Influence of fluorescent tag on the motility properties of kinesin-1 in single-molecule assays

Biophysical Journal
Stephen R NorrisKristen J Verhey

Abstract

Molecular motors such as kinesin and dynein use the energy derived from ATP hydrolysis to walk processively along microtubule tracks and transport various cargoes inside the cell. Recent advancements in fluorescent protein (FP) research enable motors to be fluorescently labeled such that single molecules can be visualized inside cells in multiple colors. The performance of these fluorescent tags can vary depending on their spectral properties and a natural tendency for oligomerization. Here we present a survey of different fluorescent tags fused to kinesin-1 and studied by single-molecule motility assays of mammalian cell lysates. We tested eight different FP tags and found that seven of them display sufficient fluorescence intensity and photostability to visualize motility events. Although none of the FP tags interfere with the enzymatic properties of the motor, four of the tags (EGFP, monomeric EGFP, tagRFPt, and mApple) cause aberrantly long motor run lengths. This behavior is unlikely to be due to electrostatic interactions and is probably caused by tag-dependent oligomerization events that appear to be facilitated by fusion to the dimeric kinesin-1. We also compared the single-molecule performance of various fluorescent SN...Continue Reading

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Nov 5, 2014·The Journal of Cell Biology·Stephen R NorrisKristen J Verhey

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Citations

Apr 20, 2016·The Journal of Cell Biology·Emma G SturgillRyoma Ohi
Dec 9, 2016·Scientific Reports·Ajit TiwariAnne K Kenworthy
Jan 21, 2018·The Journal of Cell Biology·Yang YueKristen J Verhey
Aug 12, 2017·Chemistry : a European Journal·Carolina Sánchez-RicoMichael Sattler
Jul 26, 2018·American Journal of Physiology. Renal Physiology·Ankita Bachhawat JaykumarPablo A Ortiz
Jul 10, 2018·Nature Communications·Stephen R NorrisRyoma Ohi
Mar 20, 2019·Nature Methods·Talley J Lambert
Mar 22, 2019·The Journal of Cell Biology·Anna Payne-Tobin Jost, Jennifer C Waters

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