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An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast

Yili Zhu1, Weimin Tan1, Wei-Lih Lee1

  • 1Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA.

Bio-Protocol
|February 9, 2019
PubMed

Insights

This study introduces a microscopy assay to visualize microtubule-associated protein (MAP) binding to microtubules, overcoming limitations of traditional co-sedimentation methods for MAP-microtubule interactions.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Microscopy

Background:

  • Microtubule-associated proteins (MAPs) are crucial for microtubule dynamics and function.
  • Traditional co-sedimentation assays struggle to accurately assess MAP-microtubule interactions, especially for MAPs forming large oligomers.
  • Direct visualization methods are needed to overcome the limitations of pelleting assays.

Purpose of the Study:

  • To develop and describe a simple microscopy-based protocol for assessing MAP-microtubule interactions.
  • To provide an alternative to the conventional co-sedimentation assay for studying MAP binding.
  • To enable direct visualization and quantification of microtubule bundling.

Main Methods:

  • Utilizing a microscopy-based assay with fluorescently-labeled MAPs for direct visualization of microtubule binding.
  • Employing centrifugation and SDS-PAGE analysis in traditional co-sedimentation assays.
  • Quantifying microtubule bundling by measuring the thickness of microtubule structures via microscopy.

Main Results:

  • The microscopy assay allows direct visualization of MAP-microtubule interactions, circumventing issues with MAP self-pelleting.
  • This method effectively assesses microtubule binding by fluorescently labeled MAPs.
  • Microtubule bundling can be quantified by measuring microtubule structure thickness.

Conclusions:

  • The described microscopy assay offers a more advantageous and accurate method for studying MAP-microtubule interactions compared to traditional co-sedimentation assays.
  • Direct visualization provides a robust solution for assessing MAP binding, particularly for oligomeric MAPs.
  • The protocol enables enhanced analysis of microtubule organization and dynamics.
Keywords:
Imaging chamberMicroscopy-based assayMicrotubuleMicrotubule bindingMicrotubule crosslinkingMicrotubule-associated proteinYeast

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