Real-time imaging of Ca(2+) mobilization and degranulation in mast cells

Abstract

Mast cells play a key role in allergy and inflammation processes as part of the immune response. The activation of mast cells via antigen binding and cross-linking of IgE receptors initiates the onset of dramatic calcium (Ca(2+)) mobilization dynamics that promote the release of mediators of inflammation and allergy. Ca(2+) signaling in mast cells has been studied extensively using a variety of research tools and techniques. In these studies, a large number of proteins have been identified to participate in various stages of these processes. Here we describe single-cell imaging as an important approach for examining Ca(2+) signaling and exocytosis in mast cells. Single-cell imaging tools have advanced significantly over the last 10 years, in part due to improvements in microscope technology and in part due to the development of a new generation of Ca(2+) indicators and genetically encoded Ca(2+) sensors. The single-cell imaging techniques described here provide the spatial and temporal resolution required to decipher the signaling events that are critical for mast cell functions.

Figure 1. Elongated morphology of mast cells

Image of RBL-2H3 mast cells in culture demonstrates their elongated morphology. Typically, cells will have one or two protrusions extending to various lengths away from the cell body. Morphological features of mast cells (length, angle, volume etc.) can be quantified using various overlay tools in Image J or other image processing software. Fluorescent label is Fluo4.

Figure 2. Imaging and quantifying Ca²⁺ waves and puffs

In these examples, RBL-2H3 cells expressing the Ca²⁺ sensor GCaMP2 were monitored for fluorescence intensity as a function of time before and after stimulation by antigen, and changes in Ca²⁺ concentration are presented in pseudo colors (warmer colors represent higher Ca²⁺ levels). A) Upper: image of cell, with direction of Ca²⁺ wave indicated; lower: time line analysis (“Virtual Linescan” plugin for Image J) provides both spatial and temporal information about changes in Ca²⁺ concentration. This analysis measures the changes in florescence over time along a designated line across the cell. Here the line width is 2 pixels. This visualization can be used to determine the point of origin of the wave. B) Wave propagation can be visualized and quantified by plotting the fluorescence signal from two regions of interest (ROI; 1-protrusion, 2- cell body) over time and measuring the time difference between them (Δt) as a function of distance (in μm; “Z Profiler” plug-in for Image J). The ROI quantification can be used to determine wave velocity. C) As described for waves in A, time line analysis can be used to detect and visualize the spatial attributes of Ca²⁺ puffs. In this example, the puffs are localized and confined to the cell's protrusion. D) Temporal dynamics of Ca²⁺ puffs can be quantified from fluorescence (F) vs time plots, as in the case of waves. Here we integrated the fluorescence intensity over the cell body (2) or protrusion (1) to show the transient Ca²⁺ elevation during puffs.

Figure 3. Visualizing and quantifying Ca²⁺ oscillations

RBL-2H3 cell expressing GCaMP2 was stimulated with Ag and imaged for ∼100 sec. A) Time line analysis along the cell body (upper panel) reveals repetitive Ca²⁺ oscillations (middle panel; warmer colors represent higher Ca²⁺). Lower panel illustrates a “3D” representation of the kymograph (“Interactive 3D Surface Plot” plug-in for Image J), for enhanced visualization of spatial and temporal dynamics of the oscillations. B) Integration of the fluorescence signal over the cell body plotted against time can be used to quantify the oscillation's dynamics. With this approach, one can measure the oscillation peak-to-peak interval (a, frequency) and peak width (b). Additional parameters can be extracted including oscillation rise phase (representing Ca²⁺ influx), down phase (representing Ca²⁺ clearance), and the integrated Ca²⁺ elevation during various time points.

Figure 4. Imaging of single cell degranulation events using FITC-dextran

A) Individual frames taken from ∼3 minutes of imaging RBL-2H3 cells loaded with FITC-dextran and stimulated with Ag. Arrows point to some of the degranulation events taking place in each frame. B) For initial evaluation of the total degranulation magnitude from cells, a Z projection (integrating 160 sec) can be used which shows the sum of all or a range of frames.

Figure 5. Quantification of single cell degranulation events using FITC-dextran

As for Ca²⁺ waves and oscillations, integration of fluorescence intensity over ROI or time line analysis can be used for quantification of degranulation visualized with FITC-dextran. A) Time line analysis (middle panel) over cell of interest (upper left panel, broader yellow line) reveals the spatial and temporal dynamics of degranulation events as they occur along the cell in a single kymograph. Further enhancement of visualization can be achieved by a 3D representation (lower panel). This analysis can be used, for example, to evaluate the spatial distribution of degranulation events across the cell. B) Integration of fluorescence intensity over individual degranulation events provides a simple tool for quantifying the exocytosis dynamics. Here we show a fast exocytosis event (left panel, 1) and a biphasic degranulation event where the granule undergoes two-step fusion and release of contents (right panel, 2). This method enables detection and distinction between “kiss and run”, “full” and “compound” types of exocytosis common to mast cells (Cohen, Corwith et al. 2012).

Figure 6. Simultaneous imaging of Ca²⁺ mobilization and degranulation in cells using Fura-Red and FITC-dextran

In these experiments a fluorescent Ca²⁺ indicator (synthetic or GECI) is used in combination with FITC-dextran loaded into to cells. In this case we used Fura-Red to measure Ca²⁺ changes. A) Time line analysis of Ca²⁺ concentration changes (red) or degranulation (green) channels is shown separately (left and middle lower panels) and combined to create an overlay kymograph (right panel). The overlay kymograph provides temporal and spatial view of the Ca²⁺ changes and degranulation throughout the cell. B) Integration of fluorescence over ROI in red or green channels provides high temporal resolution of correlated Ca²⁺ mobilization and degranulation events over particular regions of the cell (e.g., protrusions or cell body), as can be seen in the plot of ΔF/Fo vs time (lower panel). Black trace shows Ca²⁺ response, and individual colors show different exocytic events.