Measuring Piezo1 and Actin Polarity in Chemokine-Stimulated Jurkat Cells During Live-Cell Imaging

Abstract

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.

Keywords: Actin-GFP imaging; Angular distribution; Cell polarity; Jurkat cell line; Piezo1 mCherry; Time-lapse confocal imaging.

Figure 1.. Generation of 2D time lapse videos by performing maximum intensity Z-projection of the multi-channels/multi-time-points hyperstacks.

The multichannel 2D time hyperstack was split into two time-lapse videos corresponding to actin-GFP (green) and Piezo1 mCherry (red).

Figure 2.. Preprocessing of 2D time-lapse stack before ROI detection.

A. Gamma adjustment enhances the contrast between the object of interest and background signal. B. Background signal correction involves selecting an ROI in the background region, measuring its average intensity, and subtracting the signal from the original image. C. The maximum intensity projection along the time axis generates a comprehensive view of the cell’s overall trajectory over the course of imaging. Defining this ROI facilitates easier detection and analysis of the cell within it across all time points.

Figure 3.. Mapping out cell ROIs.

A. A generalized analysis scheme for ROI detection using thresholding and creation of binary masks (0–255). B. Resulting binary masks and outline of analyzed cell ROI at each time frame. Both actin-GFP and Piezo1-mCherry channels have been shown for the detected ROI.

Figure 4.. Bisecting cell ROI into front and back halves.

A. A cell ROI is fitted into an ellipse and its shape parameters are measured. The centroids (X_C, Y_C) of the cell ROI at subsequent time frames (T_j-1 and T_j) are used to calculate the cell displacement. The ROI is split into two halves along the minor axis (perpendicular to the major axis). θ = angle perpendicular to the major axis, φ = angle (radians) subtended by the minor axis. To determine the back and front half of the bisected ROI, dist1 and dist2 are calculated. dist1 < dist2. dist1 corresponds to the back half and dist2 corresponds to the front half of the bisected ROI. (xh1, yh1): half-centers of front half; (xh2, yh2): half-centers of back half. B. The cell ROIs across time frames (F0–F10) were fitted into an ellipse. The ellipse was bisected along the minor axis (perpendicular to the major axis) to generate two halves of the moving cell.

Figure 5.. Measuring live-cell polarity of Piezo1 mCherry and actin-GFP in chemokine-stimulated Jurkat cells.

A. Front/back (F/B) polarity of Piezo1 and actin of a single cell. Each point corresponds to F/B value at a specific time frame of a single moving cell. B. Polar plots depicting relative angles of Piezo1 (upper panel) and actin (lower panel). The relative angles correspond to a single cell against different time frames. C. Vector plot of a single cell depicting the direction of cell trajectory (blue), Piezo1 polarization (red), and actin polarization (green) at each time frame.