Actin Motility Research Articles

Electron Microscopy and 3D Reconstruction of Regulated Thin Filaments Decorated with Cardiac Myosin Binding Protein C

Myosin binding protein C (MyBP-C) is an accessory protein of striated muscle thick filaments and a modulator of cardiac muscle contraction. Defects in the cardiac isoform, cMyBP-C, cause heart disease. cMyBP-C comprises eleven 10 kDa immunoglobulin- and fibronectin-like domains, numbered C0-C10 from the N-terminus, and a MyBP-C-specific motif between C1 and C2. In vitro studies show that, in addition to binding to the thick filament via its C-terminal region, cMyBP-C can also interact with actin via its N-terminal domains, modulating actin motility. Neutron scattering and 3D EM reconstructions of F-actin decorated with N-terminal fragments of cMyBP-C suggest that cMyBP-C binds to subdomain 1 of actin close to the low Ca2+ binding site of tropomyosin. This suggests that cMyBP-C might modulate thin filament activity by physically interfering with tropomyosin regulatory movements on actin, a possibility strengthened by solution kinetics observations (White and Harris, Biophys. Soc. abstracts, 2012). To determine directly whether cMyBP-C binding affects tropomyosin position, we carried out 3D reconstruction of negatively stained thin filaments (containing F-actin, tropomyosin and troponin) decorated with the N-terminal fragment containing domains C0 to C2. Clear decoration was obtained under a variety of salt conditions. 3D reconstructions suggest that under most conditions cMyBP-C does not displace tropomyosin from its low Ca2+ position, although under certain conditions some shift of tropomyosin did appear to occur. At high Ca2+, there was little effect on tropomyosin position. The results suggest that cMyBP-C may modulate thin filament function by physically competing with tropomyosin for its low Ca2+ site on F-actin.

Factors Beyond Detachment Kinetics that Influence Unloaded Shortening Velocities of Muscle

Recent studies demonstrate the influence of actin-myosin attachment kinetics on in vitro actin filament sliding velocities (V), but it is unclear what factors determine whether velocities are detachment (d/τon) or attachment limited (Yengo et al. 2012. J Muscle Res Cell Motil.). We have developed a muscle model with molecular detail that suggests three key mechanisms by which attachment kinetics can influence unloaded shortening velocities. First, the saturation of myosin binding sites on actin by weakly bound myosin heads can result in attachment limited velocities that are slower than the detachment limit. Essentially, at high myosin densities the actin-activated ATPase activity (attachment limited) in the motility assay saturates before mechanical saturation (detachment limited). Second if actin-bound myosin heads that resist actin movement have a relatively low stiffness, unloaded shortening velocities can exceed the detachment limit. The non-linear compliance of positively and negatively strained myosin heads was demonstrated experimentally (Kaya and Higuchi. 2010. Science. 329: 686-9). Finally, our model shows that mechanically accelerated ADP release kinetics can result in velocities that exceed the detachment limit. These simulations accurately describe the observation of hypermotile velocities (Hooft et al. 2007. Biochemistry. 46:3513-20., Jackson and Baker. 2009. PCCP. 11:4808-14.). In summary, unloaded shortening velocities can be influenced by many factors other than detachment kinetics. Thus a mutation or perturbation that results in a change in V need not result from a change in only d or τon.

Measurement of Nucleotide Release from Synthetic Myosin Thick Filament along which Actin Fiaments Slide

Actin filaments move along myosin thick filaments a fast speed towards the central bare zone and at a slower speed away from the bare zone. Sellers and Kachar speculated that myosin heads rotate180 degrees to face the actin in the correct configuration and the detachment of the rotated heads from actin occurs at a slower rate. Recently, we have found that the thermal activation energy of the backward movement was remarkably higher than that of the forward movement, suggesting that the backward movement causes the myosin heads to be constrained, and increases the energy required for the ADP release step. However, there is no direct evidence for the slow ADP release rate in the backward movement. Here, in order to examine whether rate of ADP release step depends on the direction of the actin movement, we measured nucleotide turnover rates of bipolar myosin thick filament along which actin filament slides by monitoring the displacement of prebound fluorescent ATP analog, Cy3-EDA-ATP on flash photolysis of caged ATP.

The movement of actin–myosin biomolecular linear motor under AC electric fields: An experimental study

The role of actin–myosin as a biomolecular linear motor is considered a transport system at nanoscale because of their size, efficiency and functionality. To utilize the ability to transport, it is essential to control the random movement of actin filaments (F-actin) on myosin coated substrate. In the presence of an alternating current (AC) electric field, the direction of F-actin movement is regulated by electro-orientation torque and, as a result, its movement is perpendicularly toward the electrode edges. Our data confirm such aligned movement is proportional to the strength of applied electric field. Interestingly, the aligned movement is found frequency-dependent and the electrothermal effect is observed by means of the velocity measurement of aligned F-actin movement. The findings in this study may provide constructive information for manipulating actin–myosin nanotransport system to build functional nanodevices in future work.

Shape change in the receptor for gliding motility in Plasmodium sporozoites

Sporozoite gliding motility and invasion of mosquito and vertebrate host cells in malaria is mediated by thrombospondin repeat anonymous protein (TRAP). Tandem von Willebrand factor A (VWA) and thrombospondin type I repeat (TSR) domains in TRAP connect through proline-rich stalk, transmembrane, and cytoplasmic domains to the parasite actin-dependent motility apparatus. We crystallized fragments containing the VWA and TSR domains from Plasmodium vivax and Plasmodium falciparum in different crystal lattices. TRAP VWA domains adopt closed and open conformations, and bind a Mg(2+) ion at a metal ion-dependent adhesion site implicated in ligand binding. Metal ion coordination in the open state is identical to that seen in the open high-affinity state of integrin I domains. The closed VWA conformation associates with a disordered TSR domain. In contrast, the open VWA conformation crystallizes with an extensible β ribbon and ordered TSR domain. The extensible β ribbon is composed of disulfide-bonded segments N- and C-terminal to the VWA domain that are largely drawn out of the closed VWA domain in a 15 Å movement to the open conformation. The extensible β ribbon and TSR domain overlap at a conserved interface. The VWA, extensible β ribbon, and TSR domains adopt a highly elongated overall orientation that would be stabilized by tensile force exerted across a ligand-receptor complex by the actin motility apparatus of the sporozoite. Our results provide insights into regulation of "stick-and-slip" parasite motility and for development of sporozoite subunit vaccines.

Forces measured with micro-fabricated cantilevers during actomyosin interactions produced by filaments containing different myosin isoforms and loop 1 structures

BackgroundThere is evidence that the actin-activated ATP kinetics and the mechanical work produced by muscle myosin molecules are regulated by two surface loops, located near the ATP binding pocket (loop 1), and in a region that interfaces with actin (loop 2). These loops regulate force and velocity of contraction, and have been investigated mostly in single molecules. There is a lack of information of the work produced by myosin molecules ordered in filaments and working cooperatively, which is the actual muscle environment. MethodsWe use micro-fabricated cantilevers to measure forces produced by myosin filaments isolated from mollusk muscles, skeletal muscles, and smooth muscles containing variations in the structure of loop 1 (tonic and phasic myosins). We complemented the experiments with in-vitro assays to measure the velocity of actin motility. ResultsSmooth muscle myosin filaments produced more force than skeletal and mollusk myosin filaments when normalized per filament overlap. Skeletal muscle myosin propelled actin filaments in a higher sliding velocity than smooth muscle myosin. The values for force and velocity were consistent with previous studies using myosin molecules, and suggest a close correlation with the myosin isoform and structure of surface loop 1. General significanceThe technique using micro-fabricated cantilevers to measure force of filaments allows for the investigation of the relation between myosin structure and contractility, allowing experiments to be conducted with an array of different myosin isoforms. Using the technique we observed that the work produced by myosin molecules is regulated by amino-acid sequences aligned in specific loops.

Taking a translational turn

Taking a translational turn

From cycling between coupled reactions to the cross-bridge cycle: mechanical power output as an integral part of energy metabolism.

ATP delivery and its usage are achieved by cycling of respective intermediates through interconnected coupled reactions. At steady state, cycling between coupled reactions always occurs at zero resistance of the whole cycle without dissipation of free energy. The cross-bridge cycle can also be described by a system of coupled reactions: one energising reaction, which energises myosin heads by coupled ATP splitting, and one de-energising reaction, which transduces free energy from myosin heads to coupled actin movement. The whole cycle of myosin heads via cross-bridge formation and dissociation proceeds at zero resistance. Dissipation of free energy from coupled reactions occurs whenever the input potential overcomes the counteracting output potential. In addition, dissipation is produced by uncoupling. This is brought about by a load dependent shortening of the cross-bridge stroke to zero, which allows isometric force generation without mechanical power output. The occurrence of maximal efficiency is caused by uncoupling. Under coupled conditions, Hill’s equation (velocity as a function of load) is fulfilled. In addition, force and shortening velocity both depend on [Ca2+]. Muscular fatigue is triggered when ATP consumption overcomes ATP delivery. As a result, the substrate of the cycle, [MgATP2−], is reduced. This leads to a switch off of cycling and ATP consumption, so that a recovery of [ATP] is possible. In this way a potentially harmful, persistent low energy state of the cell can be avoided.

Antibodies Covalently Immobilized on Actin Filaments for Fast Myosin Driven Analyte Transport

Biosensors would benefit from further miniaturization, increased detection rate and independence from external pumps and other bulky equipment. Whereas transportation systems built around molecular motors and cytoskeletal filaments hold significant promise in the latter regard, recent proof-of-principle devices based on the microtubule-kinesin motor system have not matched the speed of existing methods. An attractive solution to overcome this limitation would be the use of myosin driven propulsion of actin filaments which offers motility one order of magnitude faster than the kinesin-microtubule system. Here, we realized a necessary requirement for the use of the actomyosin system in biosensing devices, namely covalent attachment of antibodies to actin filaments using heterobifunctional cross-linkers. We also demonstrated consistent and rapid myosin II driven transport where velocity and the fraction of motile actin filaments was negligibly affected by the presence of antibody-antigen complexes at rather high density (>20 µm−1). The results, however, also demonstrated that it was challenging to consistently achieve high density of functional antibodies along the actin filament, and optimization of the covalent coupling procedure to increase labeling density should be a major focus for future work. Despite the remaining challenges, the reported advances are important steps towards considerably faster nanoseparation than shown for previous molecular motor based devices, and enhanced miniaturization because of high bending flexibility of actin filaments.

Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions.

Membrane-Bound Myo1c Powers Asymmetric Motility of Actin Filaments

Class I myosins are molecular motors that link cellular membranes to the actin cytoskeleton and play roles in membrane tension generation, membrane dynamics, and mechanosignal transduction. The widely expressed myosin-Ic (myo1c) isoform binds tightly to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] via a pleckstrin homology domain located in the myo1c tail, which is important for its proper cellular localization. In this study, we found that myo1c can power actin motility on fluid membranes composed of physiological concentrations of PtdIns(4,5)P(2) and that this motility is inhibited by high concentrations of anionic phospholipids. Strikingly, this motility occurs along curved paths in a counterclockwise direction (i.e., the actin filaments turn in leftward circles). A biotinylated myo1c construct containing only the motor domain and the lever arm anchored via streptavidin on a membrane containing biotinylated lipid can also generate asymmetric motility, suggesting that the tail domain is not required for the counterclockwise turning. We found that the ability to produce counterclockwise motility is not a universal characteristic of myosin-I motors, as membrane-bound myosin-Ia (myo1a) and myosin-Ib (myo1b) are able to power actin gliding, but the actin gliding has no substantial turning bias. This work reveals a possible mechanism for establishing asymmetry in relationship to the plasma membrane.

Construction of Flow Chambers for Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Motility Assays

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to construct sample chambers (flow chambers) for polTIRFM motility assays. Each chamber can hold ∼20 µL of solution. To flow a solution through the chamber, the solution is added to the chamber with a pipette while wicking out the previous contents with filter paper. Each end of the coverslip should extend beyond the edge of the slide to support the pipette tip and filter paper. The flow rate can be roughly controlled by adjusting the contact area between the filter paper and the solution. The chambers can be used for investigating the motility of myosin V in vitro with the processive motility assay, as well as for assessing the motility of actin using the twirling assay.

Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N-terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS-related peptide could affect migration of macrophages, using the mouse macrophage-like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP-1 (25-100 ng/ml) or C5a (5-20 ng/ml). Cells were preincubated (15 min) with MANS or a mis-sense control peptide (RNS), both at 50 μM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP-1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP-1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.

Rho Kinase Inhibitors: Potential Treatments for Diabetes and Diabetic Complications

The small GTPase RhoA and its downstream effector, Rho kinase (ROCK), appear to mediate numerous pathophysiological signals, including smooth muscle cell contraction, actin cytoskeleton organization, cell adhesion and motility, proliferation, differentiation and the expression of several genes. Clinical interest in the RhoA/ROCK pathway has increased, due to emerging evidence that this signaling pathway is involved in the pathogenesis of several diseases, including hypertension, coronary vasospasm, stroke, atherosclerosis, heart failure and diabetes; ROCK is considered an important future therapeutic target. Several pharmaceutical companies are already actively engaged in the development of ROCK inhibitors as the next generation of therapeutic agents for these diseases. This review discusses the relationship between diabetes and hyperglycemia-induced RhoA/ROCK activation, highlights recent findings on the roles of ROCK inhibitors from experimental models of diabetes and clinical studies in cardiovascular patients, and elucidates major challenges for developing more selective ROCK inhibitors. Accumulating evidence suggests the potential of ROCK inhibitors as therapeutic agents for diabetes and its complications.

Abstract 4312: E3 ligase activity of XIAP RING domain is required for XIAP-mediated cancer cell migration but not for its RhoGDI binding activity

Abstract Although an increased expression level of XIAP is associated with cancer cell metastasis in individuals with various types of cancer, the molecular mechanism linking XIAP to cancer metastasis remains unexplored. Here we show that in XIAP depletion HCT116 cells (XIAP−/−), cell migration, invasion and actin polymerization are decreased, which could be rescued either by the introduction of XIAP RING domain or knockdown of RhoGDI. Subsequent studies demonstrate that this function of the XIAP RING domain depends on its inhibition of RhoGDI SUMOylation at K138. Furthermore, SUMO1-RhoGDI, but not un-sumoylated RhoGDI preferentially binds to Rho GTPase and downregulates the activities of GTPases, including RhoA, Cdc42 and Rac1, by which RhoGDI inhibits cancer cellular actin polymerization and motility. Taken together, our study reveals a novel post-translational modification (SUMOylation) of RhoGDI, and its physiological regulation by the RING domain of XIAP, which may account for XIAP modulation of cancer cell motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4312. doi:1538-7445.AM2012-4312

Abstract 33: Implementing CAD cells quantify the relationships between Ig-CAM-mediated adhesion, force transduction, and actin dynamics

Abstract Several neuro-degenerative diseases and motor defects have been linked to malfunctions in the motility of neurons. This project is focused on the determining whether the roles of the transmembrane proteins NCAM and L1-CAM can be studied using the CAD cell line as a model. CAD cells are a cancer cell line that come from mice and can be stimulated to grow as either neurons or glial cells. In addition, this cell line has been shown to be an easily transfected line. In order to determine usefulness of this cell line several techniques were used. Western blots were implemented in order to determine if the NCAM and/or L1-CAM and proteins known to be involved in their signaling pathways were present in the cell line. Western blots were then confirmed by immunocytochemistry staining. Next, the CAD cell line was transfected with photoactivateable actin. Finally, immuno beads were attached to the membranes of these CAD cells and then holographic optical tweezers were used to determine whether or not thsee ligands coupled to the retrograde actin flow in the cells. The combination of these experiments should provide the evidence for whether or not the CAD cells would be a feasible model to study the nature of the NCAM and L1-CAM proteins and their relationship to the movement of actin in the cytoskeleton of neurons. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 33. doi:1538-7445.AM2012-33

Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology.

Putting a bifunctional motor to work: insights into the role of plant KCH kinesins

Putting a bifunctional motor to work: insights into the role of plant KCH kinesins

Rho-Associated Kinase 2 Polymorphism of Vasospastic Angina in Korean Population

Rho-Associated Kinase 2 Polymorphism of Vasospastic Angina in Korean Population

The retromer complex – endosomal protein recycling and beyond

The retromer complex is a vital element of the endosomal protein sorting machinery that is conserved across all eukaryotes. Retromer is most closely associated with the endosome-to-Golgi retrieval pathway and is necessary to maintain an active pool of hydrolase receptors in the trans-Golgi network. Recent progress in studies of retromer have identified new retromer-interacting proteins, including the WASH complex and cargo such as the Wntless/MIG-14 protein, which now extends the role of retromer beyond the endosome-to-Golgi pathway and has revealed that retromer is required for aspects of endosome-to-plasma membrane sorting and regulation of signalling events. The interactions between the retromer complex and other macromolecular protein complexes now show how endosomal protein sorting is coordinated with actin assembly and movement along microtubules, and place retromer squarely at the centre of a complex set of protein machinery that governs endosomal protein sorting. Dysregulation of retromer-mediated endosomal protein sorting leads to various pathologies, including neurodegenerative diseases such as Alzheimer disease and spastic paraplegia and the mechanisms underlying these pathologies are starting to be understood. In this Commentary, I will highlight recent advances in the understanding of retromer-mediated endosomal protein sorting and discuss how retromer contributes to a diverse set of physiological processes.