The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold
- Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, China
- Chinese Academy of Science, China
- Chinese Academy of Sciences, China
Figures
Figure 1 with 1 supplement
The construction of B1-8-BCR-specific NP-TGT mechanical force sensor system.
(A) Schematic representation of the NP-TGT and NP-specific B1-8-BCR expressing B cells. NP-TGT molecule is immobilized on the surface of coverslip, which will get ruptured if the mechanical force …
Figure 1—figure supplement 1
The quality control of NP-TGT sensor based experimental system.
(A) The quality control of the purified NP-ssDNA by mass spectrum. (B, C) No obvious dissociation of the neutravidin was detected in a 10 min incubation time course. In B, two-color TIRF images …
Figure 2 with 2 supplements
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 …
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.
(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 …
Figure 2—figure supplement 2
The patterned dependence on the mechanical forces of IgM-BCR activation does not rely on BCR internalization.
(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 …
Figure 3
The volume of the IgM-BCR microcluster produced by different NP-TGT sensors is dependent on mechanical forces and exhibits a similar multi-threshold effect.
(A) Representative original (top panel), pseudo-colored 2D (middle panel), and 2.5D Gaussian images (bottom panel) of typical BCR microclusters induced by 12, 16, 23, 33, 43, 50, 54, and 56 pN …
Figure 4
The strength of IgM-BCR signaling is dependent on mechanical forces.
(A–C) Statistical quantification of the synaptic recruitment of pSyk (A), pPLCγ2 (B), and pTyr (C) in primary naive B cells expressing B1-8-IgM-BCR that were placed on coverslip presenting 12 pN, 43 …
Figure 5 with 1 supplement
The patterned dependence on the mechanical forces of IgM-BCR activation does not rely on LFA-1 mediated adhesion and dynein, and is only partially dependent on myosin IIA.
(A, B) The synaptic accumulation of IgM-BCRs in primary naive B cells expressing B1-8-IgM-BCR in contact with the indicated types of NP-TGT sensors with or without ICAM-1 co-tethering. Cross …
Figure 5—figure supplement 1
Functional test of pharmaceutical inhibitors.
(A–C) Positive control experiments were performed to show that each inhibitor used in Figure 5 is working. These experiments showed that FAK inhibitor significantly reduced the size of the contact …
Figure 6 with 1 supplement
The activation of isotype-switched IgG-BCRs or IgE-BCRs on memory B cells requires either no tension or a mechanical force below 12 pN.
(A, B) Statistical quantification of the synaptic accumulation of IgG-BCR and the volume of the contact area of J558L cells expressing memory B1-8-IgG-BCR encountering 12 pN, 43 pN, or 56 pN NP-TGT …
Figure 6—figure supplement 1
Quantification of the accumulation of IgG-BCR in recognition of NP-TGT sensors at different surface density.
(A–C) Quantification of the synaptic accumulation of IgG-BCRs in J558L cells expressing memory B1-8-IgG-BCR that were placed on coverslip coated with NP-TGT sensors at a density of 29.0 molecule/μm2 …
Figure 7
The lower mechanical force threshold of IgG-BCR activation is dependent on its cytoplasmic tail.
(A) Schematic illustration of the strategy of swapping the cytoplasmic tail of B1-8-IgG- or B1-8-IgM-BCRs. (B–G) Quantification of the synaptic accumulation of BCRs in J558L cells expressing memory …
Author response image 1
Videos
Video 1
Time lapse images showing the dynamics of the synaptic accumulation of BCRs from J558L cells expressing naive B1-8-IgM-BCR in contact with coverslip presenting 56 pN NP-TGT or control TGT (NC) sensor.
Scale bar is 1.5 μm. The video was recorded with a 4-s time interval and is shown at 30 frames per second. Related to Figure 1C.
Video 2
Representative time lapse 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 sensor.
Scale bar is 1.5 μm. The video was recorded with a 4-s time interval and is shown at 30 frames per second. Related to Figure 2C.
Video 3
Time lapse images showing the dynamics of the synaptic accumulation of IgG-BCRs from J558L cells expressing memory B1-8-IgG-BCR in contact with coverslip presenting 12 pN, 56 pN NP-TGT or control TGT (NC) sensor at the indicated time points.
Scale bar is 1.5 μm. The video was recorded with a 4-s time interval and is shown at 30 frames per second. Related to Figure 6G.
Additional files
-
Source code 1
Matlab supported 2D Gaussian fitting code.
- https://doi.org/10.7554/eLife.06925.018
