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  • In this study we obtained the absorbance

    2023-01-21

    In this study, we obtained the absorbance and photoluminescence (PL) of palovarotene sale polymerized with various types of actin-binding proteins in order to probe the actin structures in situ without labeling. Both optical measurements have been useful in examining biomaterials such as DNA and proteins without any detrimental indicator [20]. Absorption spectroscopy, which measures the light transmittance of a suspended sample solution in the ultraviolet (UV) to visible range, has been utilized to determine the presence and concentration of proteins in a sample solution [21], [22]. PL, which is light emission from a sample that has been excited by a photon, has been used to mark the biomolecules with fluorescent indicators. Using PL, bioimaging has been studied with various fluorescent dyes using charge-coupled devices for light detection. We measured the PL spectrum under UV excitation to determine the actin structure. G-actin, F-actin and bundled actin were regulated by adding ABPs such as gelsolin and α-actinin [7], [23], which showed remarkably different features in absorbance and PL. The actin monomer showed a PL emission peak located at 334 nm and polymerized actins provided a shifted peak at 323 nm with a higher intensity. Bundled actin exhibited the strongest PL emission among samples due to the bulk structure of the protein. We investigated the actin polymerization process according to the reaction time using PL characteristics. Furthermore, the actin structure transformation by cytochalasin D in cells, NIH-3T3s and HaCaTs, was also detected by the label-free PL spectrum, which includes the fluorescence of other biomaterials. The PL based determination in vivo was highly consistent with the result of dye-based fluorescence images.
    Methods Actin samples were prepared using the actin polymerization from G-actin and modified with regulatory proteins [9], [24]. G-actin, α-actinin and gelsolin were obtained from rabbit skeletal muscle and were purchased from Cytoskeleton Inc. Actin monomer was reconstituted at 20 μM in a fresh G-buffer (5 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM adenosine triphosphate (ATP), 0.01% (w/v) NaN3, pH 8.0) and incubated at 4 °C for 2 h. A tenth of the final volume of F-buffer (50 mM Tris-HCl, 500 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 5 mM ATP, 0.01% (w/v) NaN3, pH 7.5) was mixed with actin-binding proteins (ABPs), gelsolin or α-actinin and was added to the actin monomer to polymerize G-actin into F-actin and cross-link them. To examine the structures of the actin filaments that were polymerized with ABPs, a field emission scanning electron microscope (SEM; JEOL-7001F, JEOL) was used in conjunction with the negative staining method [25]. Specifically, a 5 μL droplet of polymerized actin solutions was laid on a Formvar Film Cu grid (TedPella Inc.) for 30 s to ensure sample’s adsorption on the grid. Afterwards, the excess of the droplet was removed by filter paper and the grid was washed using deionized water. Samples remaining on the grid were stained with 1% uranyl acetate for 15 s. The length of the filament was measured from the SEM images using the JFilament plug-in in ImageJ software [26]. A transmission electron microscope (TEM; JEOL, Ultra high resolution FE-TEM) was employed to monitor the morphology and structure of sample. Using a 1 cm path length cuvette, transmittance and absorbance of sample were measured by a UV/VIS spectrophotometer (JASCO Corporation, V-650) and the PL was obtained from fluorescence spectrophotometer (Perkin Elmer, LS 55) with an excitation and emission monochromator. Monochromators enabled us to measure the spectrum as a function of excitation and emission wavelengths. NIH-3T3 mouse fibroblasts and HaCaT human keratinocytes were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; LM 001–05, Welgene) supplemented with 10% fetal bovine serum (FBS; 16000-044, Invitrogen Corp.) and 1% penicillin/streptomycin (Pen/Strep; 15140-122, Invitrogen Corp.). BEAS–2 B human bronchial epithelial cells were maintained in a bronchial epithelial cell growth medium (BEGM; 12-707F, Lonza) containing optimized supplements (BEGM Bullet kit; CC-3170, Lonza). In experiments with chemicals such as cytochalasin D and nocodazole, cells were plated on a cover glass and incubated for 24 h at 37 °C at 5% CO2 in culture media. After a few days of culture, cells were incubated in 5–20 μM cytochalasin D for 30 min and in 0.1–10 μg/mL nocodazole for 2 h. For fluorescence imaging, cells were fixed in 4% paraformaldehyde (PFA; 15710, Electron Microscopy Science), and permeabilized in 0.1% Triton X-100 (X100-5ML, Sigma-Aldrich). Alexa-488-labelled phalloidin (A12379, Invitrogen Corp.) and Hoechst 33342 (B2261-25MG, Sigma-Aldrich) containing 1% BSA were used to stain both the actin and nucleus in cells. For microtubule immunostaining, cells were incubated with anti-tubulin-α primary (627902, BioLegend) and Alexa-568-labelled goat anti-mouse secondary (A-11004, Invitrogen Corp.) antibodies consecutively.