Lin Chi-Iou, Lau Chun-Yee, Li Dan, Jaminet Shou-Ching
Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School.
Center for Vascular Biology Research, Department of of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School;
J Vis Exp. 2014 Apr 3(86):51320. doi: 10.3791/51320.
Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 μm diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per μm and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 μm from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 °C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.
培养中的贴壁细胞维持极化状态以支持细胞运动和细胞间相互作用。纳米足是内皮细胞和癌细胞中细的、细长的、大部分F-肌动蛋白阴性的膜突起，可通过TM4SF1（跨膜4-L-六家族1）免疫荧光染色观察到。TM4SF1聚集在直径为100 - 300μm的TMED（富含TM4SF1的微结构域）中，每个TMED含有3至多达14个单个TM4SF1分子。TMED沿着纳米足以每微米1至3个TMED的规则间距间歇性排列，并将纳米足牢固地锚定在基质上。这使得纳米足能够从极化的内皮细胞或肿瘤细胞的前沿或后沿伸出超过100μm，并且当细胞移开时会在基质上留下膜残留物。由于TMED和纳米足极其脆弱且对温度敏感，它们一直被忽视。常规洗涤和固定会破坏其结构。纳米足可通过在37°C下直接用多聚甲醛（PFA）固定，然后在染色前短暂暴露于0.01% Triton X-100来保存。纳米足为细胞生物学开辟了新视野：它们有望重塑我们对细胞如何感知其环境、远距离检测和识别其他细胞、在紧密接触时启动细胞间相互作用以及参与运动、增殖和细胞间通讯的信号传导机制的理解。为研究TM4SF1衍生的纳米足而开发的方法可能对研究通过经典四跨膜蛋白（特别是普遍表达的CD9、CD81和CD151）在其他细胞类型中形成的纳米足有用。
