Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.
Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.
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DOI：
10.1083/jcb.140.5.1227
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发表时间：
1998-03-09
期刊：
影响因子：
--
通讯作者：
Kusumi A
中科院分区：
文献类型：
--
作者：
Sako Y;Nagafuchi A;Tsukita S;Takeichi M;Kusumi A
应用单粒子跟踪（SPT）和光镊（OT）技术研究上皮细胞质膜上钙依赖性细胞间粘附分子E-cadherin的平移运动及其调控机制。在L细胞中表达野生型（Wild）和三种类型的人工胞质突变体的E-cadherin，并比较它们的运动。两个突变体是在COOH末端具有缺失并且在COOH末端失去连环蛋白结合位点的E-钙粘蛋白，在胞质结构域中剩余116和21个氨基酸（野生型为152个氨基酸）;这些在本文中分别被称为连环蛋白-减和短尾。第三种突变体称为融合蛋白，是没有连环蛋白结合位点的E-钙粘蛋白和没有其NH 2末端一半的α-连环蛋白之间的融合蛋白。这些钙粘蛋白分别用40-nm φ胶体金或210-nm φ乳胶颗粒通过针对E-cadherin胞外结构域的单克隆抗体标记用于SPT或OT实验。研究了背侧细胞表面（细胞-细胞接触区域外）上的E-钙粘蛋白。Catenin minus和Short-tailed可以被OT（捕获力为0.8 pN）平均拖曳1.1和1.8 μm，平均微观扩散系数（D micro）分别为1.2 × 10 - 10和2.1 × 10 - 10 cm 2/s。大约40%的野生型、连环蛋白阴性和短尾型表现出受限型扩散。野生型和连环蛋白缺失型的限制面积为0.13 μm2，而短尾型的限制面积大4倍。相比之下，OT可以将Fusion平均拖长140 nm。通过SPT测量的Fusion的平均D micro较小（0.2 × 10−10 cm 2/s）。这些结果表明，Fusion与细胞骨架结合。Wild由两个种群组成;大约一半的行为类似于Catenin- minus，另一半则类似于Fusion。可以得出结论，野生型E-钙粘蛋白在质膜中的运动是通过胞质结构域调节的：（a）通过连环蛋白（如Fusion）束缚到肌动蛋白丝;（B）膜骨架网络的聚集效应（如Catenin-minus）。通过拖曳实验测得的有助于束缚和畜栏效应的膜骨架的有效弹簧常数分别为30和5 pN/μm，表明产生这两种效应的骨架结构存在差异。
The translational movement of E-cadherin, a calcium-dependent cell–cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and α-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm φ colloidal gold or 210-nm φ latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell–cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 μm by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (D micro) of 1.2 × 10−10 and 2.1 × 10−10 cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 μm2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average D micro for Fusion measured by SPT was small (0.2 × 10−10 cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/μm, respectively, indicating a difference in the skeletal structures that produce these two effects.
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DOI：
10.1083/jcb.113.1.187
发表时间：
1991-04
期刊：
The Journal of cell biology
影响因子：
--
作者：
Mecham RP;Whitehouse L;Hay M;Hinek A;Sheetz MP
通讯作者：
Sheetz MP
DOI：
10.1083/jcb.121.3.491
发表时间：
1993-05
期刊：
The Journal of cell biology
影响因子：
--
作者：
Itoh M;Nagafuchi A;Yonemura S;Kitani-Yasuda T;Tsukita S;Tsukita S
通讯作者：
Tsukita S
影响因子：
56.9
作者：
EDIDIN, M;KUO, SC;SHEETZ, MP
通讯作者：
SHEETZ, MP
影响因子：
7.8
作者：
MCNEILL, H;RYAN, TA;NELSON, WJ
通讯作者：
NELSON, WJ
影响因子：
3.6
作者：
ASHKIN, A;DZIEDZIC, JM;CHU, S
通讯作者：
CHU, S