In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.

  title={In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.},
  author={Yin-wei Kuo and Jonathon Howard},
  journal={Methods in molecular biology},
The dynamic architecture of the microtubule cytoskeleton is crucial for cell division, motility and morphogenesis. The dynamic properties of microtubules-growth, shrinkage, nucleation, and severing-are regulated by an arsenal of microtubule-associated proteins (MAPs). The activities of many of these MAPs have been reconstituted in vitro using microscope assays. As an alternative to fluorescence microscopy, interference-reflection microscopy (IRM) has been introduced as an easy-to-use, wide… 

Counting fluorescently labeled proteins in tissues in the spinning–disk microscope using single–molecule calibrations

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The force required to remove tubulin from the microtubule lattice by pulling on its α-tubulin C-terminal tail

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Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy.

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Label‐free high‐speed wide‐field imaging of single microtubules using interference reflection microscopy

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Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules.

It is found that images of similar quality to TIRF can be obtained using interference reflection microscopy (IRM), which suggests that IRM might be a general technique for visualizing the dynamics of large biomolecules and oligomers in vitro.

Dynamics of microtubules visualized by darkfield microscopy: treadmilling and dynamic instability.

Evidence shows that MAPs suppress the dynamic instability of microtubules, which means that treadmilling can take place in the steady state only after microtubule-associated proteins have been stabilized by MAPs.

Reconstitution of a microtubule plus-end tracking system in vitro

In vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2 is reported and it is shown that the EB1 homologue Mal3 has an enhanced affinity for growing microtubules end structures as opposed to the microtubule lattice, which allows it to track growingMicrotubule ends autonomously by an end recognition mechanism.

In vitro microtubule severing assays.

This chapter presents a detailed protocol for a light microscopy based in vitro microtubule severing assay that was instrumental in the identification and characterization of these enzymes.

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies

The data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubule can be significantly different.

Direct observation of single kinesin molecules moving along microtubules

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