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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

DOI:http://dx.doi.org/10.7554/eLife.06925.021
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fig8: DOI:http://dx.doi.org/10.7554/eLife.06925.021

Mentions: We agree with the reviewer that additionally there are two alternative methods to address the internalization question as discussed above: (1) to use a fluorophore-conjugated version of NP-TGT, for example, Cy3-NP-TGT; (2) to use NP-specific antibody to stain the quantity of the NP-TGTs on the surface of the coverslip. When revising the manuscript, we chose the second approach to re-address the internalization question. However we found that it is technically difficult to accurately stain the NP-TGT sensors by NP-specific antibodies at the interface between the B cells and coverslip presenting high-force NP-TGT (this interface was termed as B cell IS thereafter). In our tests, we performed the standard protocol to stain NP-TGT sensors in permeabilized B cells following the published studies of ours and those of others (Vascotto F, et al., The Journal of Cell Biology, 2007, 176:1007-19; Liu W, et al., The Journal of Experimental Medicine, 2010, 207:1095-111; Kumari S, et al., eLife, 2015, v.4; Tolar P, et al., Immunity, 2009, 30:44-55). We cannot detect obvious fluorescent signal for 56 pN NP-TGT sensor (fluorophore-conjugated NP-specific antibodies) at the area within the B cell IS, although such signal can be readily detected at the area without B cells (see Author response image 1A). We tested different batches of NP-specific polyclonal antibodies and consistently observed these results. Although as the reviewers have commented that “…the cell might create a hole in a substrat…,” we speculate there are two explanations for the production of these “holes” (area lack of fluorescent signal, see Author response image 1A): (1) NP-conjugated DNA strain on 56 pN NP-TGT sensors were ruptured and dissociated from the coverslip. Subsequently, these dissociated NP-DNA strains can either be internalized by the B cells or diffuse into the bulky aqueous buffer; (2) The adhesion of B cells with 56 pN NP-TGT presenting coverslip was so tight that it would greatly diminish the accessibility of NP-specific antibodies to NP-TGT molecules within the B cell IS. Through a series of further tests, we figured out that the second possibility makes more sense.10.7554/eLife.06925.021Author response image 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)

DOI:http://dx.doi.org/10.7554/eLife.06925.021
© Copyright Policy
Related In: Results  -  Collection

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

fig8: DOI:http://dx.doi.org/10.7554/eLife.06925.021
Mentions: We agree with the reviewer that additionally there are two alternative methods to address the internalization question as discussed above: (1) to use a fluorophore-conjugated version of NP-TGT, for example, Cy3-NP-TGT; (2) to use NP-specific antibody to stain the quantity of the NP-TGTs on the surface of the coverslip. When revising the manuscript, we chose the second approach to re-address the internalization question. However we found that it is technically difficult to accurately stain the NP-TGT sensors by NP-specific antibodies at the interface between the B cells and coverslip presenting high-force NP-TGT (this interface was termed as B cell IS thereafter). In our tests, we performed the standard protocol to stain NP-TGT sensors in permeabilized B cells following the published studies of ours and those of others (Vascotto F, et al., The Journal of Cell Biology, 2007, 176:1007-19; Liu W, et al., The Journal of Experimental Medicine, 2010, 207:1095-111; Kumari S, et al., eLife, 2015, v.4; Tolar P, et al., Immunity, 2009, 30:44-55). We cannot detect obvious fluorescent signal for 56 pN NP-TGT sensor (fluorophore-conjugated NP-specific antibodies) at the area within the B cell IS, although such signal can be readily detected at the area without B cells (see Author response image 1A). We tested different batches of NP-specific polyclonal antibodies and consistently observed these results. Although as the reviewers have commented that “…the cell might create a hole in a substrat…,” we speculate there are two explanations for the production of these “holes” (area lack of fluorescent signal, see Author response image 1A): (1) NP-conjugated DNA strain on 56 pN NP-TGT sensors were ruptured and dissociated from the coverslip. Subsequently, these dissociated NP-DNA strains can either be internalized by the B cells or diffuse into the bulky aqueous buffer; (2) The adhesion of B cells with 56 pN NP-TGT presenting coverslip was so tight that it would greatly diminish the accessibility of NP-specific antibodies to NP-TGT molecules within the B cell IS. Through a series of further tests, we figured out that the second possibility makes more sense.10.7554/eLife.06925.021Author response image 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