We also investigated the transmittance of actin polymerized

We also investigated the transmittance of actin polymerized with and without ABP. The presence of actin in the solution results in an increment in turbidity as other proteins provided atpase inhibitor features [21], [32]. While G-actin showed a transmittance peak positioned at 241 nm, F-actin exhibited a smaller peak at 232 nm, which is according to their optical properties (Fig. 3d). Generally, F-actins with a relatively larger structure absorbed more light than G-actins because they create an entangled net interrupting the transmission. The peaks measured from F-actin samples, including modified F-actins, decreased as the length of actin fibrin increased. The assembly of G-actin into actin filaments resulted in an enhancement in the light absorption, with an observed peak shift from 232 nm to 240 nm. In the measurements involving polymerized actin filaments, no significant peak shift was observed. In Fig. 4a, b, the absorbance and PL results were presented in order to compare the optical characteristics among F-actin, α-actinin and bundled actin. These results demonstrate the thickness dependence of the absorbance and PL. The absorbance spectra of the samples with different peak positions are shown in Fig. 4a. F-actin has a broad peak near 264 nm, a reverse of its transmittance feature. α-actinin and bundled actin exhibit one sharp peak each, with a peak at 278 nm corresponding to α-actinin and another peak at 287 nm corresponding to bundled actin. After making the bundled structure from the G-actin with α-actinin, the wide peak of F-actin at 264 nm shrank significantly and shifted to 278 nm. In the PL spectra for the excitation, as shown in Fig. 4-b, bundled actin showed a strongly enhanced peak at 290 nm when compared with F-actin, and the intrinsic α-actinin exhibited a relatively minor peak with small intensity. Likewise, the emission peak located at 334 nm also increased as the bundled actin network formed. To see a time-dependent PL intensity during actin polymerization, we measured the PL at every 10 min after initiating polymerization by adding F-buffer. In the experiments for F-actin polymerization at 37 degrees, the PL intensity increased for 100 min of the elapsed time as actin monomers were polymerized into F- actin (Fig. 4c). The PL intensity became saturated after 90 min which indicates that the F-actin polymerization was finished. When the temperature was reduced to 20 degrees, the increase of PL intensity was delayed due to the slower reaction of F-actin polymerization. Not a significant change was observed in the PL intensity measured at 4 degrees as the polymerization could not occur. Similar to the experimental results for F-actin polymerization, the PL intensity gradually increased with time during actin filament bundling [4], [22]. However, the period over which the PL intensity became saturated was smaller compared to that observed in the experiment for F-actin polymerization. There was no significant difference in the PL intensity curves obtained at 37 degrees and 20 degrees (Fig. 4-Fig. 4d) [33]. We found that the absorbance, transmittance and PL of actin depended on the molecular structure determined by the bonds between monomers and the actin-binding proteins. Type of bond between proteins played a major role in the modification of the optical properties. Alteration in the optical properties was also attributed to the structural change due to assembly of molecules. Peptide showed an improvement in its fluorescence when its morphology was reformed from powder into nanowire with its various interactions, aromatic stacking, two types of herringbone interaction networks and intermolecular hydrogen bonds [34]. The absorption of light could be regulated depending on the length of protein based adaptor and connector [35]. Carotenoid emission might be originated from the excited state of the polyenes with the optically forbidden S1 state [36]. Actin is the primary cytoskeleton protein in cells and is observed as various forms such as monomers, filaments, and filaments assembly. To see if our technique is able to probe the structural alterations of actin in cells, we tested mouse fibroblasts NIH-3T3 and human keratinocytes HaCaT which have been widely used for study of migration and differentiation. The PL spectrum was deconvoluted into two Gaussian components, the actin features and the background, which included multiple other effects caused by the cell (Fig. 5a). Specifically, the emission band in the PL spectrum from 280 to 385 nm shown in Fig. 5b focused on the characteristics of G-actin and polymerized actins and corresponded to the results in vitro described in Fig. 3, Fig. 4. To measure the PL characteristics in response to the conformation change of the actin structure in cells, we inhibited fibrous actin formation with a cytoskeleton-disrupting chemical, cytochalasin D (Fig. S2). Cytochalasin D, which is used in cell experiments, inhibits actin polymerization by capping the barbed ends of filaments and disassembles actin filaments or networks into actin monomers in cells [37], [38]. Fluorescence images of NIH-3T3s and HaCaTs with and without cytochalasin D treatment showed that the actin networks and bundles were disrupted by cytochalasin D (5–20 μM) (Fig. 5e, f). The addition of cytochalasin D decreased the PL intensity at the ∼340 nm peak on both NIH-3T3 s and HaCaTs (Fig. 5c, d). From these results, the PL peak at approximately 340 nm can be attributed to actin structures in cells. To achieve consistent results, the PL intensity was also measured in human bronchial epithelial cells, BEAS-2B, and showed the same results as the previous measurement (Fig. S3).