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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 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 showing the formation of a typical immunological synapse (IS) of a single B cell (BCR, red color) and the corresponded Alexa488-conjugated neutravidin within the B cell IS. Also given are control neutravidin TIRF images from an area without B cells. Scale bar is 5 μm. In C, the statistical analyses of the MFI of Alexa488-conjugated neutravidin from the area on coverslip without B cells (no cell region) vs the case within the area of B cell IS (beneath the cell) that were induced by 12 pN, 43 pN or 56 pN NP-TGT sensors and a negative control 56 pN TGT without NP conjugation (NC). (D) Statistical analyses for the MFI of FITC-conjugated NP-specific antibodies to show that NP-TGT can only be coated to coverslip in a neutravidin-dependent manner. (E) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR to show that non-specific NP-TGT that were tethered on coverslip in a neutravidin-independent manner cannot activate B cells. J558L cells expressing naive B1-8-IgM-BCR were either placed on neutravidin-coated coverslip that were incubated with 56 pN NP-TGT sensor or placed on neutravidin-non-coated coverslip that were incubated with indicated types of NP-TGT sensors. NC represents 56 pN TGT sensor without NP conjugation. In figure C, D, and E, bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were from at least 30 cells or 20 measurements in each group of two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.004
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fig1s1: 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 showing the formation of a typical immunological synapse (IS) of a single B cell (BCR, red color) and the corresponded Alexa488-conjugated neutravidin within the B cell IS. Also given are control neutravidin TIRF images from an area without B cells. Scale bar is 5 μm. In C, the statistical analyses of the MFI of Alexa488-conjugated neutravidin from the area on coverslip without B cells (no cell region) vs the case within the area of B cell IS (beneath the cell) that were induced by 12 pN, 43 pN or 56 pN NP-TGT sensors and a negative control 56 pN TGT without NP conjugation (NC). (D) Statistical analyses for the MFI of FITC-conjugated NP-specific antibodies to show that NP-TGT can only be coated to coverslip in a neutravidin-dependent manner. (E) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR to show that non-specific NP-TGT that were tethered on coverslip in a neutravidin-independent manner cannot activate B cells. J558L cells expressing naive B1-8-IgM-BCR were either placed on neutravidin-coated coverslip that were incubated with 56 pN NP-TGT sensor or placed on neutravidin-non-coated coverslip that were incubated with indicated types of NP-TGT sensors. NC represents 56 pN TGT sensor without NP conjugation. In figure C, D, and E, bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were from at least 30 cells or 20 measurements in each group of two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.004

Mentions: To further our understanding of how mechanical forces influence BCR activation, we constructed the NP-TGT sensors by modifying a dsDNA-based TGT system (Wang and Ha, 2013). Each NP-TGT molecule is composed of two single-stranded DNA (ssDNA) molecules with different modifications (Figure 1A,B). The first ssDNA molecule is biotin-conjugated at different positions to provide a defined range of rupture force anchoring positions as illustrated in Figure 1B. In the original version of the TGT system utilized by Ha and his colleagues (Wang and Ha, 2013), the second ssDNA molecule was conjugated with a well-characterized integrin ligand, cyclic RGDfk peptide, to provide an integrin binding site for quantifying the mechanical force spectrum (12, 16, 23, 33, 43, 50, 54, and 56 pN) in the activation of integrin molecules. As stated by the authors (Wang and Ha, 2013), TGT molecules with modifications can provide an experimental system for the study of many other types of receptors. Here, we conjugated the B1-8-BCR-specific antigen, NP, to the second ssDNA molecule (Figure 1A,B, Figure 1—figure supplement 1A). Since we just exchanged the cyclic RGDfk peptide with the NP hapten antigen and did not change the sequence and design of these dsDNA-based TGT sensors, our NP-TGT system shall have the same tension gauge scales as the one used by Ha and his colleagues (Wang and Ha, 2013). In our experimental system, each NP-TGT molecule can be recognized by the B1-8-IgM-BCRs (Figure 1A). Thus, the NP-TGT sensor that is immobilized on the surface of coverslip would be ruptured if the mechanical force applied by the B1-8-IgM-BCR is larger than the predefined tension force of a certain NP-TGT sensor. A key feature of dsDNA-based mechanical force sensor is that the specific force value of each sensor is not an absolute value but presents a distribution with a full width at half maximum (FWHM) of 5 pN for unzipping rupture mode and 15 pN for shearing rupture mode (Lang et al., 2004). So, precisely, the actual range of any given NP-TGT sensor represents a distribution with the most possible value of rupture force at 12, 16, 23, 33, 43, 50, 54, and 56 pN, respectively.10.7554/eLife.06925.003Figure 1.The construction of B1-8-BCR-specific NP-TGT mechanical force sensor system.


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 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 showing the formation of a typical immunological synapse (IS) of a single B cell (BCR, red color) and the corresponded Alexa488-conjugated neutravidin within the B cell IS. Also given are control neutravidin TIRF images from an area without B cells. Scale bar is 5 μm. In C, the statistical analyses of the MFI of Alexa488-conjugated neutravidin from the area on coverslip without B cells (no cell region) vs the case within the area of B cell IS (beneath the cell) that were induced by 12 pN, 43 pN or 56 pN NP-TGT sensors and a negative control 56 pN TGT without NP conjugation (NC). (D) Statistical analyses for the MFI of FITC-conjugated NP-specific antibodies to show that NP-TGT can only be coated to coverslip in a neutravidin-dependent manner. (E) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR to show that non-specific NP-TGT that were tethered on coverslip in a neutravidin-independent manner cannot activate B cells. J558L cells expressing naive B1-8-IgM-BCR were either placed on neutravidin-coated coverslip that were incubated with 56 pN NP-TGT sensor or placed on neutravidin-non-coated coverslip that were incubated with indicated types of NP-TGT sensors. NC represents 56 pN TGT sensor without NP conjugation. In figure C, D, and E, bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were from at least 30 cells or 20 measurements in each group of two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.004
© Copyright Policy
Related In: Results  -  Collection

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

fig1s1: 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 showing the formation of a typical immunological synapse (IS) of a single B cell (BCR, red color) and the corresponded Alexa488-conjugated neutravidin within the B cell IS. Also given are control neutravidin TIRF images from an area without B cells. Scale bar is 5 μm. In C, the statistical analyses of the MFI of Alexa488-conjugated neutravidin from the area on coverslip without B cells (no cell region) vs the case within the area of B cell IS (beneath the cell) that were induced by 12 pN, 43 pN or 56 pN NP-TGT sensors and a negative control 56 pN TGT without NP conjugation (NC). (D) Statistical analyses for the MFI of FITC-conjugated NP-specific antibodies to show that NP-TGT can only be coated to coverslip in a neutravidin-dependent manner. (E) Quantification of the synaptic accumulation of IgM-BCRs in J558L cells expressing naive B1-8-IgM-BCR to show that non-specific NP-TGT that were tethered on coverslip in a neutravidin-independent manner cannot activate B cells. J558L cells expressing naive B1-8-IgM-BCR were either placed on neutravidin-coated coverslip that were incubated with 56 pN NP-TGT sensor or placed on neutravidin-non-coated coverslip that were incubated with indicated types of NP-TGT sensors. NC represents 56 pN TGT sensor without NP conjugation. In figure C, D, and E, bars represent mean ±SEM. Two-tailed t tests were performed for the statistical comparisons. Data were from at least 30 cells or 20 measurements in each group of two independent experiments.DOI:http://dx.doi.org/10.7554/eLife.06925.004
Mentions: To further our understanding of how mechanical forces influence BCR activation, we constructed the NP-TGT sensors by modifying a dsDNA-based TGT system (Wang and Ha, 2013). Each NP-TGT molecule is composed of two single-stranded DNA (ssDNA) molecules with different modifications (Figure 1A,B). The first ssDNA molecule is biotin-conjugated at different positions to provide a defined range of rupture force anchoring positions as illustrated in Figure 1B. In the original version of the TGT system utilized by Ha and his colleagues (Wang and Ha, 2013), the second ssDNA molecule was conjugated with a well-characterized integrin ligand, cyclic RGDfk peptide, to provide an integrin binding site for quantifying the mechanical force spectrum (12, 16, 23, 33, 43, 50, 54, and 56 pN) in the activation of integrin molecules. As stated by the authors (Wang and Ha, 2013), TGT molecules with modifications can provide an experimental system for the study of many other types of receptors. Here, we conjugated the B1-8-BCR-specific antigen, NP, to the second ssDNA molecule (Figure 1A,B, Figure 1—figure supplement 1A). Since we just exchanged the cyclic RGDfk peptide with the NP hapten antigen and did not change the sequence and design of these dsDNA-based TGT sensors, our NP-TGT system shall have the same tension gauge scales as the one used by Ha and his colleagues (Wang and Ha, 2013). In our experimental system, each NP-TGT molecule can be recognized by the B1-8-IgM-BCRs (Figure 1A). Thus, the NP-TGT sensor that is immobilized on the surface of coverslip would be ruptured if the mechanical force applied by the B1-8-IgM-BCR is larger than the predefined tension force of a certain NP-TGT sensor. A key feature of dsDNA-based mechanical force sensor is that the specific force value of each sensor is not an absolute value but presents a distribution with a full width at half maximum (FWHM) of 5 pN for unzipping rupture mode and 15 pN for shearing rupture mode (Lang et al., 2004). So, precisely, the actual range of any given NP-TGT sensor represents a distribution with the most possible value of rupture force at 12, 16, 23, 33, 43, 50, 54, and 56 pN, respectively.10.7554/eLife.06925.003Figure 1.The construction of B1-8-BCR-specific NP-TGT mechanical force sensor system.

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