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Updated: Jan 19, 2026

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy
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A microtubule crosslinking protocol for integrative structural modeling activities.

Atefeh Rafiei1, David C Schriemer2

  • 1Department of Chemistry, University of Calgary, Calgary, Alberta, Canada.

Analytical Biochemistry
|September 10, 2019
PubMed
Summary
This summary is machine-generated.

This study enhances crosslinking mass spectrometry to map microtubule (MT) structures, revealing novel interactions and improving the understanding of MT regulation for cellular processes like division and transport.

Keywords:
CrosslinkingMass spectrometryMicrotubulePhoto-crosslinkingStructural biology

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Structural Biology

Background:

  • Microtubules (MTs) are crucial for eukaryotic cell structure and function, including intracellular transport and cell division.
  • Understanding MT regulation necessitates detailed structural analysis of MT lattice and protein interactions, which is challenging for existing methods.
  • Crosslinking mass spectrometry (XL-MS) offers potential for structural analysis but has shown limited representation of MTs.

Purpose of the Study:

  • To investigate the underrepresentation of microtubule lattice in crosslinking data.
  • To develop an enhanced method for mapping MT structural features using optimized reagents and fluorescence detection.
  • To improve the structural characterization of MT-protein interactions.

Main Methods:

  • Optimization of reagents for crosslinking mass spectrometry (XL-MS) applied to microtubules.
  • Integration of fluorescence detection to monitor MT structural integrity during experiments.
  • Application of stringent identification criteria for crosslink analysis.

Main Results:

  • Identified 91 unique crosslinks, with 78 uniquely mapping to 7 distinct MT lattice structural features.
  • Detected 4 crosslinks specifically for the lattice-A protofilament organization, highlighting the MT seam.
  • Demonstrated an enhanced ability to map structural features of the microtubule lattice.

Conclusions:

  • The developed methodology significantly improves the representation and structural mapping of microtubules in XL-MS datasets.
  • The findings provide new insights into the microtubule lattice structure, including the lattice-A protofilament organization.
  • This enhanced approach is broadly applicable to integrative structural studies of microtubule-protein interactions.