Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Engineered kinesin motor proteins amenable to small molecule inhibition

Martin F. Engelke, Michael Winding, Yang Yue, Shankar Shastry, Federico Teloni, Sanjay Reddy, T. Lynne Blasius, Pushpanjali Soppina, William O. Hancock, Vladimir I. Gelfand, Kristen J. Verhey
doi: https://doi.org/10.1101/042663
Martin F. Engelke
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael Winding
2Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yang Yue
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shankar Shastry
3Department of Biomedical Engineering, Penn State University, University Park, PA 16802
4Current Address: Department of Chemistry and Biochemistry, UC Santa Cruz, Santa Cruz, CA 95064
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Federico Teloni
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sanjay Reddy
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
T. Lynne Blasius
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Pushpanjali Soppina
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
William O. Hancock
3Department of Biomedical Engineering, Penn State University, University Park, PA 16802
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Vladimir I. Gelfand
2Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kristen J. Verhey
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

ABSTRACT

The human genome encodes 45 kinesins that drive cell division, cell motility, intracellular trafficking, and ciliary function. Determining the cellular function of each kinesin would be greatly facilitated by specific small molecule inhibitors, but screens have yielded inhibitors that are specific to only a small number of kinesins, likely due to the high conservation of the kinesin motor domain across the superfamily. Here we present a chemical-genetic approach to engineer kinesin motors that retain microtubule-dependent motility in the absence of inhibitor yet can be efficiently inhibited by small, cell-permeable molecules. Using kinesin-1 as a prototype, we tested two independent strategies to design inhibitable motors. First, we inserted the six amino acid tetracysteine tag into surface loops of the motor domain such that binding of biarsenic dyes allosterically inhibits processive motility. Second, we fused DmrB dimerization domains to the motor heads such that addition of B/B homodimerizer cross-links the two motor domains and inhibits motor stepping. We show, using cellular assays that the engineered kinesin-1 motors are able to transport artificial and natural kinesin-1 cargoes, but are efficiently inhibited by the addition of the relevant small molecule. Single-molecule imaging in vitro revealed that inhibitor addition reduces the number of processively moving motors on the microtubule, with minor effects on motor run length and velocity. It is likely that these inhibition strategies can be successfully applied to other members of the kinesin superfamily due to the high conservation of the kinesin motor domain. The described engineered motors will be of great utility to dynamically and specifically study kinesin function in cells and animals.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted March 07, 2016.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Engineered kinesin motor proteins amenable to small molecule inhibition
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Engineered kinesin motor proteins amenable to small molecule inhibition
Martin F. Engelke, Michael Winding, Yang Yue, Shankar Shastry, Federico Teloni, Sanjay Reddy, T. Lynne Blasius, Pushpanjali Soppina, William O. Hancock, Vladimir I. Gelfand, Kristen J. Verhey
bioRxiv 042663; doi: https://doi.org/10.1101/042663
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
Engineered kinesin motor proteins amenable to small molecule inhibition
Martin F. Engelke, Michael Winding, Yang Yue, Shankar Shastry, Federico Teloni, Sanjay Reddy, T. Lynne Blasius, Pushpanjali Soppina, William O. Hancock, Vladimir I. Gelfand, Kristen J. Verhey
bioRxiv 042663; doi: https://doi.org/10.1101/042663

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Cell Biology
Subject Areas
All Articles
  • Animal Behavior and Cognition (4079)
  • Biochemistry (8750)
  • Bioengineering (6467)
  • Bioinformatics (23314)
  • Biophysics (11719)
  • Cancer Biology (9135)
  • Cell Biology (13227)
  • Clinical Trials (138)
  • Developmental Biology (7404)
  • Ecology (11360)
  • Epidemiology (2066)
  • Evolutionary Biology (15078)
  • Genetics (10390)
  • Genomics (14001)
  • Immunology (9109)
  • Microbiology (22025)
  • Molecular Biology (8773)
  • Neuroscience (47317)
  • Paleontology (350)
  • Pathology (1419)
  • Pharmacology and Toxicology (2480)
  • Physiology (3701)
  • Plant Biology (8044)
  • Scientific Communication and Education (1427)
  • Synthetic Biology (2206)
  • Systems Biology (6009)
  • Zoology (1247)