scholarly journals Cofilin drives rapid turnover and fluidization of entangled F-actin

2019 ◽  
Vol 116 (26) ◽  
pp. 12629-12637 ◽  
Author(s):  
Patrick M. McCall ◽  
Frederick C. MacKintosh ◽  
David R. Kovar ◽  
Margaret L. Gardel
Keyword(s):  
Stress Relaxation ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Low Frequency ◽  
Animal Cells ◽  
Single Filament ◽  
Actin Cortex ◽  
Spatial Reorganization ◽  
Actin Regulatory Proteins

The shape of most animal cells is controlled by the actin cortex, a thin network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and polymer turnover: Molecular motors drive deformations required for cell morphogenesis, while actin-filament disassembly dynamics relax stress and facilitate cortical remodeling. While many aspects of actin-cortex mechanics are well characterized, a mechanistic understanding of how nonequilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system of entangled F-actin, wherein the steady-state length and turnover rate of F-actin are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and assemble filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. We demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number despite sustained severing.

scholarly journals Cofilin Drives Rapid Turnover and Fluidization of Entangled F-actin

2017 ◽  
Author(s):  
Patrick M. McCall ◽  
Frederick C. MacKintosh ◽  
David R. Kovar ◽  
Margaret L. Gardel
Keyword(s):  
Steady State ◽  
Stress Relaxation ◽  
Actin Filaments ◽  
Atp Hydrolysis ◽  
Animal Cell ◽  
Animal Cells ◽  
Single Filament ◽  
Actin Cortex ◽  
Non Equilibrium ◽  
Entangled Solutions

AbstractThe shape of most animal cells is controlled by the actin cortex, a thin, isotropic network of dynamic actin filaments (F-actin) situated just beneath the plasma membrane. The cortex is held far from equilibrium by both active stresses and turnover: Myosin-II molecular motors drive deformations required for cell division, migration, and tissue morphogenesis, while turnover of the molecular components of the actin cortex relax stress and facilitate network reorganization. While many aspects of F-actin network viscoelasticity are well-characterized in the presence and absence of motor activity, a mechanistic understanding of how non-equilibrium actin turnover contributes to stress relaxation is still lacking. To address this, we developed a reconstituted in vitro system wherein the steady-state length and turnover rate of F-actin in entangled solutions are controlled by the actin regulatory proteins cofilin, profilin, and formin, which sever, recycle, and nucleate filaments, respectively. Cofilin-mediated severing accelerates the turnover and spatial reorganization of F-actin, without significant changes to filament length. Microrheology measurements demonstrate that cofilin-mediated severing is a single-timescale mode of stress relaxation that tunes the low-frequency viscosity over two orders of magnitude. These findings serve as the foundation for understanding the mechanics of more physiological F-actin networks with turnover, and inform an updated microscopic model of single-filament turnover. They also demonstrate that polymer activity, in the form of ATP hydrolysis on F-actin coupled to nucleotide-dependent cofilin binding, is sufficient to generate a form of active matter wherein asymmetric filament disassembly preserves filament number in spite of sustained severing.Significance StatementWhen an animal cell moves or divides, a disordered network of actin filaments (F-actin) plays a central role in controlling the resulting changes in cell shape. While it is known that continual turnover of F-actin by cofilin-mediated severing aids in reorganization of the cellular cytoskeleton, it is unclear how the turnover of structural elements alters the mechanical properties of the network. Here we show that severing of F-actin by cofilin results in a stress relaxation mechanism in entangled solutions characterized by a single-timescale set by the severing rate. Additionally, we identify ATP hydrolysis and nucleotide-dependent cofilin binding as sufficient ingredients to generate a non-equilibrium steady-state in which asymmetric F-actin disassembly preserves filament number in spite of sustained severing.

scholarly journals Molecular motors: forty years of interdisciplinary research

2011 ◽  
Vol 22 (21) ◽  
pp. 3936-3939 ◽  
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.

scholarly journals Converter domain mutations in myosin alter structural kinetics and motor function

2018 ◽  
Vol 294 (5) ◽  
pp. 1554-1567 ◽  
Author(s):  
Laura K. Gunther ◽  
John A. Rohde ◽  
Wanjian Tang ◽  
Shane D. Walton ◽  
William C. Unrath ◽  
...  
Keyword(s):  
Motor Function ◽  
Rate Constants ◽  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Actin Binding ◽  
Power Stroke ◽  
Structural Transitions ◽  
Myosin V ◽  
Time Resolved

Myosins are molecular motors that use a conserved ATPase cycle to generate force. We investigated two mutations in the converter domain of myosin V (R712G and F750L) to examine how altering specific structural transitions in the motor ATPase cycle can impair myosin mechanochemistry. The corresponding mutations in the human β-cardiac myosin gene are associated with hypertrophic and dilated cardiomyopathy, respectively. Despite similar steady-state actin-activated ATPase and unloaded in vitro motility–sliding velocities, both R712G and F750L were less able to overcome frictional loads measured in the loaded motility assay. Transient kinetic analysis and stopped-flow FRET demonstrated that the R712G mutation slowed the maximum ATP hydrolysis and recovery-stroke rate constants, whereas the F750L mutation enhanced these steps. In both mutants, the fast and slow power-stroke as well as actin-activated phosphate release rate constants were not significantly different from WT. Time-resolved FRET experiments revealed that R712G and F750L populate the pre- and post-power–stroke states with similar FRET distance and distance distribution profiles. The R712G mutant increased the mole fraction in the post-power–stroke conformation in the strong actin-binding states, whereas the F750L decreased this population in the actomyosin ADP state. We conclude that mutations in key allosteric pathways can shift the equilibrium and/or alter the activation energy associated with key structural transitions without altering the overall conformation of the pre- and post-power–stroke states. Thus, therapies designed to alter the transition between structural states may be able to rescue the impaired motor function induced by disease mutations.

scholarly journals Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays

2017 ◽  
Vol 114 (29) ◽  
pp. E5815-E5824 ◽  
Author(s):  
Stefan Brühmann ◽  
Dmitry S. Ushakov ◽  
Moritz Winterhoff ◽  
Richard B. Dickinson ◽  
Ute Curth ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Actin Binding ◽  
Actin Assembly ◽  
Single Filament ◽  
Capping Protein ◽  
Filament Assembly ◽  
Show Evidence ◽  
Membrane Protrusions ◽  
Polypeptide Chains

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin–binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.

scholarly journals Self-repair protects microtubules from their destruction by molecular motors:

2018 ◽  
Author(s):  
Sarah Triclin ◽  
Daisuke Inoue ◽  
Jeremie Gaillard ◽  
Zaw Min Htet ◽  
Morgan De Santis ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Molecular Motors ◽  
Intracellular Transport ◽  
Atp Hydrolysis ◽  
Low Energy ◽  
Mechanical Work ◽  
Repair Mechanism ◽  
Tubulin Dimer ◽  
Self Repair

Microtubules are dynamic polymers that are used for intracellular transport and chromosome segregation during cell division. Their instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Microtubules function in coordination with kinesin and dynein molecular motors, which use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the forces produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that the mechanical work of molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. This effect was not observed when free tubulin dimers were present in the assay. Using fluorescently labelled tubulin dimers we found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of kinesin and dynein motors and the renewal of the microtubule lattice.

scholarly journals Markov modelling of run length and velocity for molecular motors

2019 ◽  
Author(s):  
James L Buchanan ◽  
Robert P Gilbert
Keyword(s):  
Molecular Motors ◽  
Atp Hydrolysis ◽  
Reaction Rates ◽  
Markov Modelling ◽  
Standard Result ◽  
Run Length ◽  
Current State ◽  
Cargo Transport ◽  
Glass Slides

AbstractMolecular motors are nanometer scale proteins involved in various in-tracellular processes such as cargo transport, muscle contraction and cell division. The steps in the chemo-mechanical cycle that use ATP hydrolysis to generate motion along the cytoskeletal tracks can be characterized by stepping rates depending only on the current state, permitting modeling of the cycle as a Markov process. To learn more about the nature of motor mo-tion, cell researchers have conducted in vitro experiments in which molecular motors pull beads along filaments attached to glass slides and their veloc-ity and run length until detachment from the microtubule are recorded. In this article a formula is derived for distance traveled until detachment. Combining this with a standard result for time to absorption for Markov processes gives a formula for velocity. Two kinesins, and two variants of myosin VI, for which there are run length and velocity measurements in the literature are considered. In each case the derived formulas for run length and velocity have a generalized Michaelis-Menten form as functions of ATP concentration. The degrees of the numerator and denominator polynomials increase with the complexity of chemo-mechanical cycle. The degrees of the Michaelis-Menten form determine the maximum number of of reaction rates that can be determined from experimental data on run length and velocity, however consistency conditions may reduce this number and restrict which rates can be determined.

scholarly journals Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing

2021 ◽  
Author(s):  
Zane G Moreland ◽  
Fangfang Jiang ◽  
Carlos Aguilar ◽  
Melanie Barzik ◽  
Rui Gong ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Actin Polymerization ◽  
In Vitro Assays ◽  
Progressive Hearing Loss ◽  
Single Filament ◽  
Additional Function ◽  
Actin Regulatory Proteins ◽  
Human Hearing

The assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

Viscoelasticity and its Microscopic Characterization in Semiflexible Biopolymer Solutions

1997 ◽  
Vol 489 ◽  
Author(s):  
F. C. Mackintosh ◽  
F. Gittes ◽  
B. Schnurr ◽  
P. D. Olmsted ◽  
C. F. Schmidt
Keyword(s):  
Principal Component ◽  
Soft Materials ◽  
Thermal Fluctuations ◽  
In Vitro Models ◽  
Model Systems ◽  
Animal Cells ◽  
Actin Cortex ◽  
Flexible Polymer ◽  
Biopolymer Solutions

AbstractPlant and animal cells contain a complex polymeric network known as the cytoskeleton. A principal component of this is the actin cortex, a gel-like network of F-actin protein filaments. Recently, solutions of reconstituted F-actin have provided in vitro models of the actin cortex, as well as excellent model systems in which to study semiflexible polymers. We describe models of viscoelasticity in semifexible polymers, and report theoretical and experimental results for thermal fluctuations of embedded particles, which act as local viscoelastic probes of soft materials such as biopolymer solutions. Specifically, we report high-frequency scaling behavior of the shear modulus, as the 3/4 power of frequency, in contrast with the behavior of flexible polymer systems.

Characterization of an Intermediate Filament Protein from the Platyhelminth, Dugesia japonica

2020 ◽  
Vol 27 (5) ◽  
pp. 432-446
Author(s):  
Akiko Yamamoto ◽  
Ken-ichiro Matsunaga ◽  
Toyoaki Anai ◽  
Hitoshi Kawano ◽  
Toshihisa Ueda ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Immunohistochemical Analysis ◽  
Protein Characterization ◽  
Intermediate Filament Protein ◽  
Tail Domain ◽  
Animal Cells ◽  
Dugesia Japonica ◽  
Function Relationship

Background: Intermediate Filaments (IFs) are major constituents of the cytoskeletal systems in animal cells. Objective: To gain insights into the structure-function relationship of invertebrate cytoplasmic IF proteins, we characterized an IF protein from the platyhelminth, Dugesia japonica, termed Dif-1. Method: cDNA cloning, in situ hybridization, immunohistochemical analysis, and IF assembly experiments in vitro using recombinant Dif-1, were performed for protein characterization. Results: The structure deduced from the cDNA sequence showed that Djf-1 comprises 568 amino acids and has a tripartite domain structure (N-terminal head, central rod, and C-terminal tail) that is characteristic of IF proteins. Similar to nuclear IF lamins, Djf-1 contains an extra 42 residues in the coil 1b subdomain of the rod domain that is absent from vertebrate cytoplasmic IF proteins and a nuclear lamin-homology segment of approximately 105 residues in the tail domain; however, it contains no nuclear localization signal. In situ hybridization analysis showed that Djf-1 mRNA is specifically expressed in cells located within the marginal region encircling the worm body. Immunohistochemical analysis showed that Djf-1 protein forms cytoplasmic IFs located close to the microvilli of the cells. In vitro IF assembly experiments using recombinant proteins showed that Djf-1 alone polymerizes into IFs. Deletion of the extra 42 residues in the coil 1b subdomain resulted in the failure of IF formation. Conclusions: Together with data from other histological studies, our results suggest that Djf- 1 is expressed specifically in anchor cells within the glandular adhesive organs of the worm and that Djf-1 IFs may play a role in protecting the cells from mechanical stress.

Regulation of RNA helicase activity: principles and examples

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Pascal Donsbach ◽  
Dagmar Klostermeier
Keyword(s):  
Atp Hydrolysis ◽  
Wide Spectrum ◽  
Mrna Translation ◽  
Rna Degradation ◽  
Rna Helicases ◽  
Interaction Partners ◽  
Rna Duplexes ◽  
Self Association

Abstract RNA helicases are a ubiquitous class of enzymes involved in virtually all processes of RNA metabolism, from transcription, mRNA splicing and export, mRNA translation and RNA transport to RNA degradation. Although ATP-dependent unwinding of RNA duplexes is their hallmark reaction, not all helicases catalyze unwinding in vitro, and some in vivo functions do not depend on duplex unwinding. RNA helicases are divided into different families that share a common helicase core with a set of helicase signature motives. The core provides the active site for ATP hydrolysis, a binding site for the non-sequence-specific interactions with RNA, and in many cases a basal unwinding activity. Its activity is often regulated by flanking domains, by interaction partners, or by self-association. In this review, we summarize the regulatory mechanisms that modulate the activities of the helicase core. Case studies on selected helicases with functions in translation, splicing, and RNA sensing illustrate the various modes and layers of regulation in time and space that harness the helicase core for a wide spectrum of cellular tasks.

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