Lee G M, Zhang F, Ishihara A, McNeil C L, Jacobson K A
Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill 27599.
J Cell Biol. 1993 Jan;120(1):25-35. doi: 10.1083/jcb.120.1.25.
Nanovid (video-enhanced) microscopy was used to determine whether lateral diffusion in the plasma membrane of colloidal gold-tagged lipid molecules is confined or is unrestricted. Confinement could be produced by domains within the plane of the plasma membrane or by filamentous barriers within the pericellular matrix. Fluorescein-phosphatidylethanolamine (F1-PE), incorporated into the plasma membranes of cultured fibroblasts, epithelial cells and keratocytes, was labeled with 30-nm colloidal gold conjugated to anti-fluorescein (anti-F1). The trajectories of the gold-labeled lipids were used to compute diffusion coefficients (DG) and to test for restricted motion. On the cell lamella, the gold-labeled lipids diffused freely in the plasma membrane. Since the gold must move through the pericellular matrix as the attached lipid diffuses in the plasma membrane, this result suggests that any extensive filamentous barriers in the pericellular matrix are at least 40 nm from the plasma membrane surface. The average diffusion coefficients ranged from 1.1 to 1.7 x 10(-9) cm2/s. These values were lower than the average diffusion coefficients (DF) (5.4 to 9.5 x 10(-9) cm2/s) obtained by FRAP. The lower DG is partially due to the pericellular matrix as demonstrated by the result that heparinase treatment of keratocytes significantly increased DG to 2.8 x 10(-9) cm2/s, but did not affect DF. Pericellular matrix viscosity was estimated from the frictional coefficients computed from DG and DF and ranged from 0.5 to 0.9 poise for untreated cells. Heparinase treatment of keratocytes decreased the apparent viscosity to approximately 0.1 poise. To evaluate the presence of domains or barriers, the trajectories and corresponding mean square displacement (MSD) plots of gold-labeled lipids were compared to the trajectories and MSD plots resulting from computer simulations of random walks within corrals. Based on these comparisons, we conclude that, if there are domains limiting the diffusion of F1-PE, most are larger than 5 microns in diameter.
纳米视频（视频增强）显微镜被用于确定胶体金标记的脂质分子在质膜中的侧向扩散是受限的还是不受限制的。限制可能由质膜平面内的结构域或细胞周基质内的丝状屏障产生。掺入培养的成纤维细胞、上皮细胞和角膜细胞的质膜中的荧光素 - 磷脂酰乙醇胺（F1-PE），用与抗荧光素（抗F1）偶联的30纳米胶体金进行标记。金标记脂质的轨迹用于计算扩散系数（DG）并测试受限运动。在细胞薄片上，金标记的脂质在质膜中自由扩散。由于当附着的脂质在质膜中扩散时金必须穿过细胞周基质，该结果表明细胞周基质中任何广泛的丝状屏障距离质膜表面至少40纳米。平均扩散系数范围为1.1至1.7×10（-9）平方厘米/秒。这些值低于通过荧光恢复后漂白（FRAP）获得的平均扩散系数（DF）（5.4至9.5×10（-9）平方厘米/秒）。较低的DG部分归因于细胞周基质，如角膜细胞经肝素酶处理后DG显著增加至2.8×10（-9）平方厘米/秒，但不影响DF所证明的那样。细胞周基质粘度根据由DG和DF计算出的摩擦系数估计，未处理细胞的粘度范围为0.5至0.9泊。角膜细胞经肝素酶处理后表观粘度降低至约0.1泊。为了评估结构域或屏障的存在，将金标记脂质的轨迹和相应的均方位移（MSD）图与由围栏内随机游走的计算机模拟产生的轨迹和MSD图进行比较。基于这些比较，我们得出结论，如果存在限制F1-PE扩散的结构域，大多数直径大于5微米。
