Direct observation of the actin filament by tip-scan atomic force microscopy

Abstract

Actin filaments, the actin–myosin complex and the actin–tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin–tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin–tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.

Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs

“…AFM operating in non-contact mode is capable of reaching atomic resolution on samples of small molecules [7], whereas AFM imaging of biomolecules routinely reaches nanometre resolutions in single high signal-to-noise images, and is able to characterise biological populations at a true single molecule level. This technique has been applied to a range of different bio-molecules, including membrane proteins [8], viral capsids [9] and filamentous biomolecules, such as amyloid fibrils [10], nucleic acids [11,12], and various filaments involved in the cytoskeleton [13].…”

Demonstration of correlative atomic force and transmission electron microscopy using actin cytoskeleton

“…By subtracting the height of avidin (~5 nm), the height of an actin filament was estimated to be ~8 nm, which was consistent with the height of actin filaments reported previously [ 21 ]. Using the pixel setting of the AFM at 20~30 nm per pixel, we could not identify the typical structural features of the actin filaments, such as the distance of the half-helical pitch (~37 nm) as described [ 22 , 23 ]. However, the characteristic morphological patterns, made by several actin filaments, were useful for identifying a particular area of observation.…”

Advances in Structural Biology and the Application to Biological Filament Systems

“…AFM has found many applications since its introduction, with recent developments including a high‐speed AFM (HS‐AFM), which has been used to visualize movements and structural changes in chaperons in real time . A recently developed tip‐scan AFM was able to visualize tropomyosin (diameter 20 Å) with a temporal resolution of 10 s per frame . Thus, the AFM‐based techniques promise to answer some of the outstanding questions concerning dynamic biological systems in real time at a comparatively high resolution (20–30 Å).…”