scholarly journals A rotary molecular motor that can work at near 100% efficiency

2000 ◽  
Vol 355 (1396) ◽  
pp. 473-489 ◽  
Author(s):  
Kazuhiko Kinosita ◽  
Ryohei Yasuda ◽  
Hiroyuki Noji ◽  
Kengo Adachi
Keyword(s):  
Free Energy ◽  
Actin Filament ◽  
Single Molecule ◽  
High Efficiency ◽  
Atp Hydrolysis ◽  
Molecular Motor ◽  
Constant Torque ◽  
Γ Subunit ◽  
Work Done

A single molecule of F 1 –ATPase is by itself a rotary motor in which a central γ–subunit rotates against a surrounding cylinder made of α 3 β 3 –subunits. Driven by the three βs that sequentially hydrolyse ATP, the motor rotates in discrete 120° steps, as demonstrated in video images of the movement of an actin filament bound, as a marker, to the central γ–subunit. Over a broad range of load (hydrodynamic friction against the rotating actin filament) and speed, the F motor produces a constant torque of ca . 40 pN nm. The work done in a 120° step, or the work per ATP molecule, is thus ca . 80 pN nm. In cells, the free energy of ATP hydrolysis is ca . 90 pN nm per ATP molecule, suggesting that the F 1 motor can work at near 100% efficiency. We confirmed in vitro that F 1 indeed does ca . 80 pN nm of work under the condition where the free energy per ATP is 90 pN nm. The high efficiency may be related to the fully reversible nature of the F 1 motor: the ATP synthase, of which F 1 is a part, is considered to synthesize ATP from ADP and phosphate by reverse rotation of the F motor. Possible mechanisms of F 1 rotation are discussed.

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 Myosin and gelsolin cooperate in actin filament severing and actomyosin motor activity

2020 ◽  
pp. jbc.RA120.015863
Author(s):  
Venukumar Vemula ◽  
Tamás Huber ◽  
Marko Ušaj ◽  
Beáta Bugyi ◽  
Alf Mansson
Keyword(s):  
Motor Activity ◽  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Actin Binding ◽  
In Vitro Motility Assay ◽  
Cooperative Effects ◽  
Myosin Motor ◽  
Filament Dynamics

Actin is a major intracellular protein with key functions in cellular motility, signaling and structural rearrangements. Its dynamic behavior, such as polymerisation and depolymerisation of actin filaments in response to intra- and extracellular cues, is regulated by an abundance of actin binding proteins. Out of these, gelsolin is one of the most potent for filament severing. However, myosin motor activity also fragments actin filaments through motor induced forces, suggesting that these two proteins could cooperate to regulate filament dynamics and motility. To test this idea, we used an in vitro motility assay, where actin filaments are propelled by surface-adsorbed heavy meromyosin (HMM) motor fragments. This allows studies of both motility and filament dynamics using isolated proteins. Gelsolin, at both nanomolar and micromolar Ca2+ concentration, appreciably enhanced actin filament severing caused by HMM-induced forces at 1 mM MgATP, an effect that was increased at higher HMM motor density. This finding is consistent with cooperativity between actin filament severing by myosin-induced forces and by gelsolin. We also observed reduced sliding velocity of the HMM-propelled filaments in the presence of gelsolin, providing further support of myosin-gelsolin cooperativity. Total internal reflection fluorescence microscopy based single molecule studies corroborated that the velocity reduction was a direct effect of gelsolin-binding to the filament and revealed different filament severing pattern of stationary and HMM propelled filaments. Overall, the results corroborate cooperative effects between gelsolin-induced alterations in the actin filaments and changes due to myosin motor activity leading to enhanced F-actin severing of possible physiological relevance.

Free energy and enthalpy of ATP hydrolysis in the sarcoplasm

1969 ◽  
Vol 174 (1036) ◽  
pp. 348-353 ◽  
Keyword(s):  
Free Energy ◽  
Ionic Strength ◽  
Temperature Dependence ◽  
Thermodynamic Data ◽  
Approximate Calculation ◽  
Atp Hydrolysis ◽  
Equilibrium Conditions ◽  
Thermodynamic Measurements

A rigorous calculation of the free energy available in vivo from ATP hydrolysis requires the following information which is not all available, namely: (i) intra­cellular pH, (ii) activities of all the species of reactants and products in sarcoplasm, (iii) thermodynamic data for all the reactions involved, including values for ionic strength and temperature dependence, and (iv) the extent of deviation from equilibrium conditions, i. e. during contraction. We shall discuss each of these factors in turn and state the assumptions made that allow the approximate calculation of the free energy made available by the following net reaction in the sarcoplasm: ATP +H 2 O → ADP + Pi + H + . (1) Although it can only be an approximation this calculation is useful since it will take into account recent thermodynamic measurements in vitro .

scholarly journals Dissecting the role of the γ-subunit in the rotary–chemical coupling and torque generation of F1-ATPase

2015 ◽  
Vol 112 (9) ◽  
pp. 2746-2751 ◽  
Author(s):  
Shayantani Mukherjee ◽  
Arieh Warshel
Keyword(s):  
Free Energy ◽  
Atp Hydrolysis ◽  
Mechanical Energy ◽  
Coarse Grained ◽  
Γ Subunit ◽  
Torque Generation ◽  
Chemical Coupling ◽  
Basic Features ◽  
Experimental Findings ◽  
Molecular Nature

Unraveling the molecular nature of the conversion of chemical energy (ATP hydrolysis in the α/β-subunits) to mechanical energy and torque (rotation of the γ-subunit) in F1-ATPase is very challenging. A major part of the challenge involves understanding the rotary–chemical coupling by a nonphenomenological structure–energy description, while accounting for the observed torque generated on the γ-subunit and its change due to mutation of this unit. Here we extend our previous study that used a coarse-grained model of the F1-ATPase to generate a structure-based free energy landscape of the rotary–chemical process. Our quantitative analysis of the landscape reproduced the observed torque for the wild-type enzyme. In doing so, we found that there are several possibilities of torque generation from landscapes with various shapes and demonstrated that a downhill slope along the chemical coordinate could still result in negligible torque, due to ineffective coupling of the chemistry to the γ-subunit rotation. We then explored the relationship between the functionality and the underlying sequence through systematic examination of the effect of various parts of the γ-subunit on free energy surfaces of F1-ATPase. Furthermore, by constructing several types of γ-deletion systems and calculating the corresponding torque generation, we gained previously unknown insights into the molecular nature of the F1-ATPase rotary motor. Significantly, our results are in excellent agreement with recent experimental findings and indicate that the rotary–chemical coupling is primarily established through electrostatic effects, although specific contacts through γ-ionizable residue side chains are not essential for establishing the basic features of the coupling.

scholarly journals Alginate-based microparticles coated with HPMCP/AS cellulose-derivatives enable the Ctx(Ile21)-Ha antimicrobial peptide application as a feed additive

2021 ◽  
Author(s):  
Cesar Augusto Roque Borda ◽  
Hanyeny Raiely Leite Silva ◽  
Edson Crusca Junior ◽  
Jéssica Aparecida Serafim ◽  
Andréia Bagliotti Meneguin ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Single Molecule ◽  
High Efficiency ◽  
Antibacterial Activities ◽  
Feed Additive ◽  
Resistant Bacteria ◽  
Cellulose Derivatives ◽  
Monogastric Animal ◽  
Physical Chemical

ABSTRACTMicroencapsulation is a potential biotechnological tool, which can overcome AMPs instabilities and reduce toxic side effects. Thus, this study evaluates the antibacterial activities of the Ctx(Ile21)-Ha antimicrobial peptide against MDR and non-resistant bacteria, develop and characterize peptide-loaded microparticles coated with HPMCAS and HPMCP. Ctx(Ile21)-Ha microencapsulation was performed by ionic gelation with high-efficiency, maintaining the physical-chemical stability. Ctx(Ile21)-Ha coated-microparticles were characterized, and their hemolytic activity assay demonstrated that hemolysis was decreased up to 95% compared to single molecule. In addition, in vitro release control profile simulating different portions of gastrointestinal tract was performed and showed the microcapsules’ ability to protect the peptide and release it in the intestine, aimed pathogens location, mainly by Salmonella sp. Therefore, use of microencapsulated Ctx(Ile21)-Ha can be allowed as an antimicrobial controller in monogastric animal production, being a valuable option for molecules with low therapeutic indexes or high hemolytic rates.

scholarly journals Synergy between Cyclase-associated protein and Cofilin accelerates actin filament depolymerization by two orders of magnitude

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Shashank Shekhar ◽  
Johnson Chung ◽  
Jane Kondev ◽  
Jeff Gelles ◽  
Bruce L. Goode
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
Binding Protein ◽  
Actin Dynamics ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Actin Networks ◽  
Binding Event

AbstractCellular actin networks can be rapidly disassembled and remodeled in a few seconds, yet in vitro actin filaments depolymerize slowly over minutes. The cellular mechanisms enabling actin to depolymerize this fast have so far remained obscure. Using microfluidics-assisted TIRF, we show that Cyclase-associated protein (CAP) and Cofilin synergize to processively depolymerize actin filament pointed ends at a rate 330-fold faster than spontaneous depolymerization. Single molecule imaging further reveals that hexameric CAP molecules interact with the pointed ends of Cofilin-decorated filaments for several seconds at a time, removing approximately 100 actin subunits per binding event. These findings establish a paradigm, in which a filament end-binding protein and a side-binding protein work in concert to control actin dynamics, and help explain how rapid actin network depolymerization is achieved in cells.

scholarly journals Local Actin Filament Geometry Dictates How Myosin Va Molecular Motor Teams Transport Liposomes Through 3D Actin Networks in Vitro

2019 ◽  
Vol 116 (3) ◽  
pp. 125a
Author(s):  
Sam Walcott ◽  
Andrew T. Lombardo ◽  
Kathleen M. Trybus ◽  
David M. Warshaw
Keyword(s):  
Actin Filament ◽  
Molecular Motor ◽  
Actin Networks ◽  
Myosin Va

scholarly journals Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Benjamin A Smith ◽  
Shae B Padrick ◽  
Lynda K Doolittle ◽  
Karen Daugherty-Clarke ◽  
Ivan R Corrêa ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Single Molecule Imaging ◽  
Network Growth ◽  
Cell Locomotion ◽  
Branch Formation ◽  
Membrane Bound

During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it.

scholarly journals Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

1997 ◽  
Vol 136 (6) ◽  
pp. 1307-1322 ◽  
Author(s):  
Marie-France Carlier ◽  
Valérie Laurent ◽  
Jérôme Santolini ◽  
Ronald Melki ◽  
Dominique Didry ◽  
...  
Keyword(s):  
Actin Filament ◽  
Atp Hydrolysis ◽  
Fold Increase ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Filament Dynamics ◽  
Relevant Effect

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

scholarly journals Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

2021 ◽  
Author(s):  
Clare Rogerson ◽  
Duncan J. Wotherspoon ◽  
Cristina Tommasi ◽  
Robert W. Button ◽  
Ryan F. L. O’Shaughnessy
Keyword(s):  
Actin Filament ◽  
Molecular Mechanisms ◽  
Molecular Motor ◽  
Nuclear Lamina ◽  
Nuclear Volume ◽  
Volume Reduction ◽  
Epidermal Keratinocytes ◽  
Lamin A ◽  
Myosin Iia

AbstractKeratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction.

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