Twist and writhe dynamics of stiff filaments

This letter considers the dynamics of a stiff filament, in particular the coupling of twist and bend via writhe. The time dependence of the writhe of a filament is $W_r^2\sim L t^{1/4}$ for a linear filament and $W_r^2\sim t^{1/2} / L$ for a curved filament. Simulations are used to study the relative importance of crankshaft motion and tube like motion in twist dynamics. Fuller's theorem, and its relation with the Berry phase, is reconsidered for open filamentsComment: 7 Pages with 2 figure

Thermodynamic stability, unfolding kinetics, and aggregation of the N-terminal actin-binding domains of utrophin and dystrophin.

Muscular dystrophy (MD) is the most common genetic lethal disorder in children. Mutations in dystrophin trigger the most common form of MD, Duchenne, and its allelic variant Becker MD. Utrophin is the closest homologue and has been shown to compensate for the loss of dystrophin in human disease animal models. However, the structural and functional similarities and differences between utrophin and dystrophin are less understood. Both proteins interact with actin through their N-terminal actin-binding domain (N-ABD). In this study, we examined the thermodynamic stability and aggregation of utrophin N-ABD and compared with that of dystrophin. Our results show that utrophin N-ABD has spectroscopic properties similar to dystrophin N-ABD. However, utrophin N-ABD has decreased denaturant and thermal stability, unfolds faster, and is correspondingly more susceptible to proteolysis, which might account for its decreased in vivo half-life compared to dystrophin. In addition, utrophin N-ABD aggregates to a lesser extent compared with dystrophin N-ABD, contrary to the general behavior of proteins in which decreased stability enhances protein aggregation. Despite these differences in stability and aggregation, both proteins exhibit deleterious effects of mutations. When utrophin N-ABD mutations analogous in position to the dystrophin disease-causing mutations were generated, they behaved similarly to dystrophin mutants in terms of decreased stability and the formation of cross-β aggregates, indicating a possible role for utrophin mutations in disease mechanisms

Coordination of the filament stabilizing versus destabilizing activities of cofilin through its secondary binding site on actin.

Cofilin is a ubiquitous modulator of actin cytoskeleton dynamics that can both stabilize and destabilize actin filaments depending on its concentration and/or the presence of regulatory co-factors. Three charge-reversal mutants of yeast cofilin, located in cofilin\u27s filament-specific secondary binding site, were characterized in order to understand why disruption of this site leads to enhanced filament disassembly. Crystal structures of the mutants showed that the mutations specifically affect the secondary actin-binding interface, leaving the primary binding site unaltered. The mutant cofilins show enhanced activity compared to wild-type cofilin in severing and disassembling actin filaments. Electron microscopy and image analysis revealed long actin filaments in the presence of wild-type cofilin, while the mutants induced many short filaments, consistent with enhanced severing. Real-time fluorescence microscopy of labeled actin filaments confirmed that the mutants, unlike wild-type cofilin, were functioning as constitutively active severing proteins. In cells, the mutant cofilins delayed endocytosis, which depends on rapid actin turnover. We conclude that mutating cofilin\u27s secondary actin-binding site increases cofilin\u27s ability to sever and de-polymerize actin filaments. We hypothesize that activators of cofilin severing, like Aip1p, may act by disrupting the interface between cofilin\u27s secondary actin-binding site and the actin filament

Conformational Dynamics of Actin: Effectors and Implications for Biological Function

Actin is a protein abundant in many cell types. Decades of investigations have provided evidence that it has many functions in living cells. The diverse morphology and dynamics of actin structures adapted to versatile cellular functions is established by a large repertoire of actin-binding proteins. The proper interactions with these proteins assume effective molecular adaptations from actin, in which its conformational transitions play essential role. This review attempts to summarise our current knowledge regarding the coupling between the conformational states of actin and its biological function

Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics

Uremic myopathy: Is oxidative stress implicated in muscle dysfunction in uremia?

© 2015 The Authors. Published by Frontiers Media. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.3389/fphys.2015.00102Renal failure is accompanied by progressive muscle weakness and premature fatigue, in part linked to hypokinesis and in part to uremic toxicity. These changes are associated with various detrimental biochemical and morphological alterations. All of these pathological parameters are collectively termed uremic myopathy. Various interventions while helpful can't fully remedy the pathological phenotype. Complex mechanisms that stimulate muscle dysfunction in uremia have been proposed, and oxidative stress could be implicated. Skeletal muscles continuously produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) at rest and more so during contraction. The aim of this mini review is to provide an update on recent advances in our understanding of how ROS and RNS generation might contribute to muscle dysfunction in uremia. Thus, a systematic review was conducted searching PubMed and Scopus by using the Cochrane and PRISMA guidelines. While few studies met our criteria their findings are discussed making reference to other available literature data. Oxidative stress can direct muscle cells into a catabolic state and chronic exposure to it leads to wasting. Moreover, redox disturbances can significantly affect force production per se. We conclude that oxidative stress can be in part responsible for some aspects of uremic myopathy. Further research is needed to discern clear mechanisms and to help efforts to counteract muscle weakness and exercise intolerance in uremic patients.This work has been co-financed by the European Union (European Social Fund—ESF) and Greek national funds through the Operational Program “Educational and Lifelong Learning” of the National Strategic Reference Framework (NSRF)—Research Funding Program: Thales (MuscleFun Project-MIS 377260) Investing in knowledge society through the European Social Fund.Published versio

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

In vitro, actin filament tension correlates with the binding and apparent activity of the filament-severing protein cofilin, suggesting a molecular mechanism by which cells respond to changes in mechanical force

Long-Term Storage of Surface-Adsorbed Protein Machines

The effective and simple long-term storage of complex functional proteins is critical in achieving commercially viable biosensors. This issue is particularly challenging in recently proposed types of nanobiosensors, where molecular-motor-driven transportation substitutes microfluidics and forms the basis for novel detection schemes. Importantly, therefore, we here describe that delicate heavy meromyosin (HMM)-based nanodevices (HMM motor fragments adsorbed to silanized surfaces and actin bound to HMM) fully maintain their function when stored at -20 degrees C for more than a month. The mechanisms for the excellent preservation of acto-HMM motor function upon repeated freeze thaw cycles are discussed. The results are important to the future commercial implementation of motor-based nanodevices and are of more general value to the long-term storage of any protein-based bionanodevice.</p

FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations

Major role for active extension in the formation of processes by ras -transformed fibroblasts

Expression of constitutively active Ras protein in fibroblasts results in enhanced cell motility, invasion competence and morphological changes including the formation of elongate cellular processes. These processes have been shown to resemble retraction tails formed passively behind nontransformed cells by movement relative to sites of cell-substrate attachment. However, analysis presented here reveals that active extension mechanisms also play a role in the formation of these processes. Extension of distal process ends occurs at 0.42 ± 0.44 μm/min in ras -transformed fibroblasts and accounts for 63.6 ± 27.5% of observed process lengths. Active process extension by ras -transformed fibroblasts also persists in the presence of cell-cell contacts. Studies conducted using actin or microtubule antagonists, and correlation of process behavior followed by fixation and immunostaining reveal that process extension requires intact actin and microtubule networks. Other analyses reveal that active extension plays a significantly smaller role in the formation of processes by non-transformed control fibroblasts. These observations demonstrate that constitutively active Ras enhances process extension in fibroblasts and is a causal factor in process extension by fibroblasts in the presence of cell-cell contacts. Moreover, these studies demonstrate that process extension by ras -transformed fibroblasts is accomplished through mechanisms similar to those thought to drive active extension of processes by other cell types including neurons. These findings suggest that extension of cellular processes could play an important role in the metastatic behavior of ras -transformed fibroblasts as well as the response of untransformed fibroblasts to receptor mediated signal transduction events. Cell Motil. Cytoskeleton 42:12–26, 1999. © 1999 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34964/1/2_ftp.pd