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Biophysical studies of the actin-myosin motor system and applications in nanoscience
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. (The Molecular motor and bionano-group)ORCID iD: 0000-0002-2797-2294
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 [en]
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: urn:nbn:se:lnu:diva-87498Libris ID: r297ppgzpnmxj2btISBN: 978-91-88898-82-1 (print)ISBN: 978-91-88898-83-8 (electronic)OAI: oai:DiVA.org:lnu-87498DiVA, id: diva2:1341912
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
List of papers
1. Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament
Open this publication in new window or tab >>Myosin-Induced Gliding Patterns at Varied [MgATP] Unveil a Dynamic Actin Filament
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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.

National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-58085 (URN)10.1016/j.bpj.2016.08.025 (DOI)000385471500013 ()27705769 (PubMedID)2-s2.0-85002888273 (Scopus ID)
Available from: 2016-11-11 Created: 2016-11-11 Last updated: 2019-08-12Bibliographically approved
2. Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin
Open this publication in new window or tab >>Blebbistatin Effects Expose Hidden Secrets in the Force-Generating Cycle of Actin and Myosin
2018 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 115, no 2, p. 386-397Article in journal (Refereed) Published
Abstract [en]

Cyclic interactions between myosin II motors and actin filaments driven by ATP turnover underlie muscle contraction and have key roles in the motility of nonmuscle cells. A remaining enigma in the understanding of this interaction is the relationship between the force-generating structural change and the release of the ATP-hydrolysis product, inorganic phosphate (Pi), from the active site of myosin. Here, we use the small molecular compound blebbistatin to probe otherwise hidden states and transitions in this process. Different hypotheses for the Pi release mechanism are tested by interpreting experimental results from in vitro motility assays and isolated muscle fibers in terms of mechanokinetic actomyosin models. The data fit with ideas that actomyosin force generation is preceded by Pi release, which in turn is preceded by two serial transitions after/coincident with cross-bridge attachment. Blebbistatin changes the rate limitation of the cycle from the first to the second of these transitions, uncovering functional roles of an otherwise short-lived pre-power stroke state that has been implicated by structural data.

Place, publisher, year, edition, pages
Rockville, MD: Biophysical Society, 2018
National Category
Biophysics
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-77397 (URN)10.1016/j.bpj.2018.05.037 (DOI)000438958800024 ()30021113 (PubMedID)2-s2.0-85048935566 (Scopus ID)
Conference
Biophysical-Society Thematic Meeting on Single-Cell Biophysics - Mearurement, Modulation, and Modeling, JUN, 2017, Natl Taiwan Univ, Acad Sinica, Inst Atom & Mol Sci, Taipei, TAIWAN
Available from: 2018-08-29 Created: 2018-08-29 Last updated: 2019-08-29Bibliographically approved
3. Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay
Open this publication in new window or tab >>Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay
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
Keywords
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:nbn:se:lnu:diva-82905 (URN)10.1007/s10974-019-09505-1 (DOI)000466555500004 ()30850933 (PubMedID)
Available from: 2019-05-23 Created: 2019-05-23 Last updated: 2019-08-12Bibliographically approved

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