Megrelis Laura, Delon Jérôme
Inserm U1016, Institut Cochin; Cnrs, UMR8104; Université Paris Descartes, Sorbonne Paris Cité
Inserm U1016, Institut Cochin; Cnrs, UMR8104; Université Paris Descartes, Sorbonne Paris Cité;
J Vis Exp. 2014 Dec 12(94):52140. doi: 10.3791/52140.
Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.
细胞通过在一个称为极化的过程中失去圆形形状以及改变许多蛋白质的亚细胞定位来响应趋化因子刺激。经典成像技术已被用于研究这些现象。然而，它们需要手动采集许多细胞，随后对数十个细胞的形态和染色共定位进行耗时的定量分析。在此，描述了一种快速且强大的方法，使用一种结合了显微镜和细胞仪优点的成像流式细胞术技术，对由数千个细胞组成的样本进行这些现象的研究。利用用CCL19刺激的T淋巴细胞并对MHC I类分子和丝状肌动蛋白进行染色，提出了一种门控策略，以同时测量形状改变的程度以及受CCL19信号影响的标记物的共定位程度。此外，这种门控策略使我们能够观察到CXCL12刺激后极化T细胞中丝状肌动蛋白（在前端）和磷酸化埃兹蛋白-根蛋白-莫伊辛（磷酸化-ERM）蛋白（在后端）的分离。该技术对于观察两种不同因素对极化的阻断作用也很有用：一种药理学抑制剂对肌动蛋白聚合的抑制以及Par6/非典型蛋白激酶C信号通路突变体的表达。因此，有证据表明该技术对于分析形态学改变和蛋白质重新分布都很有用。
