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Transportation of Nanoscale Cargoes by Myosin Propelled Actin Filaments
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0003-2819-3046
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Linnaeus University, Faculty of Health and Life Sciences, Department of Biology and Environmental Science.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0003-3841-4826
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 2, e55931Article in journal (Refereed) Published
Abstract [en]

Myosin II propelled actin filaments move ten times faster than kinesin driven microtubules and are thus attractive candidates as cargo-transporting shuttles in motor driven lab-on-a-chip devices. In addition, actomyosin-based transportation of nanoparticles is useful in various fundamental studies. However, it is poorly understood how actomyosin function is affected by different number of nanoscale cargoes, by cargo size, and by the mode of cargo-attachment to the actin filament. This is studied here using biotin/fluorophores, streptavidin, streptavidin-coated quantum dots, and liposomes as model cargoes attached to monomers along the actin filaments ("side-attached") or to the trailing filament end via the plus end capping protein CapZ. Long-distance transportation (> 100 mu m) could be seen for all cargoes independently of attachment mode but the fraction of motile filaments decreased with increasing number of side-attached cargoes, a reduction that occurred within a range of 10-50 streptavidin molecules, 1-10 quantum dots or with just 1 liposome. However, as observed by monitoring these motile filaments with the attached cargo, the velocity was little affected. This also applied for end-attached cargoes where the attachment was mediated by CapZ. The results with side-attached cargoes argue against certain models for chemomechanical energy transduction in actomyosin and give important insights of relevance for effective exploitation of actomyosin-based cargo-transportation in molecular diagnostics and other nanotechnological applications. The attachment of quantum dots via CapZ, without appreciable modulation of actomyosin function, is useful in fundamental studies as exemplified here by tracking with nanometer accuracy.

Place, publisher, year, edition, pages
2013. Vol. 8, no 2, e55931
National Category
Biochemistry and Molecular Biology
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-24860DOI: 10.1371/journal.pone.0055931ISI: 000315186000012OAI: oai:DiVA.org:lnu-24860DiVA: diva2:612554
Available from: 2013-03-22 Created: 2013-03-22 Last updated: 2017-02-16Bibliographically approved
In thesis
1. Characterization and optimization of the in vitro motility assay for fundamental studies of myosin II
Open this publication in new window or tab >>Characterization and optimization of the in vitro motility assay for fundamental studies of myosin II
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Myosin II is the molecular motor responsible for muscle contraction. It transforms the chemical energy in ATP into mechanical work while interacting with actin filaments in so called cross-bridge cycles. Myosin II or its proteolytic fragments e.g., heavy meromyosin (HMM) can be adsorbed to moderately hydrophobic surfaces in vitro, while maintaining their ability to translocate actin filaments. This enables observation of myosin-induced actin filament sliding in a microscope. This “in vitro motility assay” (IVMA) is readily used in fundamental studies of actomyosin, including studies of muscle contraction. The degree of correlation of the myosin II function in the IVMA with its function in muscle depends on how the myosin molecules are arranged on the surface. Therefore a multi-technique approach, including total internal reflection spectroscopy, fluorescence interference contrast microscopy and quartz crystal microbalance with dissipation, was applied to characterize the HMM surface configurations. Several configurations with varying distributions were identified depending on the surface property. The most favorable HMM configurations for actin binding were observed on moderately hydrophobic surfaces.

 

The effects on actomyosin function of different cargo sizes and amount of cargo loaded on an actin filament were also investigated. No difference in sliding velocities could be observed, independent of cargo size indicating that diffusional processive runs of myosin II along an actin filament are not crucial for actomyosin function in muscle. Furthermore, a tool for accurate velocity measurements appropriate for IVMAs at low [MgATP] was developed by utilizing the actin filament capping protein CapZ. These improvements allowed an investigation of the [MgATP]-velocity relationship to study possible processivity in fast skeletal muscle myosin II.  It is shown that the [MgATP]–velocity relationship is well described by a Michaelis-Menten hyperbola.  In addition, statistical cross-bridge modeling showed that the experimental results are in good agreement with recent findings of actomyosin cross-bridge properties, e.g., non-linear cross-bridge elasticity. However, no effect of inter-head cooperativity could be observed.

 

In conclusion, the described results have contributed to in-depth understanding of the actomyosin cross-bridge cycle in muscle contraction.

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2013
Series
Linnaeus University Dissertations, 134/2013
Keyword
Myosin II, skeletal muscle, actomyosin, in vitro motility assay, protein adsorption, cargo transportation, CapZ, cross-bridge cycle, inter-head cooperativity, processivity
National Category
Biophysics
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-25241 (URN)978-91-87427-26-8 (ISBN)
Public defence
2013-05-17, N2007, Smålandsgatan 26, Kalmar, 09:00
Opponent
Supervisors
Available from: 2013-04-26 Created: 2013-04-06 Last updated: 2017-02-16Bibliographically approved

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Persson, MalinGullberg, MariaTolf, ConnyLindberg, A. MichaelMånsson, Alf
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