lnu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0002-2797-2294
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0003-4835-0598
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0002-5889-7792
2018 (English)In: Journal of Muscle Research and Cell Motility, ISSN 0142-4319, E-ISSN 1573-2657, Vol. 39, no 5-6, p. 175-187Article in journal (Refereed) Published
Abstract [en]

The in vitro motility assay allows studies of muscle contraction through observation of actin filament propulsion by surface-adsorbed myosin motors or motor fragments isolated from muscle. A possible problem is that motility may be compromised by nonfunctional, "dead", motors, obtained in the isolation process. Here we investigate the effects on motile function of two approaches designed to eliminate the effects of these dead motors. We first tested the removal of heavy meromyosin (HMM) molecules with ATP-insensitive "dead" heads by pelleting them with actin filaments, using ultracentrifugation in the presence of 1 mM MgATP ("affinity purification"). Alternatively we incubated motility assay flow cells, after HMM surface adsorption, with non-fluorescent "blocking actin" (1 µM) to block the dead heads. Both affinity purification and use of blocking actin increased the fraction of motile filaments compared to control conditions. However, affinity purification significantly reduced the actin sliding speed in five out of seven experiments on silanized surfaces and in one out of four experiments on nitrocellulose surfaces. Similar effects on velocity were not observed with the use of blocking actin. However, a reduced speed was also seen (without affinity purification) if HMM or myosin subfragment 1 was mixed with 1 mM MgATP before and during surface adsorption. We conclude that affinity purification can produce unexpected effects that may complicate the interpretation of in vitro motility assays and other experiments with surface adsorbed HMM, e.g. single molecule mechanics experiments. The presence of MgATP during incubation with myosin motor fragments is critical for the complicating effects.

Place, publisher, year, edition, pages
Springer, 2018. Vol. 39, no 5-6, p. 175-187
Keywords [en]
Affinity purification, Blocking actin, Cross-bridge cycle, In vitro motility assay, Molecular motor, Myosin
National Category
Cell Biology
Research subject
Natural Science, Cell and Organism Biology
Identifiers
URN: urn:nbn:se:lnu:diva-82905DOI: 10.1007/s10974-019-09505-1ISI: 000466555500004PubMedID: 30850933OAI: oai:DiVA.org:lnu-82905DiVA, id: diva2:1317775
Available from: 2019-05-23 Created: 2019-05-23 Last updated: 2019-08-12Bibliographically approved
In thesis
1. Biophysical studies of the actin-myosin motor system and applications in nanoscience
Open this publication in new window or tab >>Biophysical studies of the actin-myosin motor system and applications in nanoscience
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The actin-myosin motor system plays important roles in cellular processes. In addition, actin and myosin have been used for developments towards nanotechnological applications in recent years. Therefore, fundamental biophysical studies of actin and myosin and the actomyosin force generating cycle are important both in biology and for nanotechnology where the latter applications require methodological insights for optimization. This dual goal is central in the present thesis with major focus on factors that control the function (e.g. velocity) and the effectiveness of transport of filaments (e.g. filament flexural rigidity) through nanoscale channels with supplementation of methodological insights. The thesis thus provides evidence that actin is a dynamic filament whose flexural rigidity is different at different MgATP concentrations as well as in the presence or absence of myosin binding. Furthermore, probing the myosin ATPase cycle with the myosin inhibitor blebbistatin revealed that velocity is easily modified by this drug. Our detailed studies also suggest that actin-myosin force generation is preceded by Pi release and that blebbistatin changes the rate limiting transition in the cycle from the attachment step to a step between weakly attached states. The studies of actin dynamics and of the actomyosin force generating cycle were largely performed using in vitro motility assay (IVMA) where surface adsorbed myosin motor or its proteolytic fragments propel fluorescently labeled actin filaments. The IVMA is often taken as the basis for developments towards different nanotechnological applications. However, in the IVMA, actomyosin motility is often negatively affected by the presence of “dead”, non-functional myosin heads. Therefore, in this thesis, two popular methods, that are often used to remove dead myosin heads, are analyzed and compared. It was found that after affinity purification, the in vitro actin sliding velocity is reduced compared to the control conditions, something that was not seen with the use of blocking actin. Therefore, the effects of the affinity purification method should be considered when interpreting IVMA data. This is important while using IVMA both for fundamental studies and for nanotechnological applications. Another issue in the use of IVMAs in nanotechnological applications is the requirement for expensive and time-consuming fabrication of nanostructured devices. We therefore developed a suitable method for regenerating molecular motor based bionanodevices without a need to disassemble the flow cell. Evidence is presented that, use of proteinase K with a suitable detergent (SDS or Triton X100) lead to successful regeneration of devices where both actin-myosin and microtubule-kinesin motility are used. Lastly, this thesis presents efforts to immobilize engineered light sensitive myosin motors on trimethyl chlorosilane (TMCS) derivatized surfaces for light operated switching of myosin motor in order to control actin movement in nano-networks. This has potential for developing a programmable junction in a biocomputation network. In brief, the described results have contributed both to the fundamental understanding of actin and myosin properties and the actomyosin interaction mechanisms. They have also given technical insights for molecular motor based bionanotechnology.

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2019. p. 121
Series
Linnaeus University Dissertations ; 359
Keywords
myosin II, actin, actomyosin force generating cycle, blebbistatin, in vitro motility assay, actin affinity purification, blocking actin, bionanodevices, proteinase k, SDS, triton X100, surface recycling, engineered myosin motor, programmable gate, biocomputation.
National Category
Microbiology in the medical area Biophysics Nano Technology
Research subject
Natural Science, Biomedical Sciences
Identifiers
urn:nbn:se:lnu:diva-87498 (URN)978-91-88898-82-1 (ISBN)978-91-88898-83-8 (ISBN)
Public defence
2019-09-05, Falken C305, Nygatan 18B, Kalmar, 09:30 (English)
Opponent
Supervisors
Available from: 2019-08-19 Created: 2019-08-12 Last updated: 2019-09-16Bibliographically approved

Open Access in DiVA

No full text in DiVA

Other links

Publisher's full textPubMed

Authority records BETA

Rahman, Mohammad A.Salhotra, AseemMånsson, Alf

Search in DiVA

By author/editor
Rahman, Mohammad A.Salhotra, AseemMånsson, Alf
By organisation
Department of Chemistry and Biomedical Sciences
In the same journal
Journal of Muscle Research and Cell Motility
Cell Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
pubmed
urn-nbn

Altmetric score

doi
pubmed
urn-nbn
Total: 10 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf