title: Utilization of a photoactivatable antigen system to examine B-cell probing termination and the B-cell receptor sorting mechanisms during B-cell activation.
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.
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China;
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Tang
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China;
authors.forename: Zhengpeng
authors.initials: Z
authors.lastname: Wan
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China;
authors.forename: Yiren
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China;
authors.forename: Yiyun
authors.initials: Y
authors.lastname: Cao
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China;
authors.forename: Junyang
authors.initials: J
authors.lastname: Yi
authors.affiliation: Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Si
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China;
authors.forename: Haowen
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Tsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China lliu@mail.tsinghua.edu.cn liuwanli@biomed.tsinghua.edu.cn.
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of Immunology, Tsinghua University, Beijing 100084, China; lliu@mail.tsinghua.edu.cn liuwanli@biomed.tsinghua.edu.cn.
authors.forename: Wanli
authors.initials: W
authors.lastname: Liu
mesh_terms: D000818:Animals; D000941:Antigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D008822:Mice, Transgenic; D011947:Receptors, Antigen, B-Cell / Q000276:immunology / Q000378:metabolism*
chemical_list: D000941:Antigens; D011947:Receptors, Antigen, B-Cell
keywords: B-cell activation; B-cell receptor; caged-NP; photoactivatable antigen; single molecule imaging