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Single-molecule anatomy by atomic force microscopy and recognition imaging.

Atomic force microscopy (AFM) has been a useful technique to visualize cellular and molecular structures at single-molecule resolution. The combination of imaging and force modes has also allowed the characterization of physical properties of biological macromolecules in relation to their structures. Furthermore, recognition imaging, which is obtained under the TREC(TM) (Topography and RECognition) mode of AFM, can map a specific protein of interest within an AFM image. In this study, we first demonstrated structural properties of purified α Actinin-4 by conventional AFM. Since this molecule is an actin binding protein that cross-bridges actin filaments and anchors it to integrin via tailin-vinculin-α actinin adaptor-interaction, we investigated their structural properties using the recognition mode of AFM. For this purpose, we attached an anti-α Actinin-4 monoclonal antibody to the AFM cantilever and performed recognition imaging against α Actinin-4. We finally succeeded in mapping the epitopic region within the α Actinin-4 molecule. Thus, recognition imaging using an antibody coupled AFM cantilever will be useful for single-molecule anatomy of biological macromolecules and structures.


Introduction
Atomic force microscopy (AFM) can be operated in two different modes; imaging and force modes. The imaging mode has been used to visualize various biological structures and processes (reviewed in Hirano et al., 2008). Nano-scale structures and the dynamics of cells, cytoskeletons (Henderson et al., 1992;Hofmann et al., 1997;Haga et al., 2000;de Jager et al., 2001;Yoshimura et al., 2003;Mucke et al., 2004), DNA-protein interactions (Nettikadan et al., 1996;de Jager et al., 2001;Schnitzler et al., 2001;Yoshimura et al., 2002;Rivetti calponin homology domains, a C-terminal EF hand, and an central rod domain composed of four spectrin repeats (Mimura and Asano, 1987;Davison and Critchley, 1988). Anti-parallel dimerization is necessary for cross-linking of actin filaments (Ylanne et al., 2001). We found that there are several different structures in dimerized Actinin-4 in vitro. In addition, by using antibody-coupled cantilevers, we could successfully map the position of the epitopic region within a single Actinin-4 molecule.

Materials and Methods
Expression and purification of full-length and coiledand actin-binding regions of α  D N A c o n s t r u c t s f o r f u l l l e n g t h A c t i n i n -4 (corresponding for 1-911 amino acids), actin binding region (1-286 amino acids) and coiled region (287-758 amino acids) were amplified by PCR from a HeLa cDNA pool and cloned into a pGEX-5X-1 vector (GE Healthcare). Escherichia coli cells (BL21) harboring the plasmid were cultured in LB medium and the expression of the protein was induced by IPTG (final concentration of 1 mM) at 30 for 4 h. The cells were harvested and resuspended in a buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM PMSF) and sonicated on ice for a total of 90 sec. After removing the cell debris by centrifugation, the supernatant was mixed with Glutathione-Sepharose 4 B beads (GE Healthcare) in a tube. After washing with the above buffer not containing PMSF several times, the proteins fused to GST were cleaved off from the GST moiety by Factor Xa protease (New England Bio Lab) by following the manufacturer s protocol. To remove free Factor Xa protease in the solution, benzamidine beads (GE Healthcare) were mixed with the solution and precipitated by centrifugation. The supernatant was dialyzed against a buffer (50 mM Hepes-NaOH, pH 7.6, 100 mM NaCl).

Specificity of anti-α Actinin-4 antibody
The amino terminal domain (1-286 amino acids), which contains the actin-binding domain, and the central rod domain (287-758 amino acids), as well as full length Actinin-4, were expressed as GST-fused proteins in E. coli and the cell lysates were subjected to SDS-PAGE followed by Western-blot analyses using the anti-Actinin-4 monoclonal antibody (Fig. 1). The results showed that the antibody specifically recognized the amino-terminal domain but not the central rod domain. Das et al., 2006;Lushnikov et al., 2006;Hizume et al., 2007), and membrane proteins (Saslowsky et al., 2002;Czajkowsky et al., 2004;Barrera et al., 2005) have been elucidated. Several technical improvements, such as carbon nanotube probe (Hohmura et al., 2000), and fast-scanning AFM (Viani et al., 2000;Ando et al., 2001;Yokokawa et al., 2006;Crampton et al., 2007) have greatly expanded the application range of AFM in molecular and cellular biology.
The force mode of AFM has contributed to the characterization of physical properties of biological macromolecules such as the elucidation of internal s t r u c t u r e s o f b i o l o g i c a l m a c r o m o l e c u l e s a n d intermolecular interaction of protein subunits, and protein-lipid/protein-DNA interactions (Hinterdorfer et al., 1996;Nevo et al., 2003;Afrin et al., 2005). This mode depends on the force measurements between the cantilever and the specimen. Unique ideas for the cantilever modification techniques have been the key for successful force measurement (Ikai et al., 2003;Gamsjaeger et al., 2004;Kamruzzahan et al., 2006;Ebner et al., 2007). Previously, we have developed a novel method for a site-specific attachment of glutathione S-transferase (GST)-fused proteins to the cantilever in a desired direction, and applied to the measurement of interaction between importin and its interacting proteins Otsuka et al., 2008).
Recent development of recognition imaging under the PicoTREC TM mode of AFM (Topography and R E C o g n i t i o n m o d e , c o m m e r c i a l i z e d b y A g i l e n t Technologies, Santa Clara, USA) has further enabled researchers to simultaneously obtain a topographic image together with a recognition signal that is based on the interaction between the specimen and the protein/ ligand-coupled cantilever, and thus, to identify a specific molecule within the topographic image. In this imaging mode, an attractive force between a molecule on the cantilever surface and its partner molecules on the specimen can be detected during the imaging (Stroh et al., 2004a). With this technique, histone proteins in reconstituted chromatin and membrane proteins on the cell surface have been successfully recognized (Stroh et al., 2004b;Bash et al., 2006;Chtcheglova et al., 2007).
In this study, we established our own procedure to covalently couple the antibody to the cantilever surface with commercially-available chemical compounds. By using this method, we attached an anti-Actinin-4 mo n o c lo n a l a n t ib o d y to t h e A F M c a n ti l e v er an d performed recognition imaging against purified Actinin-4.
Actinin-4 is an actin-binding cytoskeletal protein and contains three functional sub-domains; an N-terminal actin-binding domain composed of two mixed with antibody solution (final concentration; 100 g/ml) and EDC (final concentration; 500 g/ml; Pierce) in an activation buffer (100 mM MES-NaOH, pH 5.0) and kept for 1 h at room temperature. After the reaction, the cantilever was rinsed with PBS, then immediately used for AFM imaging.

Topographic and recognition imaging
For the recognition imaging, purified Actinin-4 was immobilized on the mica substrate previously mentioned (Lohr et al., 2007). Firstly, freshly cleaved mica was treated with APTES by vapor for 1 h at room temperature inside argon-gas-filled chamber. Then, the mica was treated with glutaraldehyde (final concentration of 1 mM) for 10 min. After washing with ultrapure water, 100 ng/ ml of purified Actinin-4 was immediately dropped on the mica and incubated for 10 min at room temperature. For the blocking of free glutaraldehyde, the mica substrate was treated with 1 mM glycine in PBS for 10 min. After washing with PBS, the mica substrate was immediately used for the recognition imaging. The AFM imaging was performed by PicoPlus AFM equipped with Pico TREC TM module (Agilent Technologies, Tempe, USA) was operated by Pico View software (Agilent Technologies). The image was taken in the magnetic Top MAC mode

AFM imaging of α Actinin-4 on the mica surface
Purified Actinin-4 was diluted to 0.1 ng/ l with HMgG buffer (5 mM Hepes-NaOH, pH 7.5, 5 mM MgCl 2 , 0.3% glutaraldehyde), incubated at 20 for 30 min, and dropped onto a freshly cleaved mica surface. After 10 min incubation at room temperature, the mica was washed with Milli Q water and dried by nitrogen gas blowing.
AFM imaging was performed with Mul ti Mode AFM (Digital Instruments, Santa Barbara, CA, USA), in air using Tapping Mode TM . The cantilever (Veeco NanoProbe RTESP7, Tucson AZ, USA) used for the imaging was 125 m in length. The scanning rate was set at 2-3 Hz, and images were captured in 512 512 pixel format. To measure the width and length of the structures in AFM images, the tip effect was removed by using the apparent size of nucleosomes as a reference (Nettikadan et al., 1996). In this study, DNA, which has the width of 2 nm, was imaged and used as a standard. For the analysis of the volume, the full width at halfmaximum (Schneider et al., 1998) and height were measured by a procedure published previously (Schneider et al., 1998). Briefly, the specific volume of an assumed globular protein (0.75 cm 3 /g) and weight of protein Molecular Mass (kDa)/6.02 10 -23 g were combined and the volume of the molecule of protein was calculated as Molecular Mass (kDa) 1.24 10 -3 nm 3 . Based on this formula, molecular mass of the protein in AFM images were estimated.

Preparation of the antibody-coupled cantilever and α Actinin-4 protein
Cantilevers made of silicon nitride (Type VI MAC lever (Agilent Technologies) for recognition imaging) were treated with vaporized 3-aminopropyltriethoxysilane (APTES) (SIGMA) and diisopropyl-ethylamine (SIGMA) for 1 hr inside an argon gas filled chamber to generate amino groups on the cantilever surface, then incubated with heterobifunctional cross linker (5 mg/ml maleimide-dPEG TM 12-N-hydroxysulfosuccinimide (NHS) ester, (Quanta BioDesign Ltd., Ohio, the USA)) which was dissolved in chloroform containing 0.7% triethylamine for 2 hrs at room temperature. Optionally, the unreacted amino groups were blocked by 5 mg/ml NHS-m-dPEG TM (Quanta BioDesign Ltd) in chloroform containing 0.7% triethylamine. After washing with chloroform, ethanol and ultrapure water, the PEG-modified cantilever was then incubated with another crosslinker carrying thiol group, 1 mg of 5-carboxy-1-pentanethiol, (Dojindo Laboratories, Kumamoto) in PBS (pH 7.2) for 1 h at room temperature. The carboxyl-coupled cantilever was then

Fig. 2. AFM analysis of the purified full-length
Actinin-4. a: SDS-PAGE gel of purified full-length Actinin-4 (F), actin binding domain (A) and central rod domain (C). The SDS-PAGE gel (12%) was stained with Coomassie Brilliant Blue. These proteins were used for AFM imaging. b: AFM images of the purified fulllength Actinin-4 imaged by conventional AFM with un-modified cantilever. Observed elliptic structures were indicated by arrowhead, and observed round structures were indicated by arrow. 34% of the all observed structures showed elliptic shape, 62% of them showed round shape without tail, and 10% of them showed round shape with tail. c: AFM images of typical elliptic structures (panel c-i). Width (c-ii), height (c -iii), and lengths (c-iv) of the elliptic structures were measured in AFM images and tabulated into a frequency distribution. Proposed model of an anti-parallel homodimer of Actinin-4 is shown in c-v. d: AFM images of typical lower round structures (d -i), taller round structures (d -ii), and round structures with tails (d -iii). Width (d -iv) and height (d -v) of the round structures were measured in AFM images and tabulated into a frequency distribution. Proposed model of the possible conformation of Actinin-4 is shown in d-vi; monomer (left), dimer (middle), and dimer with tail (right). shown in Fig. 2 c-i) and round shapes with or without a small tail (indicated by arrow in Fig. 2 b, and typical examples with or without tails are shown in Fig. 2 d -id-iii). The width and the length of the elliptic structure were 6.08 2.08 nm and 19.2 3.9 nm, respectively. This is well consistent with previous results from X-ray crystallography (Blanchard et al., 1989) that suggested that Actinin-4 forms an anti-parallel homodimer having a rod-like shape (Fig. 2 c-v). The diameter of the round shapes (Fig. 2 d-i -iii) was 6.70 3.50 nm (Fig.  2 d -iv, d-v). However, the height had two populations; 0.7 nm (Fig. 2 d-i) and 1.4 nm (Fig. 2 d-ii). 14 % of the round structures had tails as shown in Figure 2 These results indicate that a certain population of the recombinant full-length Actinin-4 forms anti-parallel homodimers (Fig. 2 c-i, c-v), but there are a significant amount of miss-folded monomers in vitro (Fig. 2 d-i, left of d-vi), dimers (Fig. 2 d-ii, middle of d-vi), and dimers with coiled-coil tails (Fig. 2 d-iii, right of d-vi).
The actin-binding domain (Fig. 3 a) and the coiled (Han et al., 1996) with Type VI MAC levers (Typical spring constant; 0.1 N/m) (Agilent Technologies) in PBS (pH 7.4). The tune of the cantilever was carried out in PBS. The peak of the amplitude oscillation, about 15-20 kHz, was mainly used for the imaging. Set point and scan speed were set at 2.0 -2.5 V and 1 Hz, respectively. The obtained images were analyzed by SPIP TM Ver. 4.6 (Image metrology, Horsholm, Denmark).

Structural properties of α Actinin-4 molecule in vitro
The overall molecular structure and dimerization properties of purified Actinin-4 in vitro were analyzed by conventional AFM with an un-modified cantilever (Fig. 2 a). As shown in Figure 2 b, full-length Actinin-4 showed two different structures, elliptic shapes (indicated as arrowheads in Fig. 2b, and typical examples are   (Fig. 3 b) showed round structures but not elliptic ones. The widths and heights were summarized in Figure  3 c and d. The actin-binding domain had the width of 3.85 1.01 nm and the height of 0.56 0.16 nm (Fig.  3 c-i and c-ii, respectively). The volume was measured to be 31.9 nm 3 by using the previously reported method (Schneider et al., 1995(Schneider et al., , 1998. Since the specific volume of an assumed globular protein is 0.75 cm 3 /g, 31.9 nm 3 corresponds to 25.7 kDa (see Materials and Methods), which is similar to the molecular weight of the actinbinding domain (32 kDa). The coiled region had the width of 7.85 2.15 nm (Fig. 3 d-i) and the height of 0.42 1.7 nm (Fig. 3 d-ii). The volume and molecular weight was calculated to be 49.4 nm 3 and 39.8 kDa, respectively, which is far from the size of a dimer (112 kDa) but close to a monomeric size of this region (56 kDa). This indicates that the coiled region does not form a dimer under these conditions.

Monoclonal antibody-coupled cantilever recognizes the actin-binding domain of α Actinin-4.
Attachment of antibodies to the AFM cantilever with low consumption has been achieved by several different methods (Kamruzzahan et al., 2006;Ebner et al., 2007;Wakayama et al., 2008). Some of these methods require a complicated synthesis of chemical compounds. However, using our method, antibodies can be attached easily to the surface of the cantilever using only commercial linkers (See Materials and Methods and Fig. 4 a). The antibody was immobilized on the cantilever by using the procedure described in the Materials and Methods, and the purified full-length Actinin-4 was covalently cross-linked to the mica surface. Before recognition imaging of Actinin-4, we confirmed the shape of Actinin-4 in PBS by AFM imaging on MAC mode (Fig. 4 b -d). To obtain a good resolution image, the imaging was performed with as high gain as possible (Integral and Proportional gain were set at 5 and 8, respectively). In the image, several dimers of Actinin-4 (indicated by white arrows) were observed within many aggregated Actinin-4 molecules (Fig 4 b).
In the magnified image of those molecules, rod-like (Fig.  4 c, proposed as Fig.2 c-v) and round shapes (Fig. 4 d, proposed as Fig. 2 d-vi) can be detected.
In the recognition imaging of Actinin-4 with the antibody-coupled MAC lever, several dimerized Actinin-4 were observed in the height image (Fig. 4 e). By overlapping the height and recognition images (Fig.  4 f), the dark brown recognition signals can be detected on the head region of Actinin-4 ( Fig. 4 e, f), which is expected to correspond to the interaction spots between the epitope region of Actinin-4 and the coupled antibody on the MAC lever. Interestingly, two recognition spots were detected in each dimerized Actinin-4 in the magnified images (Fig. 4 g -j). As interpreted in Figure  2 a, dimerized Actinin-4 molecules have two epitope regions (refer to Fig. 2) and the recognition imaging successfully mapped two epitope regions of the dimerized Actinin-4 molecules. To confirm whether the detected recognition signals were generated from a specific interaction between Actinin-4 and the antibody, Actinin-4 dimers were imaged with unmodified AFM cantilever (Fig. 4 k), and specific signals were not detected in the recognition image (Fig. 4 l). These results demonstrated that Actinin-4 forms a stable dimer with anti-parallel orientation, and the TREC TM mode can be used to identify a specific position of the epitope within a single protein complex.
This study demonstrated that Actinin-4 forms not only an anti-parallel dimer, but also a parallel dimer in vitro (Fig. 3). Among three functional sub-domains of Actinin-4 (N-terminus actin binding domain, central rod domain and C-terminus EF hand), the central rod domain has been thought to be responsible for the anti-parallel dimerization by hydrophobic binding (Blanchard et al., 1989;Imamura et al., 1988). In the AFM observation of full-length Actinin-4, the distances between the two head-like structures in the molecule varies among individual dimers, indicating that the actin-binding domains in a dimer can move flexibly around a dimerized coiled region. Since Actinin-4 is one of the actinbinding and anchoring proteins that link actin molecules to a variety of inter/extracellular structures, the flexibility may give elasticity to the cytoskeletal fibrous structures in cells.
To elucidate behavior of Actinin-4 and other cytoskeletal proteins inside the cell, we are aiming to map specific proteins within the cellular cytoskeletal structure. When the whole cell was treated with detergent such as Triton X-100, cytoskeletal fibrous network structures appeared (Yoshimura et al., 2003). Several studies have observed these cytoskeletal fibrous architectures by AFM (Hofmann et al., 1997;Yoshimura et al., 2003;Santacroce et al., 2006), however, individual proteins within the fibrous networks have not been identified. For further understanding of the cytoskeletal architecture, identification and mapping of these proteins within the skeletal structure will be important. In this sense, the recognition imaging will be a powerful tool for singlemolecule mapping on biological architectures.