Prolongation of good myosin motor function over time using in vitro motility assay
2022 (English)Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE credits
Student thesis
Abstract [en]
In vitro motility assays (IVMAs) are test systems that allow for the investigation of actin-myosin interactions using isolated proteins. Myosin motor proteins are attached to a glass slide after which fluorescently labeled actin filaments are added. After further adding assay solution with adenosine triphosphate (ATP) the myosin motor proteins will propel the actin filaments, something that is observed using a fluorescence microscope. IVMAs are important for studying the function of different classes of myosin. The IVMAs may also be used in nanodevices e.g. in biosensing and biocomputation. Particularly, in the latter case, it is often necessary for the biological system to be functioning for extended time periods. It is therefore important to maintain good motor function over time in the IVMA.
The purpose of this study was to improve myosin motor function for extended time periods. Firstly, assay solution exchanges every 30 minutes were performed to try and maintain myosin motor function. Secondly, we tested the effect of different reducing substances on IVMAs.
By exchanging the assay solution (along with addition of new actin filaments) every 30 minutes, motility was observed for a period of 8 hours instead of 1 h with no such exchange. Trolox (dissolved in v/v 3.2% methanol), 4-Nitrobenzyl alcohol (NBA; dissolved in v/v 1% DMSO), and cyclooctatetraene (COT; dissolved in v/v 1% DMSO) were the reducing substances tested along with the relevant solvents. The most severe effects on function were observed with DMSO (v/v 1%) and trolox. DMSO halved the sliding velocity compared to control conditions but did not cause any further decrease over time. Trolox produced a decrease in sliding velocity initially and further decrease over time. The fraction of motile filaments was similar to that under control conditions initially but after 1-hour in trolox the fraction was reduced. Similar results were found for methanol, but the decrease was not as severe as with trolox regarding the fraction of motile filaments and sliding velocity. Neither COT nor NBA altered the fraction of motile filaments. However, NBA led to a slight decrease in sliding velocity initially, an effect that remained static over time.
Place, publisher, year, edition, pages
2022. , p. 27
Keywords [en]
IVMA, myosin, actin, molecular motor, longevity
National Category
Physiology Medical and Health Sciences Medical Biotechnology Biochemistry and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:lnu:diva-115132OAI: oai:DiVA.org:lnu-115132DiVA, id: diva2:1680275
Educational program
Biology Programme, 180 credits
Supervisors
Examiners
2022-07-042022-07-042022-07-04Bibliographically approved