引言#

细胞膜是细胞与外界环境进行信息和物质交换的前沿阵地，膜表面的受体与配体相互作用调控着细胞粘附、迁移和信号转导等关键生理过程。虽然传统的生物化学和光学成像技术能够告诉我们这些相互作用发生的时间和位置，但一直缺乏在活细胞原位测量单个受体-配体复合物机械结合强度的有效工具。长期以来，学术界普遍将分子结合强度视为固定的生物化学常数。然而，细胞膜本身是一个高度异质且动态重构的力学环境，受体聚集、脂质微区和细胞骨架偶联都会对局部的力学稳定性产生深刻影响。为了打破这一局限，研究团队利用磁镊技术的长时稳定性与低光毒性优势，首次建立了针对活细胞表面受体-配体互作的单分子纳米力学测量平台。

Fig.1 Magnetic-tweezers-based single-molecule force measurement of integrin–RGD interactions on HeLa cells. (A) Schematic of the magnetic-tweezers assay. A dsDNA handle (1558 bp) is functionalized with an RGD ligand and attached to a 2.32 μm streptavidin-coated superparamagnetic bead. The RGD motif binds to integrin receptors on the cell membrane. An external magnetic field lifts the magnetic bead, allowing measurement of the force required to disrupt the interaction between RGD and integrins on the cell surface. (B) Representative bright-field images of DNA-bound superparamagnetic beads attached to different cellular regions. Left: bead tethered to a healthy cell main body; middle: bead attached to a healthy cell extension; right: bead attached to an unhealthy main body. Arrows highlight beads of interest. Scale bars = 5 μm. (C) Quantification of bead rotation used to confirm the single DNA tether attached to a cell, showing a circular trajectory characteristic of single-molecule tethering. (13) (D) Sequential bright-field images of a superparamagnetic bead (indicated by the black arrow) attached to the surface of a healthy HeLa cell in the microfluidic chamber inside the magnetic tweezers instrument. When a pair of magnets was brought closer to the chamber (vertical distance between the magnets and the chamber surface is marked at the top of each frame, see Figure 1A for setup), a tensile force was exerted on the magnetic bead (the magnitude of the force exerted at a specific distance is shown at the bottom of each frame). Diffraction rings of the magnetic bead changed as the bead was pulled away from the focal plane. (E) Violin plots of rupture forces to disrupt integrin–RGD interactions. Healthy cell extensions (purple, n = 92 cells) show a mean rupture force of 33.9 pN, whereas unhealthy cells (pink, n = 26) display a higher average force (44 pN). In contrast, dsDNA handles without the RGD ligand (blue, n = 85) and bare beads (green, n = 25) show nonspecific interactions with significantly lower forces (Favg = 11 pN and 2 pN, respectively). The stars indicate average forces.

主要实验及结论#

研究团队首先构建了结合力测试体系，如图1所示，他们合成了一段长为1558个碱基对的双链DNA柄，一端修饰RGD多肽用于特异性结合HeLa细胞表面的整合素受体，另一端修饰生物素并结合到微米级磁珠上。通过在微流控芯片上方精确调节磁体距离，磁镊可以对单个DNA拉绳施加方向垂直且连续增加的拉力。为了确保测量处于单分子水平，实验通过观测磁珠在xy平面内的旋转轨迹来确认单根DNA柄的系留状态。以0.7 pN/s的力加载速率加载时，当拉力超过受体与配体的结合阈值，磁珠便会脱离细胞表面。实验数据表明，健康HeLa细胞伸展端的整合素与RGD平均断裂力为33.9 pN，而缺少RGD的对照组断裂力仅为11 pN，纯磁珠的背景力为2 pN，这证实了该系统测量的是特异性受体-配体解离事件。

Fig.2 Effect of lipid rafts on the interaction between ligands and cell-surface receptors. (A) Schematic showing the role of lipid rafts in integrin clustering. In intact membranes (left), integrin α and β subunits are colocalized within cholesterol-rich lipid rafts, ready to bind RGD ligands. Treatment with MβCD (right) extracts membrane cholesterol and disrupts raft domains, leading to integrin dispersion and altered force profiles. (21) (B) Violin plots of rupture forces between integrin–RGD interactions on healthy cells with intact lipid rafts (left, n = 92 cells, Favg = 33.9 pN) and on cells with compromised lipid rafts after treatment with 10 μM methyl-β-cyclodextrin (MβCD) (right, n = 43, Favg = 30.2 pN). Stars depict average values, whereas arrowheads represent the cutoff values separating different populations in (A) and (B). (C) Violin plots of rupture forces for membrane–cholesterol interactions on healthy and MβCD-treated cells (left two plots: n = 139, Favg = 41.6 pN and n = 81, Favg = 32.7 pN, respectively) and nucleolin–AS1411 complex on healthy and MβCD-treated cells (right two plots: n = 62, Favg = 49.9 pN; and n = 30, Favg = 52.6 pN, respectively). (D) Quantification of the average number of beads bound per cell for different ligands under lipid raft-intact and MβCD-treated (lipid raft-compromised) conditions. While cholesterol-functionalized beads showed minimal change in the average number of beads bound per cell, a significant reduction was observed for RGD- and AS1411-functionalized beads after lipid raft was compromised by MβCD. “n” represents the number of cells. Error bars depict the standard deviations.

接着，研究人员重点探究了细胞膜异质性微区对分子结合力的调控作用，如图2所示。细胞膜上的脂质 rafts富含胆固醇与 sphingolipids，是受体聚集与信号传导的重要平台。当使用甲基-β-环糊精提取细胞膜胆固醇并破坏脂质 rafts后，整合素与RGD的断裂力分布发生了明显改变。未处理的完整细胞呈现出42.7 pN的高力群体与26.5 pN的低力群体，而破坏脂质 rafts后高力群体消失，整体力分布重构为30.2 pN。为了厘清力群体的分化是因为脂质微区直接加强了结合，还是因为不同整合素亚型的空间分配差异，研究团队引入了无亚型分化的核核仁素与AS1411适体系统作为对照。结果显示，AS1411与核核仁素的结合力在破坏脂质 rafts前后保持在49.9 pN和52.6 pN，并无统计学差异，这表明脂质 rafts对整合素-RGD结合力的强化主要是通过富集高亲和力的整合素亚型实现的。

Fig.3 Molecular binding strength is correlated with rupture force. Violin plots showing rupture forces for nucleolin–AS1411, integrin–RGD, and membrane–cholesterol interactions in the presence of MβCD (i.e., with compromised lipid rafts). Higher forces are observed when binding interactions are stronger. Dissociation constants (Kd) are taken from literature reports. (40−42) The averaged Kd values have broad ranges since they were measured by different methods in different systems (see Table S6). The notation “n” represents the number of molecules measured. Stars depict the average rupture forces for the bindings outside the lipid rafts. For nucleolin–AS1411 interaction, the average rupture forces within and outside lipid rafts are similar. Blue arrowheads depict the cutoff points between high and low force populations. Dotted straight line is a guide to eyes for the populations outside lipid rafts. Fig.4 Spatial and temporal evaluation of ligand–membrane interactions on HeLa cells. (A) Violin plots showing rupture forces between ligand-functionalized DNA tethers and their interacting partners located on the cell main bodies or cell extensions. RGD, main body (n = 56 cells, Favg = 34.8 pN), extension (n = 41, Favg = 32.2 pN). AS1411, main body (n = 36, Favg = 48.7 pN), extension (n = 30, Favg = 50.2 pN). Cholesterol, main body (n = 69, Favg = 42.2 pN), extension (n = 44, Favg = 41.2 pN). Results indicate ligand-specific differences in force profiles across cell regions, with RGD showing enhanced binding in the main body (with more population inside lipid rafts (46%) with respect to that in cell extensions (39%), p = 0.020, see SI Section S9 for detailed calculation) whereas AS1411 and cholesterol showing similar bindings between the main bodies and the cell extensions (p = 0.183 and 0.749, respectively, for AS1411 and cholesterol). (B) Violin plots comparing rupture forces for the ligands on HeLa cells in G1 and S cell cycle phases. RGD, G1 phase (n = 92 cells, Favg = 33.9 pN); S phase (n = 49, Favg = 40 pN). AS1411, G1 phase (n = 26, Favg = 50 pN); S phase (n = 22, Favg = 47.6 pN). Cholesterol, G1 phase (n = 54, Favg = 42.9 pN); S phase (n = 35, Favg = 40.6 pN). Stars depict the average forces for different conditions. Blue arrowheads represent the cutoff points between high and low force populations. For populations that do not have an obvious cutoff point, we used Gaussian fitting to determine (see SI Section S9).

此外，研究团队将目光投向了分子结合力的时空动态演变规律。在空间维度上，测量发现细胞主体的整合素-RGD结合力显著高于细胞突出延伸端。这一力学差异完美契合了细胞迁移的生理机制，即延伸端需要较弱且易于断裂的粘附以实现向外探索，而细胞主体则需要更强的锚定力来维持细胞形态与张力。在时间维度上，研究人员通过对比不同细胞周期阶段的测量数据发现，S期的整合素-RGD结合力明显高于G1期。这一现象揭示了细胞在DNA复制阶段会倾向于增强表面粘附并抑制迁移活动，从而将能量集中用于遗传物质的稳定合成。

Fig.5 Schematic of integrin–RGD binding measured by magnetic tweezers on HeLa cells during G1 phase (smaller nucleus) and S phase (larger nucleus). In G1 phase, the main body (quadrant 2, 1.15 beads/cell) exhibits 46% integrin population for adhesion and 54% for migration (see SI Section S10 for calculations), whereas cell extensions (quadrant 3, 1.25 beads/cell) show reduced adhesion (39%) but enhanced migration (61%). In S phase, the main body (quadrant 1, 1.12 beads/cell) displays the highest adhesion (68%) and lowest migration (32%), while binding events in cell extensions (quadrant 4) were not observed. The inset summarizes the distribution of adhesion (pink box) and migration (blue box) across different cell regions and phases.

总结与展望#

本研究成功建立了活细胞表面单分子力学谱分析的通用技术平台，证明了细胞表面分子结合强度并非一成不变，而是受到膜微领域、细胞空间定位以及细胞周期阶段的精确时空调控。这一发现打破了传统生物化学中将受体-配体结合亲和力视为静态常数的固有观念，为理解细胞粘附、机械信号转导以及生物力学响应提供了全新的物理视角。未来，该磁镊单分子力学测量技术有望进一步拓展应用于肿瘤细胞转移机制研究、靶向药物分子筛选以及细胞力学受体的精准靶向设计等领域。