Hirst WG et al. (2020), In Vitro Reconstitution and Imaging of Microtub...

XB-ART-59071

STAR Protoc 2020 Dec 18;13:100177. doi: 10.1016/j.xpro.2020.100177.

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In Vitro Reconstitution and Imaging of Microtubule Dynamics by Fluorescence and Label-free Microscopy.

Hirst WG , Kiefer C , Abdosamadi MK , Schäffer E , Reber S .

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Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy. For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).

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References [+] :

Aitken, An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. 2008, Pubmed


	Figure 1. Overall Schematic WorkflowFor each stage (dotted box), steps are annotated.
	Figure 2. Schematics and Representative Images of In Vitro Reconstituted Microtubules Imaged by TIRFM and IRM(A) Schematic of a TIRFM microtubule assembly assay using biotinylated GMPCPP-stabilized seeds (red) attached to the coverslip via neutravidin (blue) as nucleation templates for dynamic microtubules (green).(B) Dynamic Cy3-labeled Xenopus microtubules (green) nucleated from GMPCPP-stabilized Cy5-labeled tubulin seeds (red) imaged by TIRFM.(C) Schematic of dynamic microtubules (green) nucleated from rhodamine-labeled GMPCPP-stabilized tubulin seeds (red) immobilized via anti-rhodamine antibodies and imaged by IRM.(D) Xenopus microtubules nucleated from stabilized seeds as in (B) and imaged by IRM. Scale bars, 10 μm.
	Figure 3. Hydrophobicity Test of Cleaned and Silanized Glass SurfacesSchematic showing the behavior of a 10 μL water droplet on a glass surface with increasing hydrophobicity (left to right). The contact angle θ, which increases with hydrophobicity, is the angle between the glass surface and the tangent to the surface of the droplet where it meets the glass.
	Figure 4. Schematic of a Flow Chamber
	Figure 5. Schematic of the IRM Light PathThe light path and optical components necessary for IRM.
	Figure 6. Representative Kymographs of Xenopus Microtubules Imaged by TIRFM and IRM to Analyze Parameters of Dynamic Instability(A and B) Kymograph of a Xenopus microtubule produced from a series of images acquired by (A) TIRFM and (B) IRM.(C) Example of a single microtubule showing growth, shrinkage, catastrophes, and rescues.(D) How to calculate the four dynamics parameters of dynamic instability from kymograph data.