Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus.
Translational diffusion of macromolecule-sized solutes in cytoplasm and nucleus.
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大分子大小的溶质在细胞质和核中的平移扩散。
DOI：
10.1083/jcb.138.1.131
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发表时间：
1997-07-14
影响因子：
7.8
通讯作者：
Verkman, AS
中科院分区：
文献类型：
--
作者：
Seksek, O;Biwersi, J;Verkman, AS
光漂白后的荧光恢复（FRAP）被用来量化的MDCK上皮细胞和瑞士3 T3成纤维细胞的细胞质和细胞核中的微注射的FITC-葡聚糖和Ficoll的平移扩散。使用微秒分辨率FRAP装置和溶液标准测量绝对扩散系数（D）。在水性介质（粘度1 cP）中，随着葡聚糖尺寸的增加（4- 2，000 kD），FITC-葡聚糖的D从75 × 10−7 cm 2/s降低到8.4 × 10−7 cm 2/s。细胞质中相对于水中的D（D/Do）为0.26 ± 0.01（MDCK）和0.27 ± 0.01（成纤维细胞），并且与FITC-葡聚糖和Ficoll大小无关（回转半径[RG] 40-300 μ m）。通过斑点光漂白后的荧光恢复程度确定的移动的FITC-葡聚糖分子的分数（fmob）对于RG < 200 μ l为>0.75，但降低至<0.5 for RG >300 μ l。D/Do对FITC-葡聚糖和Ficoll大小的独立性不支持溶质在细胞质中“筛分”（大小依赖性扩散）的概念。使用不同光斑直径（1.5-4 μm）的光漂白测量给出了相似的D/Do，表明微区室（如果存在）具有亚微米尺寸。D/Do和fmob在浓缩的葡聚糖溶液中，以及在肿胀和收缩的细胞中的测量表明，非常大的大分子的低fmob可能与固定障碍物（如微室）或异常扩散（如渗滤）所施加的限制有关。在细胞核中，D/Do为0.25 ± 0.02（MDCK）和0.27 ± 0.03（成纤维细胞），且与溶质大小无关（RG 40-300 μ m）。我们的研究结果表明，相对自由和快速扩散的大分子大小的溶质高达约500 kD的细胞质和细胞核。
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 × 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 Å). The fraction of mobile FITC-dextran molecules (fmob), determined by the extent of fluorescence recovery after spot photobleaching, was >0.75 for RG < 200 Å, but decreased to <0.5 for RG > 300 Å. 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 μm) 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 Å). Our results indicate relatively free and rapid diffusion of macromolecule-sized solutes up to approximately 500 kD in cytoplasm and nucleus.
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DOI：
10.1083/jcb.112.4.719
发表时间：
1991-02
期刊：
The Journal of cell biology
影响因子：
--
作者：
Fushimi K;Verkman AS
通讯作者：
Verkman AS
影响因子：
4
作者：
BEREZNEY, R
通讯作者：
BEREZNEY, R
影响因子：
3.4
作者：
FARINAS, J;SIMANEK, V;VERKMAN, AS
通讯作者：
VERKMAN, AS
影响因子：
64.5
作者：
CAPCO, DG;WAN, KM;PENMAN, S
通讯作者：
PENMAN, S
影响因子：
3.4
作者：
KAO, HP;VERKMAN, AS
通讯作者：
VERKMAN, AS