Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: Cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion
Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: Cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion
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DOI：
10.1016/s0006-3495(97)78835-0
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发表时间：
1997-04-01
影响因子：
3.4
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Swaminathan, R;Hoang, CP;Verkman, AS
绿色荧光蛋白（GFP）被用作非侵入性探针来量化的流变学特性。细胞质从重组细菌中纯化GFP突变体S65 T用于溶液研究，并在CHO细胞质中表达。GFP-S65 T在溶液中是明亮的荧光（λ（ex）492 nm，λ（am）509 nm），寿命为2.9 ns，旋转相关时间（t（c））为20 ns。光漂白后GFP荧光的恢复在盐水溶液中完成，半衰期（t（1/2））为30 +/- 2 ms（5 μ m直径斑点），扩散系数为8.7 × 10（-7）cm（2）/s。t（1/2）与溶液粘度成正比，并取决于斑点直径。与荧光素相反，GFP光漂白效率不受溶液O-2含量、三重态猝灭剂、单线态氧清除剂和一般自由基猝灭剂的影响。在较高粘度的溶液中，检测到另外的快速GFP恢复过程，并归因于可逆的光漂白。可逆光漂白的t（1/2）为1.5-5.5 ms（相对粘度5-250），与斑点直径无关，不受O-2或猝灭剂的影响。在细胞质中，时间分辨显微荧光测定法表明GFP寿命为2.6 ns，t（c）为36 +/- 3 ns，相对粘度（细胞质对水）为1.5。GFP在细胞质中的光漂白恢复完成82 +/-2%，t（1/2）为83 +/-6ms，得到3.2的相对粘度。当细胞从相对体积0.5膨胀到2时，GFP翻译扩散增加4.7倍。与在葡聚糖水溶液中的GFP翻译和旋转的测量一起，在细胞质中的数据支持GFP扩散的主要障碍是GFP和大分子溶质之间的碰撞相互作用的观点。
The green fluorescent protein (GFP) was used as a noninvasive probe to quantify the rheological properties of. cell cytoplasm. GFP mutant S65T was purified from recombinant bacteria for solution studies, and expressed in CHO cell cytoplasm. GFP-S65T was brightly fluorescent in solution (lambda(ex) 492 nm, lambda(am) 509 nm) with a lifetime of 2.9 ns and a rotational correlation time (t(c)) of 20 ns. Recovery of GFP fluorescence after photobleaching was complete with a half-time (t(1/2)) in aqueous saline of 30 +/- 2 ms (5-mu m diameter spot), giving a diffusion coefficient of 8.7 x 10(-7) cm(2)/s. The t(1/2) was proportional to solution viscosity and was dependent on spot diameter. In contrast to fluorescein, GFP photobleaching efficiency was not affected by solution O-2 content, triplet state quenchers, singlet oxygen scavengers, and general radical quenchers. In solutions of higher viscosity, an additional, rapid GFP recovery process was detected and ascribed to reversible photobleaching. The t(1/2) for reversible photobleaching was 1.5-5.5 ms (relative viscosity 5-250), was independent of spot diameter, and was unaffected by O-2 or quenchers. In cell cytoplasm, time-resolved microfluorimetry indicated a GFP lifetime of 2.6 ns and a t(c) of 36 +/- 3 ns, giving a relative viscosity (cytoplasm versus water) of 1.5. Photobleaching recovery of GFP in cytoplasm was 82 +/- 2% complete with a t(1/2) of 83 +/- 6 ms, giving a relative viscosity of 3.2. GFP translational diffusion increased 4.7-fold as cells swelled from a relative volume of 0.5 to 2. Taken together with measurements of GFP translation and rotation in aqueous dextran solutions, the data in cytoplasm support the view that the primary barrier to GFP diffusion is collisional interactions between GFP and macromolecular solutes.