Analyzing kinesin motor domain translocation in cultured hippocampal neurons

Rui Yang, Marvin Bentley, Chung Fang Huang, Gary Banker

Research output: Chapter in Book/Report/Conference proceedingConference contribution

4 Scopus citations

Abstract

Neuronal microtubules are subject to extensive posttranslational modifications and are bound by MAPs, tip-binding proteins, and other accessory proteins. All of these features, which are difficult to replicate in vitro, are likely to influence the translocation of kinesin motors. Here we describe assays for evaluating the translocation of a population of fluorescently labeled kinesin motor domains, based on their accumulation in regions of the cell enriched in microtubule plus ends. Neurons lend themselves to these experiments because of their microtubule organization. In axons, microtubules are oriented with their plus ends out; dendrites contain a mixed population of microtubules, but those near the. tips are also plus end out. The assays involve the expression of constitutively active kinesins that can walk processively, but that lack the autoinhibitory domain in the tail that normally prevents their binding to microtubules until they attach to vesicles. The degree to which such motor domains accumulate at neurite tips serves as a measure of the efficiency of their translocation. Although these assays cannot provide the kind of quantitative kinetic information obtained from in vitro assays, they offer a simple way to examine kinesin translocation in living neurons. They can be used to compare the translocation efficiency of different kinesin motors and to evaluate how mutations or posttranslational modifications within the motor domain influence kinesin translocation. Changes to motor domain accumulation in these assays can also serve as readout for changes in the microtubule cytoskeleton that affect kinesin translocation.

Original languageEnglish (US)
Title of host publicationThe Neuronal Cytoskeleton, Motor Proteins, and Organelle Trafficking in the Axon, 2016
EditorsK. Kevin Pfister
PublisherAcademic Press Inc.
Pages217-232
Number of pages16
ISBN (Print)9780128033449
DOIs
StatePublished - Jan 1 2016

Publication series

NameMethods in Cell Biology
Volume131
ISSN (Print)0091-679X

    Fingerprint

Keywords

  • Axon
  • Axonal transport
  • Dendrite
  • Hippocampal culture
  • Kinesin
  • Microtubule
  • Motor protein
  • Neuronal polarity

ASJC Scopus subject areas

  • Cell Biology

Cite this

Yang, R., Bentley, M., Huang, C. F., & Banker, G. (2016). Analyzing kinesin motor domain translocation in cultured hippocampal neurons. In K. K. Pfister (Ed.), The Neuronal Cytoskeleton, Motor Proteins, and Organelle Trafficking in the Axon, 2016 (pp. 217-232). (Methods in Cell Biology; Vol. 131). Academic Press Inc.. https://doi.org/10.1016/bs.mcb.2015.06.021