Reconstitution of microtubule-based motility using cell extracts

Methods Cell Biol. 2015;128:57-68. doi: 10.1016/bs.mcb.2015.02.002. Epub 2015 Apr 8.

Abstract

Long-range intracellular transport of organelles driven by kinesin and dynein motor proteins depends on additional cellular factors including adaptors and scaffolding proteins. While single-molecule studies of the motility of purified motor proteins have been a powerful approach, these assays are not fully representative of the complex interactions that occur in a cellular environment. To gain insights into the functioning of adaptor proteins that work in concert with motors proteins, motility assays in cell extracts have been developed. These assays are an attractive means to begin to dissect the roles of additional factors in motor-driven transport. Further, this system can be easily manipulated to study this process in different physiological environments. Here we describe in vitro reconstitution of motor-driven motility along microtubules in cell extracts, followed by considerations for data analysis and how these assays can be powerful in informing our understanding of basic cellular processes.

Keywords: Cell extract; Dynein; Kinesin; Microtubule; Motility; Motor proteins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Biological Transport / physiology*
  • COS Cells
  • Cell Extracts
  • Cell Line
  • Cell Movement / physiology*
  • Chlorocebus aethiops
  • Dyneins / metabolism*
  • Kinesin / metabolism*
  • Kymography / methods
  • Microtubule-Associated Proteins / metabolism
  • Microtubules / metabolism*
  • Organelles / metabolism

Substances

  • Cell Extracts
  • Microtubule-Associated Proteins
  • Dyneins
  • Kinesin