Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
J Phys Chem B. 2013 Feb 7;117(5):1241-51. doi: 10.1021/jp310348s. Epub 2013 Jan 25.
Fluorescence recovery after photobleaching (FRAP) is widely used to interrogate diffusion and binding of proteins in live cells. Herein, we apply two-photon excited FRAP with a diffraction limited bleaching and observation volume to study anomalous diffusion of unconjugated green fluorescence protein (GFP) in vitro and in cells. Experiments performed on dilute solutions of GFP reveal that reversible fluorophore bleaching can be mistakenly interpreted as anomalous diffusion. We derive a reaction-diffusion FRAP model that includes reversible photobleaching, and demonstrate that it properly accounts for these photophysics. We then apply this model to investigate the diffusion of GFP in HeLa cells and polytene cells of Drosophila larval salivary glands. GFP exhibits anomalous diffusion in the cytoplasm of both cell types and in HeLa nuclei. Polytene nuclei contain optically resolvable chromosomes, permitting FRAP experiments that focus separately on chromosomal or interchrosomal regions. We find that GFP exhibits anomalous diffusion in chromosomal regions but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin and not crowding by macromolecules is a source of anomalous diffusion in polytene nuclei. This behavior is likely true in other cells, so it will be important to account for this type of transport physics and for reversible photobleaching to properly interpret future FRAP experiments on DNA-binding proteins.
荧光漂白后恢复（FRAP）广泛用于研究活细胞中蛋白质的扩散和结合。在此，我们应用双光子激发 FRAP 技术，其具有衍射极限的漂白和观察体积，以研究未缀合的绿色荧光蛋白（GFP）在体外和细胞中的异常扩散。在 GFP 的稀溶液上进行的实验表明，可逆荧光漂白可能被错误地解释为异常扩散。我们推导出一个包含可逆光漂白的反应扩散 FRAP 模型，并证明它可以正确地解释这些光物理。然后，我们将该模型应用于研究 GFP 在 HeLa 细胞和果蝇幼虫唾液腺多线染色体细胞中的扩散。GFP 在这两种细胞类型的细胞质中和 HeLa 核中表现出异常扩散。多线染色体核包含可分辨的染色体，允许分别聚焦于染色体或染色体间区域的 FRAP 实验。我们发现 GFP 在染色体区域中表现出异常扩散，但在没有染色质的区域中扩散正常。这一观察结果表明，在多线染色体核中，异常扩散的来源是通过染色质的受阻运输，而不是大分子的拥挤。这种行为在其他细胞中可能是真实的，因此，为了正确解释未来关于 DNA 结合蛋白的 FRAP 实验，必须考虑这种类型的运输物理学和可逆光漂白。
