PL for actin in vitro showed that its profiles

PL for j 14 in vitro showed that its profiles, including particular peak and intensity, could determine the length and thickness of an actin filament. Furthermore, we investigated if the PL intensity peak at approximately 340 nm can also be originated from another cytoskeleton filament, such as microtubule in cells. Similar to actins, microtubules form filamentous structures in cells by the polymerization of the globular protein, α- and β-tubulin. A chemical reagent called nocodazole is known to bind to tubulin with high affinity and inhibit microtubule assembly [39], [40]. In a test with different concentrations of nocodazole, we could find the disrupted microtubule networks at the concentration from 0.1 to 20 μg/mL (Fig. S4) in the fluorescent images. The PL intensity profiles showed no significant change in the spectrum from 360 to 600 nm when nocodazole was added. This result suggests that the peak at ∼340 nm was specific for actin, rather than being caused or shared with microtubules or other proteins in the cell. Compared to actin, there was no change in PL intensity corresponding to microtubules that might contribute to the different molecular configuration and measurement conditions such as limited excitation and emission ranges.
Conclusion Using the label-free PL measurements, we were able to determine the alterations in length and thickness of protein assembly consisting of actins and actin binding proteins. We revealed that G-actin and polymerized actins exhibit their intrinsic PL characteristics with the specific peak position and intensity. As G-actin were assembled into a filament and filaments bundles, the PL emission peak shifted from 323 nm to 334 nm and its intensity increased significantly. The results indicate that the PL response depend on the number of bonds in the actin assembly. In the experiment for cells in vivo, we used cytochalasin D treatment to disrupt the actin cytoskeleton that exhibits various forms depending on type of cell. We observed that, regardless of cell type, the peak intensity at 340 nm in the PL spectrum decreased when the actin structure were disrupted. Future research for study of cellular function such as migration and division accompanied by structural changes of the cytoskeleton can be built upon our method and findings. In addition, our technique can be applied to develop an alternate diagnosis tool to detect disorganization of the cytoskeleton which can be observed in diseases [41], [42], [43], [44].
Author contributions
Acknowledgements We thank F. Nakamura and for providing gelsolin proteins. This work was partially supported by Industrial Strategic Technology Development Program through the Ministry of Trade, Industry and Energy (MOTIE, Korea) (2MR4090), the National Research Foundation of Korea (NRF) (NRF-2015R1A5A1037668) funded by the Ministry of Education, Science and Technology (MEST), the ICT R&D program of MSIP/IITP (R0101-15-0034), the NRF (NRF-2015R1A2A2A01007602), Nano-Material Technology Development Program (NRF-2017M3A7B4041987), Global Top project from Korea Ministry of Environment (2016002130005), Development of diagnostic system for mild cognitive impairment due to Alzheimer’s disease (2015-11-1684), the Global Research Lab. program through the NRF (NRF-2016K1A1A2912758) of Korea, and ICONS (Institute of Convergence Science) at Yonsei University.
GMF: A Small Protein with a Big Reach Glia maturation factor (GMF) is a 17-kDa protein with clear orthologues across species as diverse as yeast, flies, zebrafish, and mammals (Figure 1A). GMF was first identified over 40 years ago as a factor in brain extracts that could induce the differentiation of cultured glioblasts, gliomas, and neuroblastoma cells 1, 2, 3, 4. Thus, GMF was studied for decades as a signaling factor controlling brain cell differentiation and function 1, 2. Inconsistent with its early assignment as a cell differentiation factor, GMF is conserved from single-celled yeast to mammals. In fungal species and invertebrates, there is typically a single GMF gene/protein. However, vertebrates express two different GMF genes, GMFβ and GMFγ. These isoforms are 82% identical in sequence [5] and have similar 3D structures [6] (Figure 1B,D). GMFβ and GMFγ show overlapping yet distinguishable expression patterns in different tissues 7, 8, 9. Although GMFβ has been described in some studies as a brain-specific isoform, its expression has also been detected at the protein level in lung, spleen, colon, and thymus [8], and even more ubiquitously as a transcript 5, 7, 10.