Analyzing kinesin motor domain translocation in cultured hippocampal neurons

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

Research output: Contribution to journalArticle

2 Citations (Scopus)

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)
JournalMethods in Cell Biology
DOIs
StateAccepted/In press - 2015

Fingerprint

Kinesin
Microtubules
Neurons
Post Translational Protein Processing
Neurites
Dendrites
Cytoskeleton
Population
Axons
Carrier Proteins
Mutation

Keywords

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

ASJC Scopus subject areas

  • Cell Biology

Cite this

Analyzing kinesin motor domain translocation in cultured hippocampal neurons. / Yang, Rui; Bentley, Marvin; Huang, Chung Fang; Banker, Gary.

In: Methods in Cell Biology, 2015.

Research output: Contribution to journalArticle

Yang, Rui ; Bentley, Marvin ; Huang, Chung Fang ; Banker, Gary. / Analyzing kinesin motor domain translocation in cultured hippocampal neurons. In: Methods in Cell Biology. 2015.
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