Chapter 9 - High-Resolution Imaging of Microtubules and Cytoskeleton Structures by Atomic Force Microscopy

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Abstract

Atomic force microscopy (AFM), which combines a nanometer-scale resolution and a unique capacity to image biomolecular interactions in liquid environment, is a promising tool for the investigation of biological samples. In contrast with nucleic acids and nucleoprotein complexes, for which AFM is now of common use and participates in the recent advances in the knowledge of DNA-related biomolecular processes, AFM investigations of cytoskeleton structures and especially microtubules remain rare. The most critical step to observe biomolecules using AFM is the spreading of the biological material on a flat surface. This issue is now better documented concerning DNA but a lot remains to be done concerning microtubules. This is a prerequisite to further document this issue for a proper and large use of AFM to study cytoskeleton structures. We present here an overview of the various procedures previously used to spread microtubules on a flat surface and advance an easy-to-use and efficient experimental protocol for microtubule imaging by AFM in air. We show application of this protocol to observe intermediate structures of microtubule assembly without using any stabilizing agent and the observation of more complex systems like proteins or messenger ribonucleoprotein particles in interaction with microtubules.

Section snippets

Introduction to AFM Imaging of Biomolecules

The atomic force microscope is a member of the family of scanning probe microscopes which has grown steadily since the invention of the scanning tunneling microscope by Binning and Rohrer (Binnig et al., 1986). In comparison with other techniques like electron microscopy, atomic force microscopy (AFM) presents unique advantage for the observation of biomolecules. AFM allows a three-dimensional imaging with molecular resolution and, in addition, experiments can be realized under aqueous

Rationale

In a first part, we briefly remind the principle of AFM and the various operating modes used to image biomolecules in air or in liquid. The second part is devoted to the variety of substrates and surface treatments which can be used to image microtubules. Among the substrates, mica, the most popular one for biomolecules, appears to be also the most suitable for microtubule imaging. We also detail the buffer composition to be used to spread microtubules on mica. Indeed, the adsorption of

Principle of AFM

AFM uses as probe an extremely sharp tip (on the order of a few nanometer) to scan a sample surface. The tip is mounted at the very end of a flexible cantilever, the position of which, while scanning over the surface sample, can be recorded as an image. The scanning of the AFM tip over the surface requires its fine control using a piezoelectric ceramic that expands or contracts in the presence of a voltage gradient, leading to a high-precision three-dimensional positioning. The interaction

Material for AFM Imaging

Imaging was performed in Tapping Mode™ with a multimode™ AFM (Veeco, Santa Barbara, CA) operating with a nanoscope IIIa™ controller. We used Olympus (Hambourg, Germany) silicon cantilever AC160TS with nominal spring constants between 36 and 75 N/m and resonant frequency of about 300 kHz. All images were collected at a scan frequency of 1.5 Hz and a resolution of 512 × 512 pixels. A first- or second-order polynomial function was used to remove the background slope.

Material and Methods for Microtubule Assembly

Tubulin is purified from sheep brain

Control of the Tubulin Purity

AFM appears as a powerful quality control tool for tubulin purification and polymerization. Indeed, AFM allows one to image a microtubule sample without using a large quantity of biological material (10 µg of tubulin is largely enough). On a large-scale image (15 × 15 µm²), the presence of small or large aggregates of tubulin can be easily detected, reflecting the purity of the sample.

In addition, tubulin polymerization in vitro is often monitored by light scattering measurements. The scattered

AFM Imaging in Air of Tubulin or Microtubule:Protein Interactions

Free tubulin and microtubules are known to interact with numerous proteins like microtubule-associated proteins or molecular motor but they are also the target of many drugs which act as microtubule-stabilizing or microtubule-destabilizing agents (taxol, vinblastine, colchicine, etc.). Many experimental approaches are used to study such interactions in vitro but high-resolution images by AFM may provide very useful information on both the localization of the partner along the microtubule wall

Conclusion

AFM is a powerful tool to study in vitro-isolated biomolecules or microtubules in interaction with partners. It allows microtubule visualization at the single microtubule level and needs only few micrograms of tubulin per sample (10 times less than a standard light scattering measurement). In less than 10 min, AFM can reveal at high resolution the molecular composition of a microtubule sample (microtubules, free tubulin, bundles, and aggregates). Due to these characteristics, AFM can be used

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