting that GFP-myosin acts as a regulated motor when expressed in cells.

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Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development.

The Journal of Cell Biology ◽

10.1083/jcb.132.1.101 ◽

1996 ◽

Vol 132 (1) ◽

pp. 101-109 ◽

Cited By ~ 48

Author(s):

M F Kolman ◽

L M Futey ◽

T T Egelhoff

Keyword(s):

Heavy Chain ◽

Kinase Activity ◽

Developmental Stages ◽

Catalytic Domain ◽

Myosin Ii ◽

Significant Similarity ◽

Dictyostelium Myosin ◽

A Cells

Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Côté, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.

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A Dictyostelium myosin II lacking a proximal 58-kDa portion of the tail is functional in vitro and in vivo.

Molecular Biology of the Cell ◽

10.1091/mbc.3.12.1455 ◽

1992 ◽

Vol 3 (12) ◽

pp. 1455-1462 ◽

Cited By ~ 26

Author(s):

E W Kubalek ◽

T Q Uyeda ◽

J A Spudich

Keyword(s):

Molecular Genetic ◽

Myosin Ii ◽

In Vitro Assays ◽

Dependent Manner ◽

Fruiting Bodies ◽

Wild Type ◽

In Vitro Motility Assay ◽

Dictyostelium Myosin

We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.

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The In Vitro Motility Assay: A Window Into the Myosin Molecular Motor

Physiology ◽

10.1152/physiologyonline.1996.11.1.1 ◽

1996 ◽

Vol 11 (1) ◽

pp. 1-7 ◽

Cited By ~ 3

Author(s):

DM Warshaw

Keyword(s):

Atp Hydrolysis ◽

Molecular Motor ◽

Muscular Contraction ◽

Mechanical Work ◽

Motility Assay ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Molecular Events ◽

Hydrolysis Of

Muscular contraction is powered by myosin, a molecular motor, that derives its energy from hydrolysis of ATP as it interacts with actin. With the development of the in vitro motility assay, molecular events that couple ATP hydrolysis to mechanical work can be probed at the level of a single myosin molecular motor.

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In Vitro Motility Assay Studies at Low [MgATP] - Evidence For Inter-Head Cooperativity in Fast Skeletal Myosin II

Biophysical Journal ◽

10.1016/j.bpj.2011.11.800 ◽

2012 ◽

Vol 102 (3) ◽

pp. 146a

Author(s):

Malin Persson ◽

Elina Bengtsson ◽

Lasse ten Siethoff ◽

Maria Gullberg ◽

Conny Tolf ◽

...

Keyword(s):

Myosin Ii ◽

Motility Assay ◽

In Vitro Motility Assay ◽

In Vitro Motility ◽

Skeletal Myosin

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

The Journal of Cell Biology ◽

10.1083/jcb.200404045 ◽

2004 ◽

Vol 167 (2) ◽

pp. 315-325 ◽

Cited By ~ 94

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.

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