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  • br Results and discussion The morphology and structure of

    2023-11-18


    Results and discussion The morphology and structure of GNE-617 samples were observed using an SEM, as shown in Fig. 1. The length and thickness of the actin filaments were regulated with ABPs to mimic the various actins observed in cells. We prepared three types of F-actin with or without gelsolin to control the length of actin: intrinsic F-actin polymerized from G-actin without gelsolin (Fig. 1b) and two different volume ratios of G-actin to gelsolin, which were 2000:1 (Fig. 1c) and 200:1 (Fig. 1d), respectively. We regulate the length of actin filament by controlling the concentration of actin binding protein, gelsolin that severs actin filaments. The previous study showed the length distribution of actin filament is altered by the presence of gelsolin [9]. Our relative concentrations of actin to gelsolin of 200:1 and 2000:1 were chosen based on the number-average length as a function of gelsolin concentration estimated in Janmey et al. [27]. We measured that the average length of F-actins decreased from 1.65 μm to 0.67 μm as the relative concentration of actin to gelsolin increased from 200:1 to 2000:1 (Fig. 1f), which is similar to the experimental result shown in Janmey et al. [27]. The actin networks per unit area, as represented in Fig. 1b–d, were reduced as the length of the actin filaments decreased since it plays a key role in constructing the actin network [12]. ABP α-actinin was used to regulate the thickness of the actin filaments by cross-linking them into bundled structures. The densely populated actin bundles formed by 0.5 μM α-actinin (G-actin: α-actinin = 10:1) are shown in Fig. 1e. SEM, TEM, and fluorescent images for F-actin and actin bundles were represented in Fig. 2. They demonstrated that G-actin were polymerized into F-actin in the absence of ABP, whereas thick bundles of actin filaments were formed in the presence of ABP α-actinin. Similar actin bundles can be observed in the dynamic filopodia, stress fibers in cells [28], [29], [30]. The PL of monomeric and filamentous actin was measured at protein concentrations ranging from 1 to 15 μM. Fig. 3 shows the PL emission excited by 264 nm light for various concentrations of actin monomers. The intensity profiles for G-actin (Fig. 3a) and F-actin (Fig. 3b) are exhibited by the PL emission peaks at 334 and 323 nm, respectively. The peak wavelengths were independent of actin concentration, while the emission intensities of both G- and F-actin increased at higher actin concentrations. These results suggested that PL intensities and emission peaks could be used as a label-free marker for actin in order to determine both its type and concentration. Under the same concentration of proteins, the PL results among samples of G-actin, length modified F-actins (200:1, 2000:1) and intrinsic F-actins are shown in Fig. 3c. Different peak positions and intensities in the PL excitation were obtained according to the type of actin and the length of the actin filament (Fig. 3c). G-actin provided a peak located at 285 nm, and F-actins incorporated in the 200:1 and 2000:1 samples showed higher intensity peaks at 290 nm. The peak intensity increases as the length of actin filament which is determined by the number of bonds between actin monomers increases during polymerization as shown in Fig. 2 and Fig. 3e. In the PL emission results excited at 264 nm represented in Fig. 3c, each G- and F-actin showed clearly one peak. G-actin exhibited a peak at 334 nm with a much smaller intensity, which is consistent with the results shown in Fig. 3a, b. The difference of emission peaks between G-actin and F-actin was approximately 10 nm corresponding to 0.12 eV of photon energy changed by the optical properties of the different actins. As the peak position in the PL profiles reflects the protein conformation, the excitation and emission spectra for actins were attributed to the transformation of the protein structure that was determined by the bonds between proteins, including the actin monomers, ATP, ABP gelsolin, and α-actinin (Fig. 3e) [31]. The PL intensity for gelsolin itself was negligible compared to that for actin polymerized with gelsolin (Fig. S1).