Movement Of Actin Filaments Research Articles

1PS048 ATP、ADPと無機リン酸存在下での運動するアクチン繊維の変形(日本生物物理学会第50回年会(2012年度))

1PS048 ATP、ADPと無機リン酸存在下での運動するアクチン繊維の変形(日本生物物理学会第50回年会(2012年度))

Mechanotransduction In Vivo by Repeated Talin Stretch-Relaxation Events Depends upon Vinculin

Author SummaryHow are mechanical forces that act on the surface of a cell transformed into biochemical signals within the cell? Studies of isolated proteins suggest that some of them can stretch, but whether this also happens in living cells remains unclear. In this study, we have been able to measure the stretching of single molecules of a cellular adhesion protein called talin in vivo by tagging each end of the protein with a different fluorescent marker and observing changes in the distance between the two markers with a new microscopic method. Talin is a large cellular protein that concentrates at sites where the cell attaches to the substratum and links integrins in the cell membrane to the actin filament network in the cell. In our study, a green tag at the integrin-binding site was close to the cell surface, whereas a red tag at the actin-binding site was displaced inward by actin flow. We observed repeated protein stretching to 5–8 times the native protein length and relaxation linked to the transduction process in living cells in culture. Individual molecules stretched for 6–16 seconds over ranges of 50–350 nm. Cell adhesion sites, where hundreds of talin molecules were displaced in concert, had similar dynamics. These cycles of stretching and relaxation required the contractile protein myosin. The head domain of vinculin—an adhesion site protein that binds strongly to the stretched talin—kept the adhesions stretched and blocked large oscillations in length. These observations indicate that there is repeated stretching of talin, and that adhesion proteins play a role in the transduction of mechanical signals into biochemical signals through binding and release of vinculin and possibly other focal adhesion proteins.

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

Fission yeast myosin-I (Myo1p) not only associates with calmodulin, but also employs a second light chain called Cam2p. cam2Δ cells exhibit defects in cell polarity and growth consistent with a loss of Myo1p function. Loss of Cam2p leads to a reduction in Myo1p levels at endocytic patches and a 50% drop in the rates of Myo1p-driven actin filament motility. Thus, Cam2p plays a significant role in Myo1p function. However, further studies indicated the existence of an additional Cam2p-binding partner. Cam2p was still present at cortical patches in myo1Δ cells (or in myo1-IQ2 mutants, which lack an intact Cam2p-binding motif), whereas a cam2 null (cam2Δ) suppressed cytokinesis defects of an essential light chain (ELC) mutant known to be impaired in binding to PI 4-kinase (Pik1p). Binding studies revealed that Cam2p and the ELC compete for Pik1p. Cortical localization of Cam2p in the myo1Δ background relied on its association with Pik1p, whereas overexpression studies indicated that Cam2p, in turn, contributes to Pik1p function. The fact that the Myo1p-associated defects of a cam2Δ mutant are more potent than those of a myo1-IQ2 mutant suggests that myosin light chains can contribute to actomyosin function both directly and indirectly (via phospholipid synthesis at sites of polarized growth).

Effects of urea and guanidine hydrochloride on the sliding movement of actin filaments with ATP hydrolysis by myosin molecules

To evaluate the role of the hydration layer on the protein surface of actomyosin, we compared the effects of urea and guanidine-HCl on the sliding velocities and ATPase activities of the actin-heavy meromyosin (HMM) system. Both chemicals denature proteins, but only urea perturbs the hydration layer. Both the sliding velocity of actin filaments and actin-activated ATPase activity decreased with increasing urea concentrations. The sliding movement was completely inhibited at 1.0 M urea, while actin filaments were bound to HMM molecules fixed on the glass surface. Guanidine-HCl (0-0.05 M) drastically decreased both the sliding velocity and ATPase activation of acto-HMM complexes. Under this condition, actin filaments almost detached from HMM molecules. In contrast, the ATPase activity of HMM without actin filaments was almost independent of urea concentrations <1.0 M and guanidine-HCl concentrations <0.05 M. An increase in urea concentrations up to 2.0 M partly induced changes in the ternary structure of HMM molecules, while the actin filaments were stable in this concentration range. Hydration changes around such actomyosin complexes may alter both the stability of part of the myosin molecules, and the affinity for force transmission between actin filaments and myosin heads.

Actin Filament Patterning by Myosin Motors

We have studied the bulk alignment of actin filament sliding movement, driven by randomly oriented myosin molecules. We performed conventional, actin filament gliding assays, but using micromolar concentrations of actin which are roughly 100,000,000 times greater than normally used in such assays, but close to the concentration of actin found in living cells. Under these conditions, actin filament movement takes up a preferred orientation. The oriented patterns of movement extend over a length scale of ∼100 micrometres, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. Fluorescence video microscopy and image analysis allowed the time course of filament alignment and the formation and disappearance of oriented domains to be measured. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. We propose that self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which the cell migration might respond to chemical cues.

Titin Domains as Mechanical Reporters of UNC-45 Mediated Myosin Folding

Myosins are protein machines in which allosteric cross-talk between ATP-binding and hydrolysis and the movement of actin filaments requires precise changes in the polypeptide fold of the motor domain. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly for myosin into muscle thick filaments in vivo. However the molecular mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach to analyze the mechanical refolding of the myosin motor domain at the single molecule level utilizing AFM techniques. By chemically coupling an I27 titin polyprotein to the motor domain of myosin, we introduced a “molecular reporter” into the motor domain, providing a specific attachment point and a well characterized mechanical fingerprint in the AFM measurements. This approach enabled us to study the folding of the motor domain and directly observe the effect of the chaperone UNC-45. The motor domain misfolded after mechanical unfolding, whereas the myosin rod refolded independently of the motor domain. The misfolded motor domain recruited the otherwise robustly folding I27 modules into a misfolded state. Thus, the I27 domains function as a folding sensor. UNC-45 prevents the detrimental interaction between the motor domain and the I27 modules, indicating that it bound to and stabilized the non-native myosin motor domain. Using truncated versions of UNC-45, we localized this protective effect to the C-terminal UCS domain of UNC-45. Our approach enables the study, for the first time, the chaperone effects of UNC-45 on myosin folding in mechanistic detail.This work was funded by NIH grants R01DK073394 (AFO), R01AR050051 (HFE), the Muscular Dystrophy Association and the Cecil and Ida Green Endowment (HFE).

3M1012 ミオシンフィラメントトラックを乗り換えるアクチンフィラメント滑り運動の観察(分子モーター4,第49回年会講演予稿集)

3M1012 ミオシンフィラメントトラックを乗り換えるアクチンフィラメント滑り運動の観察(分子モーター4,第49回年会講演予稿集)

Nonmuscle myosin IIA with a GFP fused to the N-terminus of the regulatory light chain is regulated normally

Nonmuscle myosin II plays a crucial role in a variety of cellular processes (e.g., polarity formation, cell motility, and cytokinesis). It is composed of two heavy chains, two regulatory light chains and two essential light chains. The ATPase activity of the myosin II motor domain is regulated through phosphorylation of the regulatory light chain (RLC) by myosin light chain kinase. To study myosin function and localization in cellular processes, GFP-fused RLCs are widely used; however, the exact kinetic properties of myosins with bound GFP-RLC are poorly described. More importantly, it has not been shown that a regulatory light chain fused at its N-terminus with GFP can maintain the normal phosphorylation-dependent regulation of nonmuscle myosin or serve as a substrate for myosin light chain kinase. We coexpressed N-terminal GFP-RLC with a heavy meromyosin (HMM)-like fragment of nonmuscle myosin IIA and essential light chain to characterize the phosphorylation dynamics and in vitro kinetic properties of the resulting HMM. Myosin light chain kinase phosphorylates the GFP-RLC bound to HMM IIA with the same V(max) as it does the wild type RLC bound to HMM IIA, but the K(m) is about two fold higher for the GFP fusion protein, meaning that it is a somewhat poorer substrate. The steady-state actin-activated MgATPase activity of the GFP-RLC HMM is very low in the absence of phosphorylation demonstrating that the GFP moiety does not prevent formation of the off state. The actin-activated MgATPase activity of phosphorylated GFP-RLC-HMM and is about half that of wild type phosphorylated HMM. The ability of phosphorylated GFP-RLC-HMM to move actin filaments in the actin gliding assay is also slightly compromised. These data indicate that despite some kinetic differences the N-terminal GFP fusion to the regulatory light chain is a reasonable model system for studying myosin function in vivo.

Mutating the Converter–Relay Interface of Drosophila Myosin Perturbs ATPase Activity, Actin Motility, Myofibril Stability and Flight Ability

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase ( V max) by ∼ 60% compared to wild-type myosin, but there is no change in apparent actin affinity ( K m). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by ∼ 15% or ∼ 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by ∼ 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional “cracking” of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.

Subdiffraction‐Resolution Fluorescence Microscopy of Myosin–Actin Motility

Subdiffraction-resolution imaging by subsequent localization of single photoswitchable molecules can achieve a spatial resolution in the range of approximately 20 nm with moderate excitation intensities, but have so far been too slow for imaging faster dynamics in biology. Herein, we introduce a novel approach for video-like subdiffraction microscopy based on rapid and reversible photoswitching of commercially available organic carbocyanine fluorophores. With the present concept, we demonstrate in vitro studies on the motility of fluorophore-labeled actin filaments along myosin II. Actin filaments were densely labeled with carbocyanine fluorophores, and the gliding velocity adjusted by the concentration of ATP. At imaging frame rates of approximately 100 Hz, only 100 consecutive frames are sufficient to generate a single high-resolution image of moving actin filaments with a lateral resolution of approximately 30 nm. A video-like sequence is generated from individual reconstructed images by additionally applying a sliding window algorithm. We measured velocities of individual actin filaments of up to approximately 0.18 microm s(-1), observed strong bending and disruption of filaments as well as locally immobile fragments.

Relay Loop Stabilizes the Force-Generating Region in Myosin

We have used transient time-resolved FRET (TR2FRET) to monitor the conformation of the relay helix in a myosin II functional mutant during the recovery stroke in real time. Myosin was perturbed with the F506A mutation (Dictyostelium discoideum sequence), located within the relay loop in the force-generating region. F506 is a highly conserved residue in myosin II and is a hypertrophic cardiomyopathy mutation site. Previous studies [Tsiavaliaris, EMBO Rep, 2002, 3(11), 1099] showed a significant effect of the F506A mutation on myosin function. Actin affinity in the presence of ATP was increased, and the mutant did not move actin filaments in in vitro motility assays. A small decrease in intrinsic fluorescence was observed upon addition of excess ATP, but ATP binding and hydrolysis were not affected by the mutation. It was proposed that the F506A disrupts the communication between the active site and the lever arm. We engineered a double-Cys myosin mutant (A639C:K498C) in the Cys-less background with the F506A functional mutation, and labeled the mutant with optical probes. We used TR-FRET to determine the interprobe distance, and TR2FRET measurements after rapid mixing with ATP revealed changes in the relay helix conformation during the recovery stroke in real time. The mutation induced significant disorder of the relay helix in the force-generating region, but myosin still produces a recovery stroke, changing the relay helix conformation from straight to bent. We conclude that (a) the relay helix is disordered in myosin functional mutant F506A, which demonstrates the importance of the relay loop - relay helix interaction in the relay helix stabilization, and (b) the relay helix is the major structural element in the force-generating region of myosin, responsible for communication from the active site to the converter domain and the lever arm.

Switch-2 Dependent Modulation of the Myosin Power Stroke

Central to the mechanism of myosins are two conserved sequence motifs, switch-1 and switch-2, which contact nucleotide and Mg2+ at the rear of the nucleotide-binding pocket. They act as gamma-phosphate sensors, thus controlling hydrolysis and product release. The movement of switch-2 from a ‘closed’ to an ‘open’ conformation is translated via the relay region into a rotation of the converter domain, which drives the force generating power stroke. To investigate the molecular details of this coupling mechanism, we introduced mutation S456Y in switch-2 of the myosin-2 motor domain from Dictyostelium discoideum and analyzed the structural and functional consequences. Our kinetic results showed that the S456Y mutant lost the ability to bind effectively ATP, displayed a strongly decreased actin affinity in the ADP-bound state, and moved actin filaments with highly reduced velocities. It has been proposed that the exchange of S456 by a large amino acid would prevent the complete closing of switch-2 and therefore the full generation of the power stroke providing an explanation for the observed kinetic and mechanical defects of the mutant myosin. We have solved the crystal structure of the myosin motor domain with S456Y mutation in complex with ADP-VO4. The overall crystal structure and conformation of the nucleotide binding region resembles that of the wild-type revealing that switch-2 indeed can adopt the ‘closed’ conformation. We therefore conclude that not the complete ‘closing’ but the complete ‘opening’ of switch-2 is required for the full power stroke. Additional conformational changes in the crystal structure of S456Y, e.g. the actin binding loop-2 and loop-4, explain the disturbed actin binding properties of the mutant construct.

A Comparison of Polymer Blocking Agents in the in vitro Motility Assay

Blocking agents are used in in vitro motility assays to stabilize the motor proteins myosin or heavy meromyosin (HMM) and to prevent non-specific binding of actin to regions of microscope coverslip that are devoid of motors. Bovine serum albumin (BSA) or casein is typically used for blocking, but there is occasional need for non-protein blocking agents. We compared skeletal myosin and HMM function in motility assays as a function of the blocking agent that was used; these blocking agents included polysorbate (Tween) 20 and six different molecular weights of polyvinyl pyrrolidone (PVP) ranging from 10 kDa to 1.3 MDa, as well as BSA and β-casein as controls. In vitro motility assays were preformed and actin filament movement was quantified using automated particle tracking algorithms. PVPs of all molecular weights supported the motility of both HMM and myosin, though there was a slight downward trend in mobility at the highest molecular weights. When HMM was used in the motility assay, Tween showed poor mobility (1.7 um/s) compared to BSA (9.4 um/s). In contrast, full length myosin showed high mobility when blocked with Tween (8.3 um/s) compared to BSA (6.6 um/s). To determine whether Tween is a direct inhibitor of HMM function, NH4-activated ATPase assays were performed in solution with either BSA or Tween. There was no significant difference in ATPase rates between these two conditions. However, when the NH4-activated ATPase assay was repeated with HMM bound to a flow cell, Tween inhibited the ATPase activity. Thus while Tween does not directly inhibit the motor domains of HMM, it may adversely alter its binding to hydrophobic surfaces. In conclusion, low- to mid-molecular weight PVPs are excellent polymer blocking agents for actin-myosin or actin-HMM motility assays.

1P172 ミオシンフィラメント双極トラックに沿ったアクチンフィラメント滑り運動の活性化エネルギー(分子モーター,第48回日本生物物理学会年会)

1P172 ミオシンフィラメント双極トラックに沿ったアクチンフィラメント滑り運動の活性化エネルギー(分子モーター,第48回日本生物物理学会年会)

Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers

Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

An Alternatively Spliced Isoform of Non-muscle Myosin II-C Is Not Regulated by Myosin Light Chain Phosphorylation

We report a novel isoform of non-muscle myosin II-C (NM II-C), NM II-C2, that is generated by alternative splicing of an exon, C2, encoding 41 amino acids in mice (33 in humans). The 41 amino acids are inserted into loop 2 of the NM II-C heavy chain within the actin binding region. Unlike most vertebrate non-muscle and smooth muscle myosin IIs, baculovirus-expressed mouse heavy meromyosin (HMM) II-C2 demonstrates no requirement for regulatory myosin light chain (MLC(20)) phosphorylation for maximum actin-activated MgATPase activity or maximum in vitro motility as measured by the sliding actin filament assay. In contrast, noninserted HMM II-C0 and another alternatively spliced isoform HMM II-C1, which contains 8 amino acids inserted into loop 1, are dependent on MLC(20) phosphorylation for both actin-activated MgATPase activity and in vitro motility ( Kim, K. Y., Kovacs, M., Kawamoto, S., Sellers, J. R., and Adelstein, R. S. (2005) J. Biol. Chem. 280, 22769-22775 ). HMM II-C1C2, which contains both the C1 and C2 inserts, does not require MLC(20) phosphorylation for full activity similar to HMM II-C2. These constitutively active C2-inserted isoforms of NM II-C are expressed only in neuronal tissue. This is in contrast to NM II-C1 and NM II-C0, both of which are ubiquitously expressed. Full-length NM II-C2-GFP expressed in COS-7 cells localizes to filaments in interphase cells and to the cytokinetic ring in dividing cells.

1P-118 Cy3蛍光標識されたアクチン繊維の運動の光感受性阻害(筋肉(筋蛋白質・収縮),第47回日本生物物理学会年会)

1P-118 Cy3蛍光標識されたアクチン繊維の運動の光感受性阻害(筋肉(筋蛋白質・収縮),第47回日本生物物理学会年会)

1P-115 アクチンフィラメントの滑り運動と形態の歪みの関係(筋肉(筋蛋白質・収縮),第47回日本生物物理学会年会)

1P-115 アクチンフィラメントの滑り運動と形態の歪みの関係(筋肉(筋蛋白質・収縮),第47回日本生物物理学会年会)

Structure of a Complete Integrin Ectodomain in a Physiologic Resting State and Activation and Deactivation by Applied Forces

The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.