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Rahman, M. A., Reuther, C., Lindberg, F. W., Mengoni, M., Salhotra, A., Heldt, G., . . . Månsson, A. (2019). Regeneration of Assembled, Molecular-Motor-Based Bionanodevices. Nano letters (Print), 19(10), 7155-7163
Open this publication in new window or tab >>Regeneration of Assembled, Molecular-Motor-Based Bionanodevices
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2019 (English)In: Nano letters (Print), ISSN 1530-6984, E-ISSN 1530-6992, Vol. 19, no 10, p. 7155-7163Article in journal (Refereed) Published
Abstract [en]

The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/microstructured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5% SDS or 0.05% Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/microstructured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results substantiate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2019
Keywords
Nano/microdevice, regeneration, protein desorption, molecular motor, proteinase K, detergent
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-89868 (URN)10.1021/acs.nanolett.9b02738 (DOI)000490353500058 ()31512480 (PubMedID)
Available from: 2019-10-31 Created: 2019-10-31 Last updated: 2019-10-31Bibliographically approved
Fischer, B., Meier, A., Dehne, A., Salhotra, A., Tran, T. A., Neumann, S., . . . Gentile, L. (2018). A complete workflow for the differentiation and the dissociation of hiPSC-derived cardiospheres. Stem Cell Research, 32, 65-72
Open this publication in new window or tab >>A complete workflow for the differentiation and the dissociation of hiPSC-derived cardiospheres
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2018 (English)In: Stem Cell Research, ISSN 1873-5061, E-ISSN 1876-7753, Vol. 32, p. 65-72Article in journal (Refereed) Published
Abstract [en]

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) are an invaluable tool for both basic and translational cardiovascular research. The potential that these cells hold for therapy, disease modeling and drug discovery is hampered by several bottlenecks that currently limit both the yield and the efficiency of cardiac induction. Here, we present a complete workflow for the production of ready-to-use hiPSC-CMs in a dynamic suspension bioreactor. This includes the efficient and highly reproducible differentiation of hiPSCs into cardiospheres, which display enhanced physiological maturation compared to static 3D induction in hanging drops, and a novel papain-based dissociation method that offers higher yield and viability than the broadly used dissociation reagents TrypLE and Accutase. Molecular and functional analyses of the cardiomyocytes reseeded after dissociation confirmed both the identity and the functionality of the cells, which can be used in down-stream applications, either as monolayers or spheroids.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
hiPSCs, Cardiomyocytes, Cardiac induction, 3D bioreactor, Papain dissociation
National Category
Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-78614 (URN)10.1016/j.scr.2018.08.015 (DOI)000447301200010 ()30218895 (PubMedID)2-s2.0-85053209652 (Scopus ID)
Available from: 2018-11-01 Created: 2018-11-01 Last updated: 2019-08-29Bibliographically approved
Rahman, M. A., Salhotra, A. & Månsson, A. (2018). Comparative analysis of widely used methods to remove nonfunctional myosin heads for the in vitro motility assay. Journal of Muscle Research and Cell Motility, 39(5-6), 175-187
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
Lindberg, F. W., Norrby, M., Rahman, M. A., Salhotra, A., Takatsuki, H., Jeppesen, S., . . . Månsson, A. (2018). Controlled Surface Silanization for Actin-Myosin and Biocompatibility of New Polymer Resists. Langmuir, 34(30), 8777-8784
Open this publication in new window or tab >>Controlled Surface Silanization for Actin-Myosin and Biocompatibility of New Polymer Resists
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2018 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 34, no 30, p. 8777-8784Article in journal (Refereed) Published
Abstract [en]

Molecular motor-based nanodevices require organized cytoskeletal filament guiding along motility-promoting tracks, confined by motility-inhibiting walls. One way to enhance motility quality on the tracks, particularly in terms of filament velocity but also the fraction of motile filaments, is to optimize the surface hydrophobicity. We have investigated the potential to achieve this for the actin myosin II motor system on trimethylchlorosilane (TMCS)-derivatized SiO2 surfaces to be used as channel floors in nanodevices. We have also investigated the ability to supress motility on two new polymer resists, TU7 (for nanoimprint lithography) and CSAR 62 (for electron beam and deep UV lithography), to be used as channel walls. We developed a chemical-vapor deposition tool for silanizing SiO2 surfaces in a controlled environment to achieve different surface hydrophobicities (measured by water contact angle). In contrast to previous work, we were able to fabricate a wide range of contact angles by varying the silanization time and chamber pressure using only one type of silane. This resulted in a significant improvement of the silanization procedure, producing a predictable contact angle on the surface and thereby predictable quality of the heavy meromyosin (HMM)-driven actin motility with regard to velocity. We observed a high degree of correlation between the filament sliding velocity and contact angle in the range 10-86 degrees, expanding the previously studied range. We found that the sliding velocity on TU7 surfaces was superior to that on CSAR 62 surfaces despite similar contact angles. In addition, we were able to suppress the motility on both TU7 and CSAR 62 by plasma oxygen treatment before silanization. These results are discussed in relation to previously proposed surface adsorption mechanisms of HMM and their relationship to the water contact angle. Additionally, the results are considered for the development of actin-myosin based nanodevices with superior performance with respect to actin-myosin functionality.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Biophysics Biochemistry and Molecular Biology
Research subject
Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-77391 (URN)10.1021/acs.langmuir.8b01415 (DOI)000440768400007 ()29969272 (PubMedID)2-s2.0-85049637573 (Scopus ID)
Available from: 2018-08-30 Created: 2018-08-30 Last updated: 2019-08-29Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-4835-0598

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