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. 2015 Aug 10;4:e06925. doi: 10.7554/eLife.06925

Figure 2. The synaptic accumulation of the IgM-BCRs is dependent on mechanical forces and exhibits a multi-threshold effect.

(A, B) Statistical quantification of the synaptic recruitment of IgM-BCR in J558L cells expressing naive B1-8-IgM-BCR (A) and primary naive B cells expressing B1-8-IgM-BCR (B). Bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 40 cells over three independent experiments. (C) Representative TIRFM images showing the dynamics of the synaptic accumulation of IgM-BCRs from J558L cells expressing naive B1-8-IgM-BCR in contact with coverslip presenting 12 pN, 43 pN or 56 pN NP-TGT sensors at the indicated time points. Scale bar is 1.5 μm. (D, E) Comparisons of averaged traces showing the dynamic accumulation of naive IgM-BCRs into the immunological synapse (D) and the growing features of the size of contact area (E) for J558L cells expressing naive B1-8-IgM-BCR as demonstrated in (C) in a 10 min TIRFM imaging time course. Bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data are from at least 20 cells over two independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.06925.006

Figure 2.

Figure 2—figure supplement 1. The contact area after IgM-BCR activation is dependent on mechanical forces with multi-threshold effects and such a pattern is still evident at low density of NP-TGT sensor.

Figure 2—figure supplement 1.

(A) Statistical analyses of the size of the contact area of primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice when encountering of the indicated types of NP-TGT sensors. (B) The representative image of NP-TGT molecule indicated by FITC-conjugated NP-specific antibodies within a counting area (473.1 μm2). Scale bar is 1.5 μm. (C) The conversion and strong linear correlation between the MFI and the density of FITC-conjugated NP-specific antibody on coverslip. NP-specific antibody was used to indicate the density of NP-TGT sensor on coverslip. The surface density is quantified at the appropriate incubation concentration of NP-TGT sensor to achieve well-separated and approximately round spots in TIRF imaging (B), which were subsequently analyzed by a Matlab supported 2D Gaussian fitting code (Source code 1) to perform the counting as reported in our previous studies (Liu et al., 2010a). The equation of the fitted linear regression is: Surface density (per counting area, about 473.1 μm2) = 2.42 × MFI, R square value for the linear fitting is 0.99. (D) Quantification of the MFI of NP-specific antibody on the coverslip at different incubation concentration of NP-TGT sensor. (E) Surface density of NP-TGT sensors at different incubation concentration when were coated on coverslip as calculated by combining the data in C and D. (F, G) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR (F) or primary naive B cells expressing B1-8-IgM-BCR (G) that were placed on coverslip coated with surface density of 4.0 molecule/μm2 of 12 pN, 43 pN, and 56 pN NP-TGT sensors. Bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were from at least 30 cells over three independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.06925.007
Figure 2—figure supplement 2. The patterned dependence on the mechanical forces of IgM-BCR activation does not rely on BCR internalization.

Figure 2—figure supplement 2.

(A) Representative confocal images showing the efficient internalization of BCR and antigen molecules in primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice that were interacted with soluble NP8-BSA for 10 min. Scale bar is 1.5 μm. (B) Side view confocal images showing the lack of internalization of the pre-stained BCR molecules in primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice that were placed on coverslip presenting 12 pN, 43 pN or 56 pN NP-TGT sensors for 10 min. B cells were pre-stained with DyLight 649 AffiniPure Fab Fragment Goat Anti-Mouse IgM, µ Chain Specific antibodies before the imaging experiment. Scale bar is 2 μm. (C) Statistical quantification of the percentage of B cells with internalization of BCRs as represented in A and B. (D) Statistical quantification to show that B cell internalization can be blocked by MDC inhibitor in the condition that the B cells were pretreated by MDC before were activated for 10 min by soluble NP8-BSA as represented in A. (E, F) The synaptic accumulation of IgM-BCRs in primary naive B cells expressing B1-8-IgM-BCR from B1-8 Tg mice in contact with the indicated types of NP-TGT sensors. In this experiment, B cells were pretreated with either DMSO or MDC following a protocol as detailed in ‘Materials and methods’ section. Cross comparison strategy were used in these figures. In all of these plots, bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were at least from 30 cells or 15 measurements in each group of three independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.06925.008