Seksek O, Biwersi J, Verkman A S
Department of Medicine, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521, USA.
J Cell Biol. 1997 Jul 14;138(1):131-42. doi: 10.1083/jcb.138.1.131.
Fluorescence recovery after photobleaching (FRAP) was used to quantify the translational diffusion of microinjected FITC-dextrans and Ficolls in the cytoplasm and nucleus of MDCK epithelial cells and Swiss 3T3 fibroblasts. Absolute diffusion coefficients (D) were measured using a microsecond-resolution FRAP apparatus and solution standards. In aqueous media (viscosity 1 cP), D for the FITC-dextrans decreased from 75 to 8.4 x 10(-7) cm2/s with increasing dextran size (4-2,000 kD). D in cytoplasm relative to that in water (D/Do) was 0.26 +/- 0.01 (MDCK) and 0.27 +/- 0.01 (fibroblasts), and independent of FITC-dextran and Ficoll size (gyration radii [RG] 40-300 A). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >>0.75 for RG << 200 A, but decreased to <<0.5 for RG >> 300 A. The independence of D/Do on FITC-dextran and Ficoll size does not support the concept of solute "sieving" (size-dependent diffusion) in cytoplasm. Photobleaching measurements using different spot diameters (1.5-4 micron) gave similar D/Do, indicating that microcompartments, if present, are of submicron size. Measurements of D/Do and fmob in concentrated dextran solutions, as well as in swollen and shrunken cells, suggested that the low fmob for very large macromolecules might be related to restrictions imposed by immobile obstacles (such as microcompartments) or to anomalous diffusion (such as percolation). In nucleus, D/Do was 0.25 +/- 0.02 (MDCK) and 0.27 +/- 0.03 (fibroblasts), and independent of solute size (RG 40-300 A). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.
采用光漂白后荧光恢复技术（FRAP）对微量注射的异硫氰酸荧光素标记葡聚糖（FITC - dextrans）和聚蔗糖在MDCK上皮细胞及瑞士3T3成纤维细胞的细胞质和细胞核中的平移扩散进行定量分析。使用微秒级分辨率的FRAP仪器和溶液标准品测量绝对扩散系数（D）。在水性介质（粘度1厘泊）中，随着葡聚糖大小（4 - 2000kD）增加FITC - dextrans的D值从75降至8.4×10⁻⁷cm²/s。细胞质中的D相对于水中的D（D/Do）在MDCK细胞中为0.26±0.01，在成纤维细胞中为0.27±0.01，且与FITC - dextrans和聚蔗糖大小无关（回转半径[RG] 40 - 300 Å）。通过点光漂白后荧光恢复程度确定的可移动FITC - dextran分子分数（fmob），对于RG << 200 Å时>>0.75，但对于RG >> 300 Å时降至<<0.5。D/Do对FITC - dextran和聚蔗糖大小的独立性不支持细胞质中溶质“筛分”（大小依赖性扩散）的概念。使用不同光斑直径（1.5 - 4微米）进行的光漂白测量得到相似的D/Do，表明如果存在微区室，其大小为亚微米级。在浓缩葡聚糖溶液以及肿胀和收缩细胞中对D/Do和fmob进行测量，结果表明对于非常大的大分子，低fmob可能与固定障碍物（如微区室）施加的限制或异常扩散（如渗滤）有关。在细胞核中，D/Do在MDCK细胞中为0.25±0.02，在成纤维细胞中为0.27±0.03，且与溶质大小无关（RG 40 - 300 Å）。我们的结果表明，在细胞质和细胞核中，高达约500kD的大分子大小溶质具有相对自由且快速的扩散。
