Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation

Jing Wang; Shan Tang; Zhengpeng Wan; Yiren Gao; Yiyun Cao; Junyang Yi; Yanyan Si; Haowen Zhang; Lei Liu; Wanli Liu

doi:10.1073/pnas.1517612113

New Research In

Edited by Ulrich von Andrian, Harvard Medical School, Boston, MA, and accepted by the Editorial Board December 10, 2015 (received for review September 4, 2015)

Significance

B-cell receptor (BCR) and antigen engagement induces several responses resulting in B-cell activation. However, it has been difficult to study these responses due to their dynamic nature. To solve this problem, a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP), was developed. B cells contacting caged-NP exhibited probing behaviors that are cell intrinsic with strict dependence on F-actin remodeling. B-cell probing behaviors were terminated within 4 s after the photoactivation of caged-NP. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. The analysis of temporally segregated single molecule images demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

Abstract

Antigen binding to the B-cell receptor (BCR) induces several responses, resulting in B-cell activation, proliferation, and differentiation. However, it has been difficult to study these responses due to their dynamic, fast, and transient nature. Here, we attempted to solve this problem by developing a controllable trigger point for BCR and antigen recognition through the construction of a photoactivatable antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This photoactivatable antigen system in combination with live cell and single molecule imaging techniques enabled us to illuminate the previously unidentified B-cell probing termination behaviors and the precise BCR sorting mechanisms during B-cell activation. B cells in contact with caged-NP exhibited probing behaviors as defined by the unceasing extension of membrane pseudopods in random directions. Further analyses showed that such probing behaviors are cell intrinsic with strict dependence on F-actin remodeling but not on tonic BCR signaling. B-cell probing behaviors were terminated within 4 s after photoactivation, suggesting that this response was sensitive and specific to BCR engagement. The termination of B-cell probing was concomitant with the accumulation response of the BCRs into the BCR microclusters. We also determined the Brownian diffusion coefficient of BCRs from the same B cells before and after BCR engagement. The analysis of temporally segregated single molecule images of both BCR and major histocompatibility complex class I (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to acquire antigens.

Footnotes

↵¹J.W. and S.T. contributed equally to this work.
↵²To whom correspondence may be addressed. Email: lliu{at}mail.tsinghua.edu.cn or liuwanli{at}biomed.tsinghua.edu.cn.

Author contributions: L.L. and W.L. designed research; J.W., S.T., Z.W., and Y.G. performed research; J.W., S.T., J.Y., Y.S., and H.Z. contributed new reagents/analytic tools; J.W., Y.C., J.Y., and H.Z. analyzed data; and W.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. U.v.A. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1517612113/-/DCSupplemental.

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