Chapter 9 - High-Resolution Imaging of Microtubules and Cytoskeleton Structures by Atomic Force Microscopy
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
References (35)
- et al.
Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer
Protein Expr. Purif.
(2003) - et al.
Atomic force microscopy reveals binding of mRNA to microtubules mediated by two major mRNP proteins YB-1 and PABP
FEBS Lett.
(2008) - et al.
Chemical treatment of mica for atomic force microscopy can affect biological sample conformation
Biophys. Chem.
(2004) - et al.
Pathways for mRNA localization in the cytoplasm
Trends Biochem. Sci.
(2006) - et al.
DNA binding to mica correlates with cationic radius: Assay by atomic force microscopy
Biophys. J.
(1996) - et al.
Microtubule-dependent oligomerization of tau. Implications for physiological tau function and tauopathies
J. Biol. Chem.
(2003) Estimation of the diffusion-limited rate of microtubule assembly
Biophys. J.
(1997)- et al.
Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules
J. Struct. Biol.
(2007) - et al.
Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5
J. Biol. Chem.
(2006) - et al.
Immobilizing and imaging microtubules by atomic force microscopy
Ultramicroscopy
(1995)
Immobilization strategies for biological scanning probe microscopy
FEBS Lett.
RNA granules
J. Cell Biol.
A high-speed atomic force microscope for studying biological macromolecules in action
Chemphyschem
High-speed AFM and nano-visualization of biomolecular processes
Pflugers Arch
Atomic force microscope
Phys. Rev. Lett.
Circular DNA molecules imaged in air by scanning force microscopy
Biochemistry
Real-time observations of microtubule dynamic instability in living cells
J. Cell Biol.
Cited by (19)
Direct visualization of the oligomeric state of hemagglutinins of influenza virus by high-resolution atomic force microscopy
2018, BiochimieCitation Excerpt :The main complication of AFM when used to study proteins is the high resolution required. Broadening of an object is an inherent feature of AFM [10–13]. A simple explanation is given in Fig. S1 (see Supplementary materials).
Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion
2023, NeuroendocrinologyAtomic Force Microscopy Stiffness Mapping in Human Aortic Smooth Muscle Cells
2022, Journal of Biomechanical EngineeringAtomic force microscopy reveals distinct protofilament-scale structural dynamics in depolymerizing microtubule arrays
2022, Proceedings of the National Academy of Sciences of the United States of America