An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy

Abstract

An improved unroofing method enabled the cantilever of an atomic force microscope (AFM) to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in phosphate-buffered saline (PBS) at a higher resolution than conventional electron microscopy. All of the actin filaments clearly exhibited a short periodicity of approximately 5–6 nm, which was derived from globular actins linked to each other to form filaments, as well as a long helical periodicity. The polarity of the actin filaments appeared to be determined by the shape of the periodic striations. Microtubules were identified based on their thickness. Clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes. The area containing clathrin molecules and their terminal domains was directly visualized. Characteristic ridge structures located at the surface of the caveolae were observed at high resolution, similar to those observed with electron microscopy (EM). Overall, unroofing allowed intracellular AFM imaging in a liquid environment with a level of quality equivalent or superior to that of EM. Thus, AFMs are anticipated to provide cutting-edge findings in cell biology and histology.

Single-cell unroofing: probing topology and nanomechanics of native membranes

“…The environment is highly diverse, therefore the biophysical parameters that was obtained here consider the overall contribution of lipids and proteins, leading to more robust values for computational lipidomics. To my knowledge, the sample preparation has always been problematic in previous unroofing techniques [13]. The single-cell method proposed here minimizes the sources of variability like large scale sonication or blotting, giving a success rate around 80%.…”

“…AFM has a big advantage compared to light or electron microscopy, in fact the sample doesnt require neither fluorescent labels nor metal coating or cryo-fixation. Some recent studies [12][13][14][15][16] unraveled a detailed architecture of the cytoskeleton on the inner face of fixed membranes with a resolution of5nm. However, the preparation of these samples requires a considerable know-how to be successful and to become reproducible in large scale [13]; moreover the aforementioned treatments act on the entire cultures without any fine control of the process.…”

“…Some recent studies [12][13][14][15][16] unraveled a detailed architecture of the cytoskeleton on the inner face of fixed membranes with a resolution of5nm. However, the preparation of these samples requires a considerable know-how to be successful and to become reproducible in large scale [13]; moreover the aforementioned treatments act on the entire cultures without any fine control of the process. In fact, the studies that required the manipulation of lipid membranes and vesicles concentrated mostly on synthetic preparations of mixtures of lipids [17][18][19] .…”

Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina

“…The cells were prepared by washing the coverslips first with Ringer's solution (+2 mM CaCl2) followed by Ringer's solution without CaCl2. The coverslips were then exposed to hypotonic Ringer's solution (one part of calcium-free Ringer's solution was diluted in two parts deionized water) to swell the cells and facilitate easy opening 77 . To open the nuclei, a two-step procedure was developed.…”

Keeping the focus on biophysics and actin filaments in Nagoya: A report of the 2016 “now in actin” symposium

“…In the image of the actin‐tropomyosin complex, each tropomyosin molecule on the actin filament was directly observed (Narita et al, ). Cells were prepared for AFM by an unroofing technique, by which a part of plasma membrane was broken by gentle sonication and the intracellular region was exposed by removing soluble components from the cytoplasm (Usukura, Narita, Yagi, Ito, & Usukura, ). The actin filaments, microtubules, clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes at nanometer‐order resolution.…”