Robert Philippe, Biarnes-Pelicot Martine, Garcia-Seyda Nicolas, Hatoum Petra, Touchard Dominique, Brustlein Sophie, Nicolas Philippe, Malissen Bernard, Valignat Marie-Pierre, Theodoly Olivier
LAI, Aix-Marseille University, CNRS, INSERM U1067 Adhésion Cellulaires et lnflammation, Turing Center for Living Systems, Marseille, France.
Aix-Marseille University, CNRS, INSERM U1104 Centre d'immunologie de Marseille-Luminy, Marseille, France.
Front Bioeng Biotechnol. 2021 Apr 7;9:625366. doi: 10.3389/fbioe.2021.625366. eCollection 2021.
Immune cells have the ubiquitous capability to migrate disregarding the adhesion properties of the environment, which requires a versatile adaptation of their adhesiveness mediated by integrins, a family of specialized adhesion proteins. Each subtype of integrins has several ligands and several affinity states controlled by internal and external stimuli. However, probing cell adhesion properties on live cells without perturbing cell motility is highly challenging, especially . Here, we developed a novel method using micron-size beads pulled by flow to functionally probe the local surface adhesiveness of live and motile cells. This method allowed a functional mapping of the adhesiveness mediated by VLA-4 and LFA-1 integrins on the trailing and leading edges of live human T lymphocytes. We show that cell polarization processes enhance integrin-mediated adhesiveness toward cell rear for VLA-4 and cell front for LFA-1. Furthermore, an inhibiting crosstalk of LFA-1 toward VLA-4 and an activating crosstalk of VLA-4 toward LFA-1 were found to modulate cell adhesiveness with a long-distance effect across the cell. These combined signaling processes directly support the bistable model that explains the emergence of the versatile guidance of lymphocyte under flow. Molecularly, Sharpin, an LFA-1 inhibitor in lymphocyte uropod, was found involved in the LFA-1 deadhesion of lymphocytes; however, both Sharpin and Myosin inhibition had a rather modest impact on adhesiveness. Quantitative 3D immunostaining identified high-affinity LFA-1 and VLA-4 densities at around 50 and 100 molecules/μm in basal adherent zones, respectively. Interestingly, a latent adhesiveness of dorsal zones was not grasped by immunostaining but assessed by direct functional assays with beads. The combination of live functional assays, molecular imaging, and genome editing is instrumental to characterizing the spatiotemporal regulation of integrin-mediated adhesiveness at molecular and cell scales, which opens a new perspective to decipher sophisticated phenotypes of motility and guidance.
免疫细胞具有无视环境黏附特性而进行迁移的普遍能力，这需要通过整合素（一类特殊的黏附蛋白家族）介导对其黏附性进行多种适应性调节。整合素的每个亚型都有几种配体以及由内部和外部刺激控制的几种亲和力状态。然而，在不干扰细胞运动性的情况下探测活细胞的黏附特性极具挑战性，尤其是……在此，我们开发了一种新方法，利用流动牵拉的微米级珠子对活的运动细胞的局部表面黏附性进行功能探测。该方法能够对活的人类T淋巴细胞的后缘和前缘由VLA - 4和LFA - 1整合素介导的黏附性进行功能图谱绘制。我们发现细胞极化过程增强了VLA - 4整合素介导的对细胞后部的黏附性以及LFA - 1整合素介导的对细胞前部的黏附性。此外，还发现LFA - 1对VLA - 4的抑制性串扰以及VLA - 4对LFA - 1的激活性串扰可通过细胞远距离效应调节细胞黏附性。这些联合信号传导过程直接支持双稳态模型，该模型解释了淋巴细胞在流动条件下多功能导向的出现。在分子层面，发现淋巴细胞尾足中的LFA - 1抑制剂Sharpin参与淋巴细胞的LFA - 1去黏附过程；然而，Sharpin和肌球蛋白抑制对黏附性的影响都相当有限。定量三维免疫染色确定在基底黏附区域高亲和力LFA - 1和VLA - 4的密度分别约为50和100个分子/μm²。有趣的是，背侧区域的潜在黏附性无法通过免疫染色检测到，而是通过珠子直接功能测定来评估。活细胞功能测定、分子成像和基因组编辑的结合有助于在分子和细胞尺度上表征整合素介导的黏附性的时空调节，这为解读复杂的运动性和导向表型开辟了新视角。
