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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

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 area of B cells that were placed on coverslip presenting antigens, consistent with the reported function of FAK inhibitor (Mlinaric-Rascan and Yamamoto, 2001) (A); Myosin IIA inhibitor dramatically reduced the proportion of B cells with internalized Alexa488-conjugated NP8-BSA antigen in the soluble format after reaction for 30 min, consistent with the reported function of Myosin IIA inhibitor (Vascotto et al., 2007) (B); Dynein inhibitor dramatically reduced the formation of cSMAC structure in B cell that were placed on planar lipid bilayers presenting antigens for 20 min, consistent with the reported function of Dynein inhibitor (Schnyder et al., 2011) (C). 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 at least 15 measurements in each group of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.013
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fig5s1: 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 area of B cells that were placed on coverslip presenting antigens, consistent with the reported function of FAK inhibitor (Mlinaric-Rascan and Yamamoto, 2001) (A); Myosin IIA inhibitor dramatically reduced the proportion of B cells with internalized Alexa488-conjugated NP8-BSA antigen in the soluble format after reaction for 30 min, consistent with the reported function of Myosin IIA inhibitor (Vascotto et al., 2007) (B); Dynein inhibitor dramatically reduced the formation of cSMAC structure in B cell that were placed on planar lipid bilayers presenting antigens for 20 min, consistent with the reported function of Dynein inhibitor (Schnyder et al., 2011) (C). 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 at least 15 measurements in each group of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.013

Mentions: We explored whether the conventional mechanosensor integrin LFA-1 expressed on the surface of B cells influences the patterned dependence on the mechanical forces in B cell activation. To address this question, we co-tethered both NP-TGT sensors and the adhesion molecule, intercellular adhesion molecule 1 (ICAM-1, which is a ligand for integrin LFA-1) on the surface of coverslip and similarly performed TIRFM imaging experiments. The addition of ICAM-1 dramatically enhanced the synaptic accumulation of the BCRs (Figure 5A), suggesting the increased sensitivity to antigen stimulation in the initiation of B cell activation, consistent with the published studies in both the BCR (Carrasco et al., 2004) and TCR system (Bachmann et al., 1997; Dustin, 2009). However, it was clear that the outside-in activation of integrin did not change the fact that B cell activation is dependent on mechanical forces with a similar multi-threshold effect (Figure 5B). To further confirm this conclusion, we inactivated the function of focal adhesion kinase (FAK), a member of the non-receptor protein-tyrosine kinase family, that is known to play a key role in the activation of integrin signaling pathways (Slack-Davis et al., 2007; Yu et al., 2012; Bashour et al., 2014). We pretreated B cells with FAK-specific inhibitor PF573-228 (Slack-Davis et al., 2007). We found that the pretreated B cells still maintained the general patterned dependence on mechanical forces with a multi-threshold effect (Figure 5C,D, Figure 5—figure supplement 1A). However, it was clear that the high-end (56 pN) and medium-level (43 pN) but not low-end (12 pN) mechanical force threshold was more influenced by the inactivation of integrin, suggesting that the breakthroughs of the medium-level and high-end threshold of mechanical forces are partially supported by the inside-out activation of integrin.10.7554/eLife.06925.012Figure 5.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.


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)

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 area of B cells that were placed on coverslip presenting antigens, consistent with the reported function of FAK inhibitor (Mlinaric-Rascan and Yamamoto, 2001) (A); Myosin IIA inhibitor dramatically reduced the proportion of B cells with internalized Alexa488-conjugated NP8-BSA antigen in the soluble format after reaction for 30 min, consistent with the reported function of Myosin IIA inhibitor (Vascotto et al., 2007) (B); Dynein inhibitor dramatically reduced the formation of cSMAC structure in B cell that were placed on planar lipid bilayers presenting antigens for 20 min, consistent with the reported function of Dynein inhibitor (Schnyder et al., 2011) (C). 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 at least 15 measurements in each group of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.013
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555871&req=5

fig5s1: 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 area of B cells that were placed on coverslip presenting antigens, consistent with the reported function of FAK inhibitor (Mlinaric-Rascan and Yamamoto, 2001) (A); Myosin IIA inhibitor dramatically reduced the proportion of B cells with internalized Alexa488-conjugated NP8-BSA antigen in the soluble format after reaction for 30 min, consistent with the reported function of Myosin IIA inhibitor (Vascotto et al., 2007) (B); Dynein inhibitor dramatically reduced the formation of cSMAC structure in B cell that were placed on planar lipid bilayers presenting antigens for 20 min, consistent with the reported function of Dynein inhibitor (Schnyder et al., 2011) (C). 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 at least 15 measurements in each group of three independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.013
Mentions: We explored whether the conventional mechanosensor integrin LFA-1 expressed on the surface of B cells influences the patterned dependence on the mechanical forces in B cell activation. To address this question, we co-tethered both NP-TGT sensors and the adhesion molecule, intercellular adhesion molecule 1 (ICAM-1, which is a ligand for integrin LFA-1) on the surface of coverslip and similarly performed TIRFM imaging experiments. The addition of ICAM-1 dramatically enhanced the synaptic accumulation of the BCRs (Figure 5A), suggesting the increased sensitivity to antigen stimulation in the initiation of B cell activation, consistent with the published studies in both the BCR (Carrasco et al., 2004) and TCR system (Bachmann et al., 1997; Dustin, 2009). However, it was clear that the outside-in activation of integrin did not change the fact that B cell activation is dependent on mechanical forces with a similar multi-threshold effect (Figure 5B). To further confirm this conclusion, we inactivated the function of focal adhesion kinase (FAK), a member of the non-receptor protein-tyrosine kinase family, that is known to play a key role in the activation of integrin signaling pathways (Slack-Davis et al., 2007; Yu et al., 2012; Bashour et al., 2014). We pretreated B cells with FAK-specific inhibitor PF573-228 (Slack-Davis et al., 2007). We found that the pretreated B cells still maintained the general patterned dependence on mechanical forces with a multi-threshold effect (Figure 5C,D, Figure 5—figure supplement 1A). However, it was clear that the high-end (56 pN) and medium-level (43 pN) but not low-end (12 pN) mechanical force threshold was more influenced by the inactivation of integrin, suggesting that the breakthroughs of the medium-level and high-end threshold of mechanical forces are partially supported by the inside-out activation of integrin.10.7554/eLife.06925.012Figure 5.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.

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