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Heavy meromyosin molecules extending more than 50 nm above adsorbing electronegative surfaces.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.ORCID iD: 0000-0003-2819-3046
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
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2010 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 12, 9927-9936 p.Article in journal (Refereed) Published
Abstract [en]

In the in vitro motility assay, actin filaments are propelled by surface-adsorbed myosin motors, or rather, myosin motor fragments such as heavy meromyosin (HMM). Recently, efforts have been made to develop actomyosin powered nanodevices on the basis of this assay but such developments are hampered by limited understanding of the HMM adsorption geometry. Therefore, we here investigate the HMM adsorption geometries on trimethylchlorosilane- [TMCS-] derivatized hydrophobic surfaces and on hydrophilic negatively charged surfaces (SiO(2)). The TMCS surface is of great relevance in fundamental studies of actomyosin and both surface substrates are important for the development of motor powered nanodevices. Whereas both the TMCS and SiO(2) surfaces were nearly saturated with HMM (incubation at 120 microg mL(-1)) there was little actin binding on SiO(2) in the absence of ATP and no filament sliding in the presence of ATP. This contrasts with excellent actin-binding and motility on TMCS. Quartz crystal microbalance with dissipation (QCM-D) studies demonstrate a HMM layer with substantial protein mass up to 40 nm above the TMCS surface, considerably more than observed for myosin subfragment 1 (S1; 6 nm). Together with the excellent actin transportation on TMCS, this strongly suggests that HMM adsorbs to TMCS mainly via its most C-terminal tail part. Consistent with this idea, fluorescence interference contrast (FLIC) microscopy showed that actin filaments are held by HMM 38 +/- 2 nm above the TMCS-surface with the catalytic site, on average, 20-30 nm above the surface. Viewed in a context with FLIC, QCM-D and TIRF results, the lack of actin motility and the limited actin binding on SiO(2) shows that HMM adsorbs largely via the actin-binding region on this surface with the C-terminal coiled-coil tails extending >50 nm into solution. The results and new insights from this study are of value, not only for the development of motor powered nanodevices but also for the interpretation of fundamental biophysical studies of actomyosin function and for the understanding of surface-protein interactions in general.

Place, publisher, year, edition, pages
2010. Vol. 26, no 12, 9927-9936 p.
National Category
Medical and Health Sciences
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-6446DOI: 10.1021/la100395aPubMedID: 20337414OAI: oai:DiVA.org:lnu-6446DiVA: diva2:326306
Available from: 2010-06-22 Created: 2010-06-22 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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