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Molecular motors on lipid bilayers and silicon dioxide: different driving forces for adsorption
Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. (Bionanogroup)
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.
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2010 (English)In: Soft Matter, ISSN 1744-683X, E-ISSN 1744-6848, Vol. 6, no 14, p. 3211-3219Article in journal (Refereed) Published
Abstract [en]

Understanding how different types of interactions govern adsorption of the myosin motor fragment heavy meromyosin (HMM) onto different substrates is important in functional studies of actomyosin and for the development of motor powered lab-on-a-chip applications. In this study, we have combined in vitro motility assays and quartz crystal microbalance with dissipation (QCM-D) monitoring to investigate the underlying adsorption mechanisms of HMM onto supported lipid bilayers in comparison with pure and silanized SiO2. The QCM-D results, combined with data showing actin transportation by HMM adsorbed onto positively charged supported lipid bilayers, suggest reversible HMM surface adsorption via the negatively charged coiled-coil tail region. In contrast, the QCM-D data for HMM adsorption onto negatively charged lipids support a model according to which HMM adsorbs onto negatively charged surfaces largely via the positively charged actin binding regions. Adsorption studies at low (30-65 mM) and high (185-245 mM) ionic strengths onto piranha cleaned SiO2 surfaces (contact angle < 20 degrees) support this general model. However, unlike the situation for charged lipids, rinsing in high ionic strength solution caused only partial HMM desorption from SiO2, without restoration of actin propulsion by the remaining HMM molecules. This suggests that mechanisms other than electrostatic interactions are involved in the tethering of HMM heads to SiO2 surfaces. An expanded model for HMM adsorption is formulated on the basis of the data and the potential of the results for nanotechnological applications of actomyosin is discussed.

Place, publisher, year, edition, pages
2010. Vol. 6, no 14, p. 3211-3219
National Category
Natural Sciences
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-7222DOI: 10.1039/c0sm00019aScopus ID: 2-s2.0-77954610067OAI: oai:DiVA.org:lnu-7222DiVA, id: diva2:343519
Available from: 2010-08-13 Created: 2010-08-13 Last updated: 2017-12-12Bibliographically 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
Keywords
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
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Supervisors
Available from: 2013-04-26 Created: 2013-04-06 Last updated: 2017-02-16Bibliographically approved

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Albet-Torres, NuriaPersson, MalinBalaz, MartinaMånsson, Alf

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