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Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Karolinska Institutet ; McGill Univ, Canada.ORCID iD: 0000-0003-2819-3046
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. Delhi Technol Univ, India.
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2016 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 111, no 7, p. 1465-1477Article in journal (Refereed) Published
Abstract [en]

Actin filaments have key roles in cell motility but are generally claimed to be passive interaction partners in actin-myosin -based motion generation. Here, we present evidence against this static view based on an altered myosin-induced actin filament gliding pattern in an in vitro motility assay at varied [MgATP]. The statistics that characterize the degree of meandering of the actin filament paths suggest that for [MgATP] >= 0.25 mM, the flexural rigidity of heavy meromyosin (HMM)-propelled actin filaments is similar (without phalloidin) or slightly lower (with phalloidin) than that of HMM-free filaments observed in solution without surface tethering. When [MgATP] was reduced to <= 0.1 mM, the actin filament paths in the in vitro motility assay became appreciably more winding in both the presence and absence of phalloidin. This effect of lowered [MgATP] was qualitatively different from that seen when HMM was mixed with ATP-insensitive, N-ethylmaleimide-treated HMM (NEM-HMM; 25-30%). In particular, the addition of NEM-HMM increased a non-Gaussian tail in the path curvature distribution as well as the number of events in which different parts of an actin filament followed different paths. These effects were the opposite of those observed with reduced [MgATP]. Theoretical modeling suggests a 30-40% lowered flexural rigidity of the actin filaments at [MgATP] <= 0.1 mM and local bending of the filament front upon each myosin head attachment. Overall, the results fit with appreciable structural changes in the actin filament during actomyosin-based motion generation, and modulation of the actin filament mechanical properties by the dominating chemomechanical actomyosin state.

Place, publisher, year, edition, pages
2016. Vol. 111, no 7, p. 1465-1477
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
URN: urn:nbn:se:lnu:diva-58085DOI: 10.1016/j.bpj.2016.08.025ISI: 000385471500013PubMedID: 27705769Scopus ID: 2-s2.0-85002888273OAI: oai:DiVA.org:lnu-58085DiVA, id: diva2:1046108
Available from: 2016-11-11 Created: 2016-11-11 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)
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Supervisors
Available from: 2019-08-19 Created: 2019-08-12 Last updated: 2019-09-16Bibliographically approved

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Bengtsson, ElinaPersson, MalinRahman, Mohammad A.Kumar, SarojTakatsuki, HideyoMånsson, Alf

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