Journal of Surface Engineered Materials and Advanced Technology, 2013, 3, 13-19 http://dx.doi.org/10.4236/jsemat.2013.34A1002 Published Online October 2013 (http://www.scirp.org/journal/jsemat) Copyright © 2013 SciRes. JSEMAT 13 AFM Investigation of the Organization of Actin Bundles Formed by Actin-Binding Proteins Jamie L. Gilmore, Masahiro Kumeta, Kunio Takeyasu Graduate School of Biostudies, Kyoto University, Kyoto, Japan. Email: takeyasu@lif.kyoto-u.ac.jp Received June 21 st , 2013; revised July 20 th , 2013; accepted August 6 th , 2013 Copyright © 2013 Jamie L. Gilmore et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT AFM is a powerful technique for revealing the morphological features of various biological systems at high resolution. However, one of the complications of AFM is that samples must be attached to a flat surface in order to obtain images. This often requires the development of specialized methods depending on the sample which is being used. In this study, we developed a novel technique to image actin bundles on the mica surface. Using this technique, we were able to image molecular assemblies of F-actin with two actin remodeling proteins: α-actinin and Caprice. High resolution AFM images of F-actin fibers and bundle organization depicted two different types of molecular assemblies: F-actin bundles forming an elongated “zipper” structure in the presence of α-actinin, and bundles forming a perpendicularly crossing the mesh structure in the presence of Caprice. Keywords: F-Actin; Caprice; α-Actinin 1. Introduction The biological applications of atomic force microscopy (AFM) [1] were realized immediately after the instru- ment was invented in 1986 [2]. Since AFM was useful for examining the surface properties of relatively solid specimens, it was initially used in the field of material sciences. The physical properties of DNA, proteins, lip- ids and carbohydrates have been extensively studied by using AFM [3-6]. There have been a number of limitations associated with AFM technology. However, the development of new scanning methods [7] and cantilever production [8] has facilitated the applications of AFM for studying bio- logical macromolecules in physiological conditions. The most fascinating technical endeavor has been the inven- tion of high-speed AFM by Ando et al., which has en- abled one to capture the motion of DNA [9-12] and pro- teins [13-17] and to monitor enzymatic reactions [9,11, 18] in solution. However, one of the inherent limitations associated with AFM other than the instrument technology is the specimen preparation procedure, which needs to be taken into account the physicochemical properties of the speci- men against substrates such as mica and glass surfaces. Here we describe procedures to image the most abundant protein in the cell: actin. We developed specimen pre- paration procedures for imaging single filamentous actin (F-actin) and actin-binding protein-promoted F-actin bundles. AFM imaging of these samples provided high resolution images of bundle nanostructures in addition to the ultrastructure of actin networks. The data suggest that F-actin bundles form an elongated “zipper” structure when the α-actinin bundling protein is added and the bundles form a perpendicularly crossing meshwork structure when the Caprice protein is added. These structures pro- vide insight into the molecular mechanisms of forming distinct actin-based structures by different actin-binding proteins. 2. Materials and Methods 2.1. Chemicals and G-Actin All chemicals used were first grade from Sigma and Na- calai Tesque. The non-muscle human actin was obtained from Cytoskeleton, Inc. (APHL99). 2.2. Actin-Binding Proteins The α-actinin from rabbit skeletal muscle, which is a mixture of the actinin-1, 2, 3, and 4 isoforms, was ob- tained from Cytoskeleton, Inc. (AT01). Caprice (C19orf21