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The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold.

Wan Z, Chen X, Chen H, Ji Q, Chen Y, Wang J, Cao Y, Wang F, Lou J, Tang Z, Liu W - Elife (2015)

Bottom Line: We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect.Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold.These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Protein Sciences, Tsinghua University, Beijing, China.

ABSTRACT
B lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs.

No MeSH data available.


Related in: MedlinePlus

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 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
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fig2s1: 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 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

Mentions: (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 applied by the B1-8-BCR is larger than the predefined tension force of a certain NP-TGT (for example 56 pN is depicted in the figure). FITC-conjugated anti-NP antibody is used to quantify the molecule density of each different type of NP-TGT sensors tethered on coverslip. (B) The dsDNA geometries and predefined tension force of all eight NP-conjugated TGT sensors and one control TGT without NP conjugation. (C) Representative TIRFM images showing the dynamics of the synaptic accumulation of BCRs from J558L cells expressing B1-8-IgM-BCR in contact with coverslip presenting 56 pN NP-TGT sensor or control TGT (NC) at the indicated time points. Scale bar is 1.5 μm. (D) Comparisons of averaged traces showing the dynamic accumulation of BCRs as demonstrated in (C) in a 13 min TIRFM imaging time course. Bars represent mean ±SEM. Data were from at least 20 cells over three independent experiments. (E) Primary mature naive B cells from wild-type C57BL/6 mice expressing non-NP-specific IgM-BCR did not initiate the activation when encountering 56 pN NP-TGT sensor compared to the response of the same B cells encountering 56 pN TGT sensor without NP conjugation. Biotin-conjugated goat anti-mouse IgM surrogate antigens were used as a positive control to efficiently drive the synaptic accumulation of IgM-BCRs in B cell activation. 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. (F) Quantification of the mean fluorescence intensity (MFI) of FITC-conjugated NP-specific antibodies on the surface of coverslip tethering the same amount of NP-TGT sensors. Bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. The surface density is 29.0 molecule/µm2, seeing more in Figure 2—figure supplement 1.


The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold.

Wan Z, Chen X, Chen H, Ji Q, Chen Y, Wang J, Cao Y, Wang F, Lou J, Tang Z, Liu W - Elife (2015)

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 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
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fig2s1: 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 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
Mentions: (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 applied by the B1-8-BCR is larger than the predefined tension force of a certain NP-TGT (for example 56 pN is depicted in the figure). FITC-conjugated anti-NP antibody is used to quantify the molecule density of each different type of NP-TGT sensors tethered on coverslip. (B) The dsDNA geometries and predefined tension force of all eight NP-conjugated TGT sensors and one control TGT without NP conjugation. (C) Representative TIRFM images showing the dynamics of the synaptic accumulation of BCRs from J558L cells expressing B1-8-IgM-BCR in contact with coverslip presenting 56 pN NP-TGT sensor or control TGT (NC) at the indicated time points. Scale bar is 1.5 μm. (D) Comparisons of averaged traces showing the dynamic accumulation of BCRs as demonstrated in (C) in a 13 min TIRFM imaging time course. Bars represent mean ±SEM. Data were from at least 20 cells over three independent experiments. (E) Primary mature naive B cells from wild-type C57BL/6 mice expressing non-NP-specific IgM-BCR did not initiate the activation when encountering 56 pN NP-TGT sensor compared to the response of the same B cells encountering 56 pN TGT sensor without NP conjugation. Biotin-conjugated goat anti-mouse IgM surrogate antigens were used as a positive control to efficiently drive the synaptic accumulation of IgM-BCRs in B cell activation. 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. (F) Quantification of the mean fluorescence intensity (MFI) of FITC-conjugated NP-specific antibodies on the surface of coverslip tethering the same amount of NP-TGT sensors. Bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. The surface density is 29.0 molecule/µm2, seeing more in Figure 2—figure supplement 1.

Bottom Line: We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect.Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold.These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Protein Sciences, Tsinghua University, Beijing, China.

ABSTRACT
B lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs.

No MeSH data available.


Related in: MedlinePlus