Understanding the initiation of B cell signaling through live cell imaging

Abstract

Antibody responses are initiated by the binding of antigens to clonally distributed cell surface B cell receptors (BCRs) that trigger signaling cascades resulting in B cell activation. Using conventional biochemical approaches, the components of the downstream BCR signaling pathways have been described in considerable detail. However, far less is known about the early molecular events by which the binding of antigens to the BCRs initiates BCR signaling. With the recent advent of high resolution, high speed, live cell, and single molecule imaging technologies, these events are just beginning to be elucidated. Understanding the molecular mechanisms underlying the initiation of BCR signaling may provide new targets for therapeutics to block dysregulated BCR signaling in systemic autoimmune diseases and in B cell tumors and to aid in the design of protein subunit vaccines. In this chapter, we describe the general procedures for using these new imaging techniques to investigate the early events in the initiation of BCR signaling.

Figure 1

System Design. Shown is a generalized diagram one of the TIRFM rigs used in the experiments described. The pertinent sections that discuss the specific components are noted beneath the figure. All optical components to the right of the fiber optic cable (FO) are mounted on a metal breadboard with ¼” screws on standard post mounts. Three lasers (boxes on right) are used to provide five usable excitation lines (wavelengths in nm indicated). After reflection on a primary mirror (1°), the argon laser lines are directed to the AOTF via a dichroic mirror (DC1). The krypton-argon lines pass through both DC1 and DC2. After reflection on a primary mirror (1°), the 440 nm diode laser line is directed to the AOTF via DC2. Power at the 440 nm laser head is computer controlled. Control boxes are required to interface with the computer software and are depicted as black boxes. Thin black lines at the bottom of the figure indicate computer connections. The AOTF and excitation filter wheel (FW) are linked to their respective control boxes, which in turn, are coupled to the PC workstation and are controlled by MetaMorph acquisition software. The user selects the laser line via the AOTF and blocks unneeded lines with the filter wheel. The selected line is directed to the laser launch (LL) lens to allow entry into the fiber optic cable. The fiber optic cable is coupled to the TIRF illuminator (TIRF IL). The TIRF angle is controlled through the software via a motorized actuator (ACT).

Figure 2

Single molecule TIRF imaging. (A) Trajectories of individual BCR molecules accumulated over the entire time course of Supplementary Movie 1. (B) Cumulative probability plots of the diffusion coefficients of individual BCR molecules were obtained from time-lapse TIRF movies (such as those shown in Supplementary Movie 1) of human peripheral blood B cells labeled with DyLight 649-Fab anti-IgM and placed on bilayers with (blue curve) or without (red curve) goat anti-human IgM F(ab’)₂ Ag. The trajectories used to construct each probability curve were collected from two independent experiments (n = 1871 with Ag; n = 3622 without Ag). Also given are the percent of mobile and immobile BCRs for cells with and without Ag..

Figure 3

Two-color time-lapse TIRF images capture the response of B cells upon initial contact with Ag-containing PLBs. Upper Panel: IgM-High J558L B cells labeled with AlexaFluor 568-Fab anti-IgM were placed on PLBs containing NIP1-His12 Ag and were examined by TIRFM over 120 s by imaging Igα-YFP (green) and AlexaFluor 568-Fab anti-IgM (red). Bars, 1.5 μm. Bottom panel: pseudo-color 2.5D Gaussian images of one representative Alexa Fluor 568–Fab anti-IgM microcluster are shown at the indicated times.

Figure 4

Imaging BCR signaling. (A) Two-color TIRF images show the distribution of the BCR and accumulation of pSyk on the membrane of human peripheral blood B cells that were placed on bilayers without (top panels) or with (bottom panels) goat anti-human IgM F(ab’)₂ Ag for 10 min, fixed and labeled as described in Section 3.3. Specifically, BCRs and pSyk are visualized using TIRFM by imaging DyLight 649-Fab anti-IgM (red) and AlexaFluor 488-labeled pSyk, respectively. Bars, 1.5 μm. (B) pSyk mean FI and pSyk cluster number quantified from several TIRF images of IgM-expressing B cells placed on bilayers without (gray circles) or with Ag (black triangles) as shown in (A). Each data point represents one cell analyzed in one of three independent experiments and the bars indicate the mean ± SD.