Liu Chinky Shiu Chen, Biswas Parijat, Ganguly Dipyaman
IICB-Translational Research Unit of Excellence, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Department of Biological Sciences, Indian Association for Cultivation of Science, Kolkata, India.
Bio Protoc. 2024 Oct 5;14(19):e5079. doi: 10.21769/BioProtoc.5079.
The process of T-lymphocyte migration involves a complex interplay of chemical and mechanical signals. Mechanotransduction mechanisms in T lymphocytes enable them to efficiently navigate through diverse architectural and topographical features of the dynamic tissue macro- and micro-niches encountered during immune responses. Piezo1 mechanosensors are crucial for driving optimal T-cell migration by driving actin-cytoskeletal remodeling. Chemokine-stimulated T lymphocytes demonstrate significant asymmetry or polarity of Piezo1 and actin along the cell axis. The establishment and maintenance of polarity in migrating cells are paramount for facilitating coordinated and directional movements along gradients of chemokine signals. Live-cell imaging techniques are widely employed to study the trajectories of migrating cells. Our approach expands upon current methodologies by not only tracking migrating cells but also imaging fluorescently labeled cellular components. Specifically, our method enables measurement of protein enrichment in the front and rear halves of the moving cell by analyzing the temporal direction of cell trajectories, subsequently bisecting the cell into front-back halves, and measuring the intensities of the fluorescent signals in each cell half at each time frame. Our protocol also facilitates the quantification of the angular distribution of fluorescent signals, enabling visualization of the spatial distribution of signals relative to the direction of cell migration. The protocol describes the examination of polarity in chemokine-treated Jurkat cells transfected with Piezo1-mCherry and actin-GFP constructs. This approach can be extended to live-cell imaging and polarity assessment of other fluorescently labeled proteins. Key features • This experimental protocol allows real-time imaging of Jurkat cells expressing two fluorescent proteins (Piezo1 mCherry and actin-GFP). • Measures cell polarity by examining spatial enrichment of Piezo1 and actin proteins within the front and rear halves of a moving Jurkat cell. • The protocol enables analysis of cell polarity in 2D tracks of moving cells. • Polarity analysis includes measuring fluorescent signal intensities in front-rear halves of a moving cell and calculation of signal polarization angles relative to the cell trajectory.
T淋巴细胞迁移过程涉及化学信号和机械信号的复杂相互作用。T淋巴细胞中的机械转导机制使其能够在免疫反应中遇到的动态组织宏观和微观生态位的各种结构和地形特征中高效导航。Piezo1机械传感器通过驱动肌动蛋白细胞骨架重塑，对驱动最佳T细胞迁移至关重要。趋化因子刺激的T淋巴细胞在细胞轴上表现出Piezo1和肌动蛋白的显著不对称或极性。迁移细胞中极性的建立和维持对于促进沿趋化因子信号梯度的协调和定向运动至关重要。活细胞成像技术被广泛用于研究迁移细胞的轨迹。我们的方法不仅通过跟踪迁移细胞，还通过对荧光标记的细胞成分进行成像，对现有方法进行了扩展。具体而言，我们的方法通过分析细胞轨迹的时间方向，随后将细胞分为前后两半，并在每个时间帧测量每个细胞半部分的荧光信号强度，从而能够测量移动细胞前后两半中的蛋白质富集情况。我们的方案还便于对荧光信号的角度分布进行量化，从而能够可视化信号相对于细胞迁移方向的空间分布。该方案描述了对用Piezo1-mCherry和肌动蛋白-GFP构建体转染的趋化因子处理的Jurkat细胞中的极性进行检测。这种方法可以扩展到对其他荧光标记蛋白的活细胞成像和极性评估。关键特性•本实验方案允许对表达两种荧光蛋白（Piezo1 mCherry和肌动蛋白-GFP）的Jurkat细胞进行实时成像。•通过检查移动的Jurkat细胞前后两半内Piezo1和肌动蛋白蛋白的空间富集情况来测量细胞极性。•该方案能够分析移动细胞二维轨迹中的细胞极性。•极性分析包括测量移动细胞前后两半中的荧光信号强度，以及计算相对于细胞轨迹的信号极化角度。
