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

Jing Wanga,1, Shan Tangb,1, Zhengpeng Wana, Yiren Gaoa, Yiyun Caoa, Junyang Yia, Yanyan Sib, Haowen Zhanga, Lei Liub,2, and Wanli Liua,2

aKey Laboratory of Protein Sciences (Ministry of Education), Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Life Sciences, Institute of , Tsinghua University, Beijing 100084, China; and bTsinghua-Peking Center for Life Sciences, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China

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) Antigen binding to the B-cell receptor (BCR) induces several re- initiation of B-cell activation process the information of antigen sponses, resulting in B-cell activation, proliferation, and differentia- specificity, density, affinity, valency, mobility, substrate stiffness, tion. However, it has been difficult to study these responses due to and mechanical forces in such an efficient way? To attempt to their dynamic, fast, and transient nature. Here, we attempted to solve address this intriguing question, a detailed understanding of the this problem by developing a controllable trigger point for BCR and precise BCR sorting mechanisms within the B-cell IS during the antigen recognition through the construction of a photoactivatable initiation of B-cell activation is required. However, it is technically antigen, caged 4-hydroxy-3-nitrophenyl acetyl (caged-NP). This pho- difficult to accurately capture these events due to their fast and toactivatable antigen system in combination with live cell and single dynamic nature. It is challenging to capture an entire molecular molecule imaging techniques enabled us to illuminate the previously event from the same B-cell before and immediately after antigen unidentified B-cell probing termination behaviors and the precise BCR recognition, and it is more difficult to capture multiple events in sorting mechanisms during B-cell activation. B cells in contact with parallel from multiple cells in a synchronized manner. An attractive caged-NP exhibited probing behaviors as defined by the unceasing solution for this dilemma is to develop a precisely controllable trigger extension of membrane pseudopods in random directions. Further point for BCR and antigen recognition by using photoactivatable analyses showed that such probing behaviors are cell intrinsic , which are initially inactive but become immediately active with strict dependence on F-actin remodeling but not on tonic on the illumination of UV light. Indeed, photoactivatable systems BCR signaling. B-cell probing behaviors were terminated within 4 s have been used to investigate the kinetics of second-messenger after photoactivation, suggesting that this response was sensitive activity through caged calcium (22) and caged cAMP (23). Ad- and specific to BCR engagement. The termination of B-cell probing ditionally, the T-cell receptor was studied using major histo- was concomitant with the accumulation response of the BCRs into compatibility complex (MHC) presenting photoactivatable peptide the BCR microclusters. We also determined the Brownian diffusion (24, 25). coefficient of BCRs from the same B cells before and after BCR In this report, we dissected the dynamic responses during the engagement. The analysis of temporally segregated single molecule initiation of B-cell activation by using a photoactivatable antigen images of both BCR and major histocompatibility complex class I based experimental system in combination with high-resolution high (MHC-I) demonstrated that antigen binding induced trapping of BCRs into the BCR microclusters is a fundamental mechanism for B cells to Significance acquire antigens. B-cell receptor (BCR) and antigen engagement induces several B-cell receptor | photoactivatable antigen | caged-NP | B-cell activation | responses resulting in B-cell activation. However, it has been single molecule imaging difficult to study these responses due to their dynamic nature. To solve this problem, a photoactivatable antigen, caged he immune system uses immune receptors to sense and ac- 4-hydroxy-3-nitrophenyl acetyl (caged-NP), was developed. B cells Tquire antigens. Antigen binding induces a series of dynamic contacting caged-NP exhibited probing behaviors that are cell changes in the biophysical behaviors and biochemical features of intrinsic with strict dependence on F-actin remodeling. B-cell the immune receptors, and these changes determine the fate of a probing behaviors were terminated within 4 s after the pho- cell (1–3). However, it has been difficult to accurately capture toactivation of caged-NP. The termination of B-cell probing was and thus comprehensively investigate these changes because they concomitant with the accumulation response of the BCRs into usually occur very quickly after immune recognition (1, 4). For the BCR microclusters. The analysis of temporally segregated example, recent live cell imaging studies showed that the B lym- single molecule images demonstrated that antigen binding in- phocytes swiftly accumulate the surface-expressed B-cell receptors duced trapping of BCRs into the BCR microclusters is a funda- (BCRs) into the contact interface between the B cells and the mental mechanism for B cells to acquire antigens. antigen-presenting substrates to form a specialized membrane structure, the B-cell immunological synapse (IS) (1, 4). More- Author contributions: L.L. and W.L. designed research; J.W., S.T., Z.W., and Y.G. per- formed research; J.W., S.T., J.Y., Y.S., and H.Z. contributed new reagents/analytic tools; over, both our studies and those of others showed that these J.W., Y.C., J.Y., and H.Z. analyzed data; and W.L. wrote the paper. accumulation events are sensitive to the biochemical and bio- The authors declare no conflict of interest. physical features of the antigens that B cells likely encounter in This article is a PNAS Direct Submission. U.v.A. is a guest editor invited by the Editorial vivo (4, 5). These features include but are not limited to antigen Board. – density (6, 7), antigen affinity (6, 7), antigen valency (8 13), the 1J.W. and S.T. contributed equally to this work. – mobility of the antigen (14 17), the stiffness of the substrates 2To whom correspondence may be addressed. Email: [email protected] or liuwanli@ presenting the antigen (18, 19) and the mechanical forces de- biomed.tsinghua.edu.cn. livered to the BCRs by the antigens (20, 21). These facts high- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. light a long-standing question in immunology: how can the 1073/pnas.1517612113/-/DCSupplemental.

E558–E567 | PNAS | Published online January 13, 2016 www.pnas.org/cgi/doi/10.1073/pnas.1517612113 Downloaded by guest on October 6, 2021 speed total internal reflection fluorescence microscopy (TIRFM) AB PNAS PLUS imaging techniques. We caged the widely used model antigen caged-NP WT-NP 4-hydroxy-3-nitrophenyl acetyl (caged-NP) that is only con- verted to its antigenic form after exposure to UV photons. The O2N O caged-NP O2N photoactivation of caged-NP in contact with NP-specific B1-8- O – O O BCR expressing B cells provides a precisely controllable trigger a) point to perform high resolution temporal analyses of the for- HN caging group mation of BCR microclusters and the B-cell IS in response to an- NP O 5 b) tigen stimulation. By combining the unique strengths of the caged- – Ala-Ser-Thr-Gly-Lys-Thr-Ala-Ser-Ala-Cys- 15 20 25 30 NP based photoactivatable antigen system with TIRFM-based live Thr-Ser-Gly-Ala-Ser-Ser-Thr-Gly-Ser-His Retention time (min) cell and single molecule imaging techniques, we examined the basal 6 response of a quiescent B cell exposed to coverslips presenting the C D caged-NP for 360 s and then examined the changes in the responses a) 916.1 of the same B cell immediately after the recognition of the pho- 1373.7 100 687.3 toactivated-NP antigen for another360s.Toourknowledge,this 80 system represents the first temporally seamless imaging ex- 600 1100 1600 m/z 60 perimental procedure for the study of the molecular events during caged-NP peptide 981.2 40 photolyzed-NP peptide the initiation of B-cell activation. We illuminated the probing be- b) haviors in quiescent B cells as defined by the unceasing extension 20

1471.1 % total of peptide of membrane pseudopods in random directions. We found that 736.1 0 BCR and antigen recognition promptly terminated the probing 0 20 40 60 responses. We also dissected the sophisticated BCR sorting mech- 600 1100 1600 m/z Time (s) anism within the B-cell IS during the initiation of B-cell activation. E 1.5 WT-NP-0 s Results WT-NP-30 s 1.0 WT-NP-120 s The Development of a Caged-NP–Based Photoactivatable Antigen WT-NP-300 s System. The NP is specifically recognized by B1-8 or caged-NP-0 s – 0.5 caged-NP-30 s

B1-8-BCR expressing B cells mainly due to the hydroxyl (-OH) OD 490 nm group in the phenol ring (26). To mask the antigenicity of NP, we caged-NP-120 s 0.0 caged-NP-300 s conjugated a UV-sensitive moiety, 4,5-dimethoxy-2-nitrobenzyl 0 5 10 15 20 (DMNB) to its -OH group to generate DMNB-NP (Fig. 1A Antigen concentration (μg/mL) 1 13 and Fig. S1). Analyses by H- and C-NMR verified the correct con- – jugation of DMNB to NP (Fig. S2 A and B). To facilitate the Fig. 1. The development of a caged-NP based photoactivatable antigen system. (A) The schematic presentation of the conjugation of a UV-sensitive downstream imaging experiments, a carrier peptide with His6-tag, moiety, DMNB (caging group), to the -OH group of the NP hapten antigen to ASTGKTASACTSGASSTGS-His6, was further conjugated to ei- generate DMNB-NP. (B) The RP-HPLC analysis of WT-NP or caged-NP. The re- ther WT NP (WT-NP) or DMNB-NP (caged-NP) (Fig. 1A)atthe tention time is shown in minutes. (C) The ESI-MS analysis of WT-NP or caged- N terminus through a 6-aminocaproic acid linker as characterized NP. The mass-to-charge ratio is shown in m/z.(D) The photolysis kinetics of by reverse-phase HPLC (RP-HPLC) (Fig. 1B) and electrospray the caged-NP peptide (1 mg/mL) on photoactivation with UV photons (365 nm, ionization-MS (ESI-MS) (Fig. 1C). To examine the photo-cleavage 18 mW/cm2) for different exposure times (0, 5, 10, 20, 30, and 60 s) as examined efficiency, the photolysis kinetics of the caged-NP was examined by RP-HPLC to quantify the percentage of photolyzed peptides in the total 2 on exposure to UV photons (365 nm, 18 mW/cm ). We used peptide population. Bars represent mean ± SD of three independent experi- RP-HPLC to quantify the photolysis kinetics of the caged-NP by ments. (E) The ELISA evaluation of the binding capacity of anti-NP determining the percentage of photolyzed peptides in a population to WT-NP- or caged-NP-peptide before (0 s) or after photoactivation with UV of total peptides including the caged-NP peptides and the photo- photons (365 nm, 18 mW/cm2) for different exposure time (30, 120, and 300 s). lyzed-NP peptides (Fig. 1D). We found that the caged-NP had rapid Each peptide (20 μg/mL) was photoactivated for the indicated amount of time photolysis kinetics with greater than 40% photolyzed peptides and was then diluted to 10 or 5 μg/mL in the binding detection. detected in 10 s. We also evaluated the antigenicity of WT-NP, caged-NP, and caged-NP after photoactivation (referred to as photoactivated-NP hereafter) using NP-specific B1-8 antibodies in the molecular events of the same B cell in a sufficient temporal an antigen dose-dependent ELISA (Fig. 1E). As a system control, domain (e.g., an examination of 360 s in the quiescent status im- mediately followed by an examination of 360 s in the activated anti–His6-tag antibodies (the carrier peptide contains a His6 tag) confirmed that all these NP-conjugated peptides were similarly status) in response to antigen recognition. coated onto the ELISA plate (Fig. S3A). WT-NP showed robust In all of the following photoactivation-based seamless imaging binding capability to NP-specific antibodies, whereas caged-NP lost experiments, NP-specific B cells prelabeled with Dylight 647- the binding abilities (Fig. 1E). On photoactivation, we observed the conjugated Fab fragment anti-mouse IgM antibodies were first recovered binding of the photoactivated-NP to NP-specific anti- placed on coverslips presenting the caged-NP antigen for 10 min bodies, and this recovery efficiency was positively correlated to the to blunt any potential behavior changes of the B cells that were coating concentration of the caged-NP peptide (Fig. 1E). Thus, a introduced into the system by the acute landing and adhesion caged-NP–based photoactivatable antigen system was developed. responses of the B cells. Thus, imaging experiments were only performed in the condition that the B cells formed steady-state Photoactivation Promptly Terminates the Probing Behaviors of Quiescent contact with the coverslips after the 10-min incubation time. We

BCells.We combined the unique strengths of the photoactivatable found that the NP-specific B cells in contact with caged-NP INFLAMMATION NPantigensystemwiththeTIRFM-basedlivecellimagingsystem exhibited the unceasing extension of membrane pseudopods in IMMUNOLOGY AND to examine the precise behavior changes of NP-specific B cells random directions, for which we termed as the probing behavior before and after photoactivation. We first imaged the basal be- hereafter in this report. This probing behavior of quiescent haviors of a single B cell in its quiescent state on coverslips pre- B cells can be readily captured in both J558L-B1-8-IgM cells senting the caged-NP for a sufficient amount of time (e.g., 360 s) (Fig. 2A, as shown by the white arrowheads; see Movie S1 for the and then examined the behavior changes of the very same B cell best visional effects with full frame information) and B1-8 primary − − immediately on photoactivation and thereafter for another 360 s. BcellsfromIgH B1-8/B1-8 Igκ / transgenic mice (26, 27) (Fig. 2B and To the best of our knowledge, this represents the first design of a Movie S2 for the best visional effects). Further experiments showed seamless imaging experimental approach to capture the changes in that these probing behaviors were not induced by caged-NP as

Wang et al. PNAS | Published online January 13, 2016 | E559 Downloaded by guest on October 6, 2021 contrast, these probing behaviors were independent of tonic BCR A photo- activation signaling as B cells pretreated with the Src family kinase inhibitor PP2 or the Syk kinase inhibitor piceatannol still maintained these Time (s) -360 -320 -280 -240 -200 -160 -120 -80 -40 0 behaviors (Fig. S4C and D). Thus, B-cell probing behaviors are cell intrinsic with dependence on F-actin remodeling but not on tonic J558L-B1-8 -IgM cell 4 40 80 120 160 200 240 280 320 360 BCR signaling. On conversion of caged-NP to photoactivated-NP, the behavior of the same NP-specific B cells changed drastically. TIRFM imag- ing demonstrated that in as short as 4 s after photoactivation B (see the 0- vs. 4-s TIRFM images in Fig. 2 or examine Movies Time (s) -360 -340 -300 -280 -260 -240 -220 -200 -180 -160 S1 and S2), the same B cells that were originally probing promptly terminated this behavior as quantified by the significantly reduced number of membrane pseudopods per cell per TIRFM photo- activation image (Fig. 2 C and D and Fig. S3F). In contrast, the photo- B1-8 primary activation triggered B-cell probing termination events were not B cell -140 -120 -100 -80 -60 -40 -20 -10 -4 0 observed in B1-8 primary B cells that were placed on coverslips alone (Fig. S5A). Similarly, photoactivation did not induce the probing termination event of primary B cells from C57BL/6-WT 4 40 80 120 160 200 240 280 320 360 mice that were placed on coverslips presenting caged-NP (Fig. S5B). These experiments suggested that the probing termination behavior was induced by the recognition of BCR and antigen. Furthermore, NP-specific B cells that have been exposed to bio- J558L-B1-8 IgM cell B1-8 primary B cell CDactive WT-NP antigen for 10 min before TIRFM imaging did not 8 p<0.001 8 p<0.001 exhibit the probing behaviors in the 360-s time courses before and 6 6 after photoactivation (Fig. S5 C and D). All these additional ex- 4 4 periments consistently suggested that photoactivation-induced phototoxicity does not contribute to the probing cessation event. 2 2 pseudopods / cell pseudopods / cell 0 0 Number membrane of Number membrane of The Termination of B-Cell Probing Is Concomitant with the Synaptic

hoto- Accumulation of the BCRs. Next, we precisely quantified the changes photo-ed-NP p ted-NP caged-NP caged-NP in the size of the contact area of the B cells on the coverslips and activat activa the mean fluorescence intensity (mFI) of the BCR molecules within Fig. 2. Photoactivation promptly terminates the probing behavior of quies- the contact area using the abovementioned seamless imaging ex- cent B cells. (A and B) Shown are the representative TIRFM images at the in- perimental approaches. We calculated the mFI instead of the total dicated time points of the same J558L-B1-8-IgM cells (A) or B1-8 primary B cells FI of BCRs within the B-cell contact area because the former can (B) prelabeled with DyLight 649-conjugated Fab fragment anti-IgM that were better reflect the changes in the density (or concentration) of the placed on coverslips coated with caged-NP before and after photoactivation. BCRs at the contact interface, whereas the latter can increase if the The basal responses of quiescent B cells on coverslips presenting caged-NP B cells simply spread over the coverslips. Thus, the mFI value can were first examined for 360 s by TIRFM imaging. Immediately starting with better represent the efficiency of the accumulation of the BCRs photoactivation (time 0 s as indicated), the behaviors of the same B cells were within the B-cell IS. The results showed that both the size of the continuously examined for another 360 s. See Movies S1 and S2 for complete B-cellcontactareaandthemFIoftheBCRsdidnotchangein time-lapse TIRFM images. The white arrowheads indicate the membrane quiescent B cells in contact with the caged-NP in the whole time protrusions of the probing J558L-B1-8-IgM cells (A)orB1-8primaryBcells course of 360 s (Fig. 3 A–E and Movie S1). Strikingly, both values (B) before the photoactivation. (Scale bar, 1.5 μm.) (C and D) Statistical com- rapidly and drastically increased in the very same J558L-B1-8-IgM parison with quantification for the number of membrane pseudopods per B cells immediately after photoactivation (Fig. 3 B–E). The same TIRFM image in each J558L-B1-8-IgM B cell (C) or B1-8 primary B cell (D) conclusion was acquired in B1-8 primary B cells (Fig. 3 F and G and before and after photoactivation. Bars represent mean ± SD of 20 cells from one representative of three independent experiments. Two-tailed t tests were Movie S2). In contrast, photoactivationdidnotdrivethesynaptic performed for statistical comparisons in C and D. accumulation of BCR molecules in the experiments where B1-8 primary B cells were placed on coverslips alone (Fig. S5E)orthe experiments where primary B cells from C57BL/6-WT mice were similar results were captured from B1-8 primary B cells that were placed on coverslips presenting caged-NP (Fig. S5F). These results placed on control coverslips without caged-NP (Fig. S3B and Movie and the additional results from NP-specific B cells that have been S3). These probing behaviors were not induced by nonspecific exposed to bioactive WT-NP antigen for 10 min before the pho- stimulation from the glass to the cells as the B1-8 primary B cells toactivation (Fig. S5 G and H) suggested that the BCR accumula- that were placed on coverslips presenting fluid planar lipid bilayers tion event was not induced by the potential phototoxicity after photoactivation. To further exclude the possibility that the increase (PLBs), which were used to insulate the direct contact of the cell in the BCR mFI resulted from the distance changes in the z di- membrane to the glass, similarly exhibited the probing behaviors mension of B cells with coverslips after photoactivation, we also (Fig. S3C and Movie S4). To further exclude the possibility that similarly quantified these changes from B cells that were placed on these probing behaviors might reflect the membrane projections coverslips presenting both caged-NP and anti–MHC-I antibodies. that are transiently entering into the TIRFM imaging plane, we Anti–MHC-I antibodies have been used in the literature to uni- imaged B1-8 primary B cells that were placed on coverslips pre- formly pretether B cells to the surface of coverslips (29). In this – sentingeitherICAM-1orantiMHC-I antibodies, both of which system, we similarly captured the drastic BCR accumulation event have been used to pretether and preadhere B cells to the surface of on photoactivation (Fig. S6A). coverslips in the literature (28, 29). The probing behaviors were Our recent studies and those of others showed that the BCR readily observed in both cases (Fig. S3 D and E and Movies S5 and microcluster is the most basic unit during the initiation of B-cell S6). Furthermore, a series of pharmaceutical inhibitor experiments activation (15, 29–34). However, it has been difficult to accu- showed that the probing behaviors were terminated in B cells rately capture and then investigate the dynamic changes of the pretreated with cytochalasin D to disrupt F-actin or with jasplaki- BCR microclusters in the same B cells before and after the rec- nolide to stabilize F-actin, suggesting that B-cell probing behaviors ognition of antigens. In all those published studies, a strategy was were dependent on the remodeling of F-actin (Fig. S4 A and B). In used to directly monitor the responses after loading B cells onto

E560 | www.pnas.org/cgi/doi/10.1073/pnas.1517612113 Wang et al. Downloaded by guest on October 6, 2021 We showed that B cells in contact with coverslips presenting PNAS PLUS A photoactivation caged-NP did not form stable and persistent BCR microclusters, Time (s) -360 -240 -120 -60 0 60 120 240 360 although we frequently observed the formation of dynamic and J558L-B1-8 transient puncta structures of BCR molecules that were con- -IgM cell comitant with the probing behaviors of the B cells (Figs. 2 and 3 and Movies S1–S6). Immediately after photoactivation, the same B C B cells terminated the probing behavior and began to form truly 40 caged-NP 4 caged-NP prominent BCR microclusters (Fig. 4A and Movie S8). To ac-

) photoactivated-NP photoactivated-NP 2 30 3

m curately quantify the spatial-temporal changes of the biophysical μ 20 2 features of the BCR microclusters, we placed control beads in 10 1 the same imaging field of the B cells to precisely calibrate the Area ( 0 0 vibration of the whole TIRFM imaging system (Fig. 4A and

-360 -240 -120 0 120 240 360 (Area) Normalized -360 -240 -120 0 120 240 360 Time (s) Time (s) Movie S8). Then we analyzed these time-lapse images following D E our published protocol (6), using a 2D Gaussian function based mathematical fitting method to accurately quantify the mFI 5000 caged-NP 10 caged-NP (integrated FI/size) and the position in the x and y coordinates of 4000 photoactivated-NP 8 photoactivated-NP both the BCR microclusters and the calibration bead (Fig. 4 B– 3000 6 E). First, we examined the lateral motility of both the calibration 2000 4 1000 2 BCR (mFI) 0 0 -360 -240 -120 0 120 240 360 -360 -240 -120 0 120 240 360

Time (s) (BCR mFI) Normalized Time (s) F G A

6 1000 caged-NP caged-NP 800 photoactivated-NP photoactivated-NP 4 600 400 2 200 BCR (mFI) 0 0 -360 -240 -120 0 120 240 360 -360 -240 -120 0 120 240 360

Time (s) (BCR mFI) Normalized Time (s) B CD Fig. 3. The termination of the B-cell probing behavior is concomitant with the synaptic accumulation of the BCRs. (A) Shown are the TIRFM images at the indicated time points of the same J558L-B1-8-IgM cells that were placed on coverslips coated with the caged-NP before and after photoactivation. The basal responses of the B cell in its quiescent state in contact with the caged-NP were first examined for 360 s by TIRFM imaging. Immediately starting with photo- activation (time 0 s as indicated), the behavioral changes of the same B cells were continuously examined for another 360 s. See Movie S1 for the complete time-lapse TIRFM images. (Scale bar, 1.5 μm.) (B–E) Statistical comparison with E quantification for the size of the contact area (B)andthemFIoftheBCRs(D) from the same B cells before and after photoactivation as depicted in A.The curve from −360 to 0 s showed the dynamic changes of the J558L-B1-8-IgM cells in contact with the caged-NP, whereas the curve from 0 to 360 s showed the changes in the responses of the same cells in contact with the photoactivated- NP. The corresponding curves following a normalization step to the value at time 0 s are also shown in C (for B)andE (for D). Bars represent mean ± SEM of 15 cells from one representative of three independent experiments. (F and G)A similar experiment was performed as described for D and E. Shown are the mFI (F) and the normalized mFI (G) of the BCR molecules within the contact area of the same B1-8 primary B cells before and after photoactivation of the caged-NP. Bars represent mean ± SEM of 10 cells from one representative of three in- dependent experiments.

coverslips presenting bioactive antigens. However, that strategy will inevitably lead to the activationoftheBCRsandtheformationof BCR microclusters immediately on contact of B-cell membrane Fig. 4. The antigen engaged BCRs are accumulated into stationary BCR pseudopods with active antigen-presenting surfaces. Thus such an microclusters. (A) Representative TIRFM images show the response of the same experimental strategy makes it difficult to examine the true basal BCR puncta before (−300 to 0 s) and after (0–300 s) photoactivation of caged- responses in quiescent B cells and the conversion from the quies- NP (time 0 s as indicated). (Scale bar, 1.5 μm.) Indicated by the white boxes in cent to the activated state in the same B cells. It is also difficult to the −240 s TIRFM image are one representative typical BCR punctum (Left)and one representative calibration bead (Right). A fraction of the entire time-lapse separate the acute landing and adhesion events of B cells from INFLAMMATION the BCR engagement induced B-cell spreading response. Thus, TIRFM images from −12 to 36 s is shown in a refined temporal manner. IMMUNOLOGY AND these conventional imaging procedures will likely overestimate the See Movie S8 for complete time-lapse TIRFM images. (B–D)ThenormalizedmFI − extent of B-cell responses that are truly induced by antigen-BCR (B) of the BCR microclusters from the whole time course ( 300 to 300 s) are recognition. For example, TIRFM imaging showed that the nor- shown along with the trajectories by means of the x vs. y footprints accumu- lated in the time course of −300 to 0 s (C)andof0–300 s (D) as depicted in A.In malized mFI of a fluorescent bead, which should be a fixed value, B, bars represent mean ± SEM of 435 BCR microclusters analyzed from 15 cells drastically increased during its stochastic landing event on the surface in one representative of three independent experiments. (E) The pseudocolor of the coverslips (Fig. S6 B and C and Movie S7). We propose that 2.5D Gaussian images of the same BCR microcluster at the indicated times from the photoactivated antigen-based seamless imaging approaches can B cells in contact with caged-NP (Upper)orphotoactivated-NP(Lower)are overcome these obstacles. shown. (Scale bars, 1.5 μm.)

Wang et al. PNAS | Published online January 13, 2016 | E561 Downloaded by guest on October 6, 2021 bead and the BCR puncta structure by generating the x and y A–E and Movie S1), probing termination event was only mild in the coordinates from the entire TIRFM imaging time course, which case of J558L-B1-8-IgM-Low cells, where attenuated but constant was presented as a typical trajectory plot. It was clear that the probing behaviors always occurred throughout the imaging time calibration bead did not move beyond one pixel (150 nm) in all course after photoactivation (Fig. S7 A and B and Movie S10). of the time course (Fig. 4 C and D and Movie S8). In marked Concomitant with the termination of the probing behaviors, high- contrast, the BCR puncta structures in the same TIRFM imaging affinity J558L-B1-8-IgM cells formed a more stable cell body, which field showed highly motile behavior in quiescent B cells contacting overtly facilitated the BCR microclusters to become prominently caged-NP (Fig. 4C). After photoactivation, the BCR puncta struc- stationary. All these early events resulted in an increased size of the tures became stationary and exhibited a trajectory range of less than B-cell contact area and the synaptic accumulation of the BCRs in one pixel (150 nm) in 300 s, comparable to a control bead (Fig. 4D high affinity J558L-B1-8-IgM cells (Fig. 3 A–E and Movie S1). and Movie S8). Next, we quantified the dynamic changes in the mFI However, J558L-B1-8-IgM-Low cells did not show a drastic in- of the BCR puncta structures in quiescent B cells in contact with crease in the size of the B-cell contact area nor was there an obvious caged-NP and in the same B cells upon photoactivation. The mFI synaptic accumulation of the BCRs (Fig. S7 A and B and Movie of the BCR puncta structures in contact with the caged-NP did not S10). Thus, the termination of the probing behavior of B cells on increase in the whole 300-s time course (Fig. 4 A and B), suggesting BCR engagement is an event that is sensitive to BCR and the lack of enrichment of BCR molecules within these puncta antigen affinity. structures in quiescent B cells. However, immediately after photo- activation, we readily observed the formation of prominent and The B-Cell IS Is a Dynamically Open yet Highly Selective Membrane stable BCR microclusters that drastically increased the mFI over Structure. The observation that the accumulation of BCR mole- time in the same B cell (Fig. 4 A–D). cules into the B-cell IS was driven by BCR engagement raised To make sure that the above observations were not induced by several intriguing questions. Is the B-cell IS a dynamic or steady the immobile nature of the antigens that were tethered to cov- structure? Is the B-cell IS an open or closed structure? Is the erslips, we examined the response of the NP-specific B cells that B-cell IS a selective or promiscuous structure? To answer these were placed on PLBs presenting caged-NP antigens. B cells questions, we performed a two-color TIRFM imaging experi- exhibited the typical probing behaviors in contacting with caged- ment in combination with the photoactivation system to simul- NP antigens on PLBs and BCR puncta structures from these taneously examine the spatial and temporal changes of the BCRs B cells similarly showed highly motile behaviors (Fig. S4 E–G and control molecules (MHC-I or lipid molecules) within the and Movie S9). Photoactivation of the antigens on PLBs also B-cell IS. We observed that both BCR and MHC-I molecules triggered B-cell probing termination response that was con- were transiently expressed in the TIRFM images in a highly dy- comitant with the formation of prominent BCR microclusters (Fig. namic manner in quiescent B cells contacting caged-NP (Fig. 5A S4 E–H and Movie S9). The newly formed BCR microclusters were and Movie S11). Furthermore, Pearson’s correlation index (PCI) still lack of obvious mobility immediately after photoactivation, al- analysis suggested that the BCR and MHC-I molecules only weakly though at the later stage some but not all BCR microclusters codistributed in a quiescent B cell (Fig. S8 A and B). We continued exhibited the obvious centripetal movement toward the central re- to capture the dynamic changes of these two molecules in the same gion of the B-cell IS (Fig. S4 E–H, as shown by the yellow arrow- B cell on photoactivation and readily observed robust accumulation heads for stationary BCR microclusters and blue arrowheads for of BCRs with very mild but reproducible accumulation of MHC-I the ones showing centripetal movements; see Movie S9 for the best molecules within the B-cell IS (Fig. 5 A–C and Movie S11). We visional effects). These results are largely consistent with our pub- further validated this observation using Dil-stained membrane lipid lished studies and those of others showing that there was a lag time molecules (Fig. S8 C–E and Movie S12). These data suggested that to allow B cells to reach the maximal spreading before the BCR the B-cell IS is a dynamically open and selective membrane struc- microcluster exhibited an obvious centripetal movement toward the ture. To further confirm these observations, we performed a long- center of B-cell IS (6, 7). In either the stationary BCR microclusters term (30 min) two-color TIRFM imaging experiment to examine (Fig. S4 E and F, as shown by the yellow arrowheads) or the BCR the accumulation and spatial and temporal dynamics of both the microclusters retrograding to the center of the B-cell IS (Fig. S4 E BCRs and the MHC-I molecules within the B-cell IS (Fig. 5 D–F and F, as shown by the blue arrowheads; see Movie S9 for the best and Movie S13). As expected, we observed a robust and steady visional effects), it was evident that the BCR microclusters in- accumulation of the BCRs into the B-cell IS with an initial expo- creasedtheirmFIovertime(Fig. S4I, or see the representative nential growth pattern followed by a linear growth pattern (Fig. 5 D BCR microclusters by arrowheads in Fig. S4 E and F and Movie and E). We also observed a synaptic accumulation of MHC-I in S9). These data suggested that photoactivation promptly induced a response to the photoactivation of caged-NP in the long-term ex- rapid and drastic accumulation of the BCRs into the BCR micro- periments, although the accumulation was obviously much weaker clusters. Because the BCR microclusters could be stationary in compared with the case of the BCRs (Fig. 5 D and F). PCI analysis of these events, we propose that the BCR molecules could be accu- the long-term TIRFM images examining the synaptic codistribution mulated into the BCR microclusters in a passive trapping manner. of the BCR and MHC-I molecules showed that the PCI value slightly increased in the first 5 min after photoactivation and then The Termination of B-Cell Probing Is Sensitive to the Affinity Between decreased over time (Fig. S8 A and B). Interestingly, the synaptic the BCR and the Antigen. B cells show excellent capability to dis- accumulation of the MHC-I molecules mainly occurred at the criminate the affinity between the BCR and the antigen during central but not the peripheral region of the B-cell IS (Fig. S8B). the initiation of B-cell activation. We thus investigated if the These results defined the unexpected heterogeneity in terms of photoactivation triggered termination of probing would be sensi- “molecule crowding” within the B-cell IS (Discussion). tive to the BCR and antigen affinity. The J558L-B1-8-IgM cells that were used in the above experiments in this report expressed a The Decreased Lateral Mobility of Antigen-Engaged BCRs Accounts 6 −1 B1-8-IgM-High BCR with high affinity to NP (Ka = 5 × 10 M ); for Their Synaptic Trapping. A unique advantage of the photo- in contrast, B1-8-IgM-Low BCRs showed a much low affinity activatable NP system is that we can accurately generate temporal 5 −1 (Ka = 1.25 × 10 M ) to NP as described in detail in our previous segments at an unprecedented resolution immediately after BCR studies and those of others (6, 35). Thus, we continued to image the engagement for dozens of B cells with a highly synchronized trigger responses of J558L-B1-8-IgM-Low cells using the photo- point. The schematic (Fig. 6A) depicts our experimental strategy activatable antigen-based seamless imaging system. J558L-B1- for the single molecule imaging (SMI) experiment for the BCR and 8-IgM-Low cells in contact with the caged-NP also showed the MHC-I molecules. Each B cell was subjected to SMI of 600 frames typical B-cell probing response. In marked contrast to the obser- in 18 s (30 ms/frame) for the first SMI reading. The second SMI vation that high-affinity J558L-B1-8-IgM cells terminated the reading was taken after an interval of 10 s followed by the third probing behavior to accumulate the BCRs into the B-cell IS (Fig. 3 SMI reading and another 10-s interval until the sixth reading.

E562 | www.pnas.org/cgi/doi/10.1073/pnas.1517612113 Wang et al. Downloaded by guest on October 6, 2021 MSD plots from the same batch of B cells suggested that, on PNAS PLUS A photoactivation photoactivation, the Brownian motility of the BCR molecules Time (s) -240 -120 -12 -4 0 4 12 36 120 240 360 became increasingly confined over time (Fig. 6D). Moreover, the BCR short-range diffusion coefficients of each individual BCR mole- cule were calculated and their distribution was analyzed and MHC-I displayed as a cumulative distribution probability (CDP) plot (Fig. 6E). It was quite clear that the Brownian diffusion coeffi- cients of the BCRs in the same batch of B cells gradually de- Merge creased over time (Fig. 6E). The mean diffusion coefficient of B BCR C the BCRs in the same batch of B cells also decreased (Fig. MHC-I 6F). Moreover, we performed subpopulation analysis to mathe- 8 caged-NP 6 caged-NP photoactivated-NP photoactivated-NP matically sort thousands of BCRs into either fast or slow pop- 6 4 ulations by a maximum likelihood estimation approach as detailed 4 2 in Materials and Methods. The results confirmed that the slow 2 BCR population in the same batch of B cells increased, whereas 0 0 Normalized (mFI) Normalized Normalized (mFI) Normalized -360 -240 -120 0 120 240 360 -360 -240 -120 0 120 240 360 the fast population decreased over time on photoactivation (Fig. 7 Time (s) Time (s) A–F, Fig. S9A,andMovie S14). In contrast, we did not observe – D photoactivation these dynamic changes for the MHC-I molecules (Fig. 6 C and G I, Figs. S9B and S10 A–F,andMovie S15). Because the overall trend Time (s) -490 -280 -14 -7 0 7 14 28 602 1197 1799 of the Brownian motion of the BCRs (but not the control MHC-I BCR molecules) was to slow down after the photoactivation of caged-NP (Figs. 6 and 7, Figs. S9 and S10,andMovies S14 and S15), we MHC-I propose that the decreased lateral mobility of antigen engaged BCRs shall account for their trapping and accumulation within the Merge B-cell IS. We also propose that antigen engagement induced freezing of the lateral mobility of the BCRs is the fundamental E F mechanism for the growth feature of the BCR microclusters BCR MHC-I (Discussion). 25 caged-NP 25 caged-NP photoactivated-NP 20 20 photoactivated-NP Discussion 15 15 In this report, by combining the photoactivatable antigen system 10 10 with TIRFM-based live cell and single molecule imaging tech- 5 5 niques, we examined the basal responses of a quiescent B cell in 0 0 Normalized (mFI) Normalized -600 0 600 1200 1800 Normalized (mFI) -600 0 600 1200 1800 contact with coverslips presenting the caged-NP for 360 s and Time (s) Time (s) then examined the response of the very same B cell immediately Fig. 5. The B-cell IS is a dynamically open yet highly selective membrane after the immune recognition of the photoactivated-NP antigen structure. Representative two-color TIRFM images at the indicated time points for another 360 s. We define our system as the first, to our show the spatial-temporal changes in the dynamic behaviors of either the BCR knowledge, temporally seamless imaging experimental procedure (green) or MHC-I (red) molecules in J558L-B1-8-IgM cells that were placed on for the study of the molecular events that occur during B-cell coverslips coated with caged-NP before and after photoactivation. The basal activation. Our approach is quite different from the conventional behaviors of both the BCR and MHC-I molecules in quiescent B cells in contact experimental system of capturing the response of a B cell that is with caged-NP were first examined for 240 s by TIRFM imaging. Immediately loaded onto the coverslips presenting bioactive antigens directly. starting with photoactivation (time 0 s as indicated), the behaviors of these two The conventional experimental systems could not accurately molecules on the same B cells were continuously examined for another 360 s. separate the landing and adhesion responses of a B cell from the μ See Movie S11 for complete time-lapse TIRFM images. (Scale bar, 1.5 m.) (B and B-cell activation responses that were truly induced by BCR and C) Statistical quantification of the normalized mFI to show the synaptic accu- antigen recognition. With the aid of this unique experimental mulation of the BCR (B)orMHC-I(C) molecules in the same B cells before and system, a series of unprecedented observations were captured to after photoactivation of caged-NP as depicted in A. The curve from −360 to 0 s showed the dynamic changes of the J558L-B1-8-IgM cells in contact with caged- help us better understand B-cell and BCR behavioral changes NP, whereas the curve from 0 to 360 s showed the changes in the responses of during the initiation of B-cell activation. the same cells in contact with photoactivated-NP. Bars represent mean ± SEM First of all, we readily recorded the probing behaviors of a of 10 cells from one representative of three independent experiments. (D–F) quiescent B cell in contact with biologically inert caged-NP, which A similar experiment was performed as described for A–C except that a long is defined by the periodical formation of membrane pseudopods TIRFM imaging time course of 30 min was performed after photoactivation. See in random directions. The probing behaviors require the remod- Movie S13 for complete time-lapse TIRFM images. (Scale bar, 1.5 μm.) eling of F-actin as B cells pretreated with latrunculin B to disrupt F-actin or with jasplakinolide to stabilize F-actin lose the probing behaviors. These probing behaviors seem to be independent of Photoactivation by 405-nm laser was only executed during the the tonic BCR signaling as B cells pretreated with the Src family ki- first and second reading as depicted in Fig. 6A. Taking advantage nase inhibitor PP2 or the Syk kinase inhibitor piceatannol main- of the photoactivatable antigen system, we can for the first time, to tain the probing behaviors. Previous studies by Brodovitch et al. our knowledge, accurately quantify the Brownian motility of single used TIRFM imaging to examine the acute adhesion response of BCR molecules in the same B cells before and immediately after T cells when landing to nonactivating surfaces. They showed that

BCR engagement in a temporally seamless manner (Fig. 6A and T cells initially touched the surfaces through highly dynamic mem- INFLAMMATION Movie S14). brane pseudopods (protrusions) of submicrometer diameter, IMMUNOLOGY AND Analyses of the single BCR trajectory footprints suggested that and substantial antigen-independent spreading was only ob- single BCR molecules were highly mobile in quiescent B cells served after multicontacts formed with a lag time of 1 min (36). contacting caged-NP. However, on photoactivation, the BCRs from Importantly, they found that these T-cell membrane pseudopods thesameBcellsbecamegraduallylessmobileovertime(Fig.6B exhibited autonomous movements with a typical duration of a few and Movie S14). Tracking hundreds of single BCR molecules seconds or less and an axial amplitude of 60–70 nm (36). In our showed that their short-range mean-square displacements (MSDs) experimental system, B cells have been placed on coverslips pre- were linearly dependent on time, indicating free diffusion movement senting inert caged-NP for 10 min before the TIRFM imaging; thus, (Fig. 6D). A simple comparison of the six temporally segregated we cannot examine the axial displacement of B-cell membrane

Wang et al. PNAS | Published online January 13, 2016 | E563 Downloaded by guest on October 6, 2021 A photo- Repeat 3 times activation 18 s 10 s 18 s 10 s 18 s Excition

Emission BC BCR MHC-I 0−18 s 56−74 s 112−130 s 0−18 s 56−74 s 112−130 s 100 100 100 100 100 100 80 80 80 80 80 80 60 60 60 60 60 60 40 40 40 40 40 40 Pixel Pixel 20 20 20 20 20 20 0 0 0 0 0 0 0 50 100 0 50 100 0 50 100 0 50 100 0 50 100 0 50 100 Pixel Pixel Pixel Pixel Pixel Pixel D EF BCR BCR BCR 1 0.15 0-18s 0-18s n=5798 28-46s 28-46s n=6033 P<0.0001 )

56-74s 0.8 56-74s n=6173 2 84-102s n=6415 P=0.0011 84-102s μ m 1.0 112-130s n=6203 ) 0.10 112-130s 2 0.6 140-158s n=6467 140-158s μ m (

D 0.4 0.5 S 0.05 0.2 0.0 s 6s 4s 0s 8s 18 4 7 02s 5 0.00 0 0- 1 13 28- 56- 2- −4 −3 −2 −1 0 84- 0.0 0.1 0.2 0.3 0.4 10 10 10 10 10 11 140-1 Single molecule diffusion ( diffusion molecule Single Time (s) Cummulative distribution probability Single molecule diffusion (μm2/s) -0.5

GHMHC-I MHC-IC-I I MHC-I 1 0.15 0-18s 0-18s n=5798 28-46s 28-46s n=6033 P=0.8309

56-74s 0.8 56-74s n=6173 ) 84-102s n=6415 2 84-102s P=0.1967

112-130s n=6203 μ m )M 0.10 112-130s 2.0 2 0.6 140-158s n=6467 140-158s 1.5 0.4

MSD ( μ m 0.05 1.0 0.2 0.5

0.00 0 0.0 −4 −3 −2 −1 0 0.0 0.1 0.2 0.3 0.4 s s 10 10 10 10 10 8 0 8s Cummulative distribution probability 1 46s 2 - 13 15 Time (s) Single molecule diffusion (μm /s) ( diffusion molecule Single 0- -0.5 28 56-74s 2- 0- 84-102s 11 14

Fig. 6. The decreased lateral mobility of antigen-engaged BCRs accounts for their synaptic trapping events. (A) A schematic representation showing the experimental strategy during the single molecule imaging (SMI) of the BCRs. Each B cell was subjected to single BCR molecule imaging of 600 frames in 18 s (30 ms/frame) for the first SMI reading. The second SMI reading was taken after an interval of 10 s followed by the third SMI reading and another 10-s interval until the sixth reading. Photoactivation was only applied during the first and second TIRFM imaging cycles. See Movies S14 (BCR) and S15 (MHC-I) for complete time-lapse TIRFM images. (B and C) The accumulated trajectory footprints of the BCR (B) or MHC-I (C) molecules from the representative B cells in three

segmented TIRFM imaging time courses of 0–18, 56–74, and 112–130 s. The instant Brownian diffusion coefficients (D0) were shown as pseudocolored tra- −4 0 2 jectories. The display range of the pseudocolor is based on the D0 value of 1e to 1e μm /s. (D–I) The D0 values from the BCR (D–F) or MHC-I (G–I) single molecules that were observed from the indicated TIRFM imaging temporal segments (0–18, 28–46, 56–74, 84–102, 112–130, and 140–158 s) from SMI imaging

experiment as depicted in A. All of the D0 values were displayed as MSD plots (D and G), CDP plots (E and H), or mean ± SD scattered plots (F and I). Data represent the single BCR or MHC-I molecules of indicated numbers (E and H) for each condition from three independent experiments. Two-tailed t tests were performed for statistical comparisons in F and I.

pseudopods that shall be only obvious during the acute landing and among a pool of protrusions with slow displacement of less than adhesion responses of lymphocytes. Instead we calculated the x and 100-nm amplitude (36). We propose that it shall be of essential y coordinates for accurate spatial position of the puncta-structured interest to investigate the mechanism accounting for the high het- membrane protrusions in quiescent B cells before photoactivation erogeneous nature of the lateral displacement of membrane pro- and determined their trajectories using single particle tracking trusions and their diverse functions for both B and T cells to survey method as reported in our published studies (14, 18, 30). Strikingly, the microenvironment. the results suggested that B cells seem to survey the microenvi- Second, we systematically compared the probing behaviors of ronments at a mean square based lateral displacement of 0.05 μm2/s the same B cell before and after photoactivation. We found that (equal to 500 nm 2/s) (Fig. S9C). If only calculating those trajec- photoactivation promptly terminated the probing behaviors. In- tories showing directed Brownian mobility and long tracking steps terestingly, the termination of the probing behavior seems to be (>30 steps), a linear lateral displacement of 76.65 ± 15.375 nm/s can dependent on the interactions between the BCR and antigen be acquired. This value is well consistent with the observation in molecules, as neither the interaction between MHC-I and anti– T-cell studies showing that the T-cell membrane protrusions only MHC-I antibodies, nor that between integrin and the adhesion displayed limited lateral movement with a value below 100-nm molecule ICAM-1, terminated the B-cell probing behaviors. This amplitude in most of the cases (36). Moreover, the relatively large highly selective and strict dependence on the interactions be- SD (76.65 ± 15.375 nm/s) indicated the diverse range of the lateral tween the BCR and antigen molecules may be linked to the well- mobility of B-cell membrane protrusions, largely consistent with the documented actin remodeling events in response to BCR and T cells studies showing that some membrane protrusions with rapid antigen recognition (15, 16, 28, 29). All these in vitro data sup- lateral displacement of hundreds of nanometers can be captured port the early intravital imaging studies, which showed that

E564 | www.pnas.org/cgi/doi/10.1073/pnas.1517612113 Wang et al. Downloaded by guest on October 6, 2021 Experimental Curve Fast Group Fitted Curve Slow Group the B-cell IS. Based on all these data, we speculate that both MHC-I PNAS PLUS A B and general lipid molecules had the freedom to diffuse into, and out 0.30 0.30 Fast=0.1070 μm2/s Fast=0.0915 μm2/s of the B-cell IS. Additionally, an interesting finding was that the 0.25 (proportion=0.8478) 0.25 (proportion=0.8052) freedom to diffuse out of the BCR microclusters is not without Slow=0.0043 μm2/s Slow=0.0033 μm2/s 0.20 (proportion=0.1521) 0.20 (proportion=0.1947) limits. When the B-cell IS matures and forms the central and pe- 0.15 0.15 ripheral structures (as examinedina30-minTIRFMimagingex- periment of live B cells in Fig. 5D and Fig. S8B), a significant Density Density 0.10 0.10 accumulation of MHC-I within the central area of the B-cell IS could 0.05 0.05 be observed. These unexpected results indicated the molecular 0 0 crowding effects that were induced by the F-actin rich cytoskeleton -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 log10 (Diffusion Rate) log10 (Diffusion Rate) structures corralling the central area of a B-cell IS. Because recent studies by Dustin and colleagues demonstrated the polarized re- CD lease of TCR-enriched vesicles at the T-cell IS (39), it shall be of 0.30 0.30 Fast=0.0854 μm2/s Fast=0.0727 μm2/s interest to examine the biological functions of the heterogeneous 0.25 (proportion=0.7691) 0.25 (proportion=0.7097) molecular crowding effects within the B-cell IS. Slow=0.0034 μm2/s Slow=0.0046 μm2/s 0.20 (proportion=0.2309) 0.20 (proportion=0.2902) Last, this report suggested that the decreased lateral mobility 0.15 0.15 of antigen engaged BCRs accounted for the synaptic accumu- Density Density lation of the BCRs into the BCR microclusters. This supporting 0.10 0.10 data were obtained by accurately quantifying the Brownian dif- 0.05 0.05 fusion coefficients of thousands of BCR molecules on the same 0 -5 -4 -3 -2 -1 0 0 -5 -4 -3 -2 -1 0 B cells before and immediately after BCR engagement in a tem- log10 (Diffusion Rate) log10 (Diffusion Rate) porally seamless manner. All these seamless single molecule ex- periments were accomplished with our photoactivatable NP system, EF which is capable of generating subminute scaled temporal segments 0.30 0.30 Fast=0.0660 μm2/s Fast=0.0784 μm2/s immediately after BCR engagement for dozens of B cells with a 0.25 (proportion=0.7166) 0.25 (proportion=0.6963) synchronized trigger point. Indeed, on photoactivation, we found Slow=0.0019 μm2/s Slow=0.0029 μm2/s 0.20 0.20 (proportion=0.2834) (proportion=0.3036) that the Brownian diffusion coefficientoftheBCRsinthesame 0.15 0.15 batch of B cells gradually decreased over time, consistent with the early end point studies showing that the mobility of BCRs de- Density Density 0.10 0.10 creased a few minutes after the activation by mobile antigens on 0.05 0.05 PLBs (6, 24, 28, 31). It is worth noting that even though the overall 0 0 -5 -4 -3 -2 -1 0 -5 -4 -3 -2 -1 0 trend of the Brownian motion feature of the BCRs was to slow log10 (Diffusion Rate) log10 (Diffusion Rate) down, this change was not absolutely monotonic as we observed that the slow-moving BCR molecules regained motile behavior. Fig. 7. The probability density function (PDF) plot and subpopulation These fluctuations fit well with the early observation by Batista and analysis of the instant diffusion coefficient of BCR molecules. The D0 values – – – – colleagues examining the mobility of BCRs upon the recognition from each temporal segment of (A)0 18, (B)28 46, (C)56 74, (D)84 102, of membrane bound antigens on PLBs (28), which depicted the (E) 112–130, and (F)140–158 s were fitted by a mixture model of two components as described in Materials and Methods. The proportion and nonmonotonic mode of regulation of the single molecule motile behavior of the BCRs by actin cytoskeleton system. The contem- absolute values of both fast and slow populations in each temporal segment “ ” – are also shown on the top left corner within each PDF plot. porary picket and fence model of the plasma membrane (40 42), where the actin cytoskeleton represents the fence and the trans- membrane proteins represent the picket, predicts that the mem- B cells terminated the migratory behavior on recognition of brane receptors will undergo oligomerization-induced trapping cognate antigens on the surface of antigen-presenting cells upon ligand binding, thus showing the decreased Brownian diffu- (APCs) and subsequently developed an enlarged contact in- sion coefficient. Interestingly, in our experimental system, this terface for the acquisition of the antigenic information (37, 38). oligomerization-induced trapping phenotype was only observed Thus, the seamless imaging system in this report may be a better with BCR molecules but not with MHC-I molecules, which sug- representation of what occurs in vivo because the probing be- gested that these events were induced by the recognition of BCR havior of B cells allows them to survey the membrane surface of and antigen. Overall, these results suggested that the decreased the APCs for the presence of cognate antigen. We speculate that lateral mobility of antigen engaged BCRs accounts for their if there is no cognate antigen, the B cells will continue with the synaptic accumulation. probing behavior. In marked contrast, the presence of BCR Thus, by using this photoactivatable antigen-supported seam- specific antigens will efficiently terminate the B-cell probing less imaging system, we can for the first time to our knowledge, behaviors and lead to the synaptic accumulation of the BCRs and readily capture the behavioral changes of the same B cell before an increase in the size of the B-cell contact area. Furthermore, the and after BCR antigen recognition. All of these results improved findings in this report demonstrated that the termination of the our understanding of the B-cell probing termination behaviors probing behavior of B cells on BCR engagement is very sensitive to and the sophisticated BCR sorting mechanisms within the B-cell the affinity between the BCR and the antigen, suggesting the po- IS during B-cell activation. tential possibility that B cells could use probing termination event to Materials and Methods discriminate antigen affinity. Mice, Cells, Antibodies, and Plasmids. High-affinity J558L-B1-8-IgM-High and Third, we captured the growth feature of the BCR microclusters INFLAMMATION in response to the immune recognition of the BCR and cognate low-affinity J558L-B1-8-IgM-Low cells were constructed and maintained IMMUNOLOGY AND antigens, consistent with our previous studies (6). We also showed as reported (6). For brevity, we used J558L-B1-8-IgM cells to indicate high- affinity J558L-B1-8-IgM-High cells in most parts of this report except in the that the BCRs could be accumulated into the stationary BCR places in comparison with J558L-B1-8-IgM-Low cells. B1-8 primary B cells were − − microclusters in a passive trapping mechanism. Using a series of isolated from the spleen of IgH B1-8/B1-8 Igκ / transgenic mice as previously two-color TIRFM imaging experiments in combination with the reported (6). DyLight 649-conjugated Fab anti-mouse IgM constant region photoactivatable system, we simultaneously examined the spatial antibodies were purchased from Jackson ImmunoResearch Laboratories. Goat and temporal changes of both the BCRs and control molecules whole IgG anti–MHC-I antibodies were purchased from BioLegend. Dil cell- (MHC-I or lipid) within the B-cell IS. We observed that only BCRs labeling was purchased from Invitrogen. ICAM-I antibodies were purchased but not MHC-I, nor lipid molecules could be stably trapped within from R&D.

Wang et al. PNAS | Published online January 13, 2016 | E565 Downloaded by guest on October 6, 2021 Chemical Reagents and Solvents. Fmoc amino acids, HCTU [2-(6-chloro-1H- (nickel salt; DOGS–Ni-NTA; Avanti Polar Lipids) in a 9:1 DOPC/DOGS–Ni-NTA benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate], DIEA ratio. Small unilamellar vesicles (SUVs) were formed by sonication of the (N,N-diisopropylethylamine), HOBt (hydroxybenzotriazole), DIC (N,N’- mixed lipids and clarified by ultracentrifugation and filtering. Acid treated diisopropylcarbodiimide), Rink amide-AM resin, TFA (trifluoroacetic acid), coverslips were first coated with Ni-NTA–containing SUVs (0.1 mM) to form

TIPS (triisopropylsilane), and phenol were acquired from GL Biochem. Pi- PLBs, and then caged-NP peptides containing a His6-Tag were tethered to peridine, DMF (dimethylformamide), and CH2Cl2 were purchased from PLBs. The coverslips were washed with PBS before the blocking step with Sinopharm Chemical Reagent Co. All reagents and solvents used for small 200 μL of 0.1% (wt/vol) casein at 37 °C for 1 h. After further washing, NP- organic compound synthesis were bought from Sinopharm Chemical Re- specific B cells were placed into the chamber and incubated for 10 min at agent Co. or Alfa Aesar with the highest commercial quality and were used 37 °C. Next, the photoactivation experiments by the 405-nm laser can be without further purification. Anhydrous solvents (THF, CH Cl )were 2 2 performed. TIRFM images were acquired using an Olympus IX-81 microscope obtained from a dry solvent system (passed through a column of alumina). equipped with a TIRF port, ANDOR iXon+ DU-897D electron-multiplying EMCCD camera, Olympus 100 × 1.45 NA objective TIRF lens, and a 405-, 568-, Peptide Synthesis. Peptide synthesis vessels were acquired from Synthware and 640-nm laser (Coherent). The acquisition was controlled by the Meta- Glass Co. All of the peptides were synthesized manually according to the morph system (MDS Analytical Technologies). All of the photoactivation and standard protocol. For the regular coupling reaction, Rink amide-AM resin TIRFM imaging were performed at 37 °C unless otherwise indicated. The was coupled with 4 eq. amino acid, 3.6 eq. HCTU in 0.4 M DIEA (8 eq.)/DMF total photoactivation time of 30 s was achieved by 10 cycles of the following solution; 20% (vol/vol) piperidine in DMF was used for deprotection. The t imaging acquisition itinerary: (i): 405 nm for caged-NP photoactivation following Fmoc amino acids were used: Fmoc-His(Trt)-OH, Fmoc-Ser( Bu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc- (exposure time, 3 s); (ii) 647 nm for BCR imaging (exposure time, 100 ms); Lys(Boc)-OH, and Fmoc-e-Acp-OH. After the N-terminal Fmoc-e-Acp-OH was and (iii) 568 nm for MHC-I or lipid imaging if necessary (exposure time, coupled to the resin, the final deprotection was performed, followed by 100 ms). Starting from the 11th cycle, the 405-nm photoactivation step was addition of 2 eq. 4-hydroxy-3-nitrophenyl acetic acid (NP-OH), 5 eq. HOBt in removed from the itinerary. The interval time between each cycle was ad- 0.2 M DIC (5 eq.)/DMF solution to afford the NP-conjugate peptide. For the justed to 4 s automatically. DyLight 649-conjugated Fab anti-mouse IgM synthesis of DMNB-NP-conjugate peptide, the corresponding 4,5-dimethoxy- constant region antibodies were used to mark the IgM BCRs. The mFI of BCRs 2-nitrobenzoyl-NP-acetic acid (DMNB-NP-OH) was added. After the assembly within the B cell’s contact interface with the coverslips tethering photo- was completed, peptides were cleaved from the resin by treatment with a activated-NP were processed and analyzed through Image J (National In-

TFA mixture [88% (vol/vol) TFA, 2% (vol/vol) TIPS, 5% (vol/vol) H2O, 5% stitutes of Health) or Matlab (MathWorks) software following our published (vol/vol) phenol]. The crude peptide was then analyzed and purified by RP- protocol (6, 18, 30, 43). HPLC, and the molecular weight of each peptide was confirmed by ESI-MS. Two-Color Time-Lapse Live-Cell Imaging by TIRFM. By using two-color TIRFM, RP-HPLC and MS. Analytical and semipreparative RP-HPLC was performed lipid or MHC-I molecules were imaged in parallel with the BCRs for 420 s on with a Prominence LC-20AT with SPD-20A UV/Vis detector. All separations J558L-B1-8-IgM cells in contact with coverslips coated with caged-NP antigen. involved a mobile phase of 0.1% (vol/vol) TFA in water (solvent A) and 0.1% Without stopping the TIRFM imaging procedure, we photoactivated caged- (vol/vol) TFA in acetonitrile (solvent B). Analytical separations were per- NP by 405-nm total reflection photons and continued to capture the behavior – formed using a linear gradient (5 95%) of buffer B in buffer A over 30 min changes of both BCRs and the other molecules in photoactivation and thereafter. after an initial isocratic phase of 5% buffer B in buffer A for 2 min with a During the acquisition, two-color TIRFM images were taken every 4 s through μ × 1 mL/min flow rate on a Vydac C18 column (5 m, 4.6 250 mm). A Vydac multiple dimensional acquisition modes controlled by the Metamorph system. C18 column (10 μm, 10 × 250 mm) with a 4 mL/min flow rate was used for TIRFM images were processed to acquire the mean fluorescence intensity using semipreparative separations. Data were recorded and analyzed by LC-Solution Image Pro Plus (Media Cybernetics) and Image J (National Institutes of Health) software. Product containing fractions were identified by ESI-MS. ESI-MS was software as mentioned above. performed on an Agilent 1200/6340 mass spectrometer in the Center of Bio- medical Analysis. The buffers for MS analysis were the same but using formic Single-Molecular Tracking and Analysis. Single BCR molecule imaging was acid instead of TFA to increase MS ionization of peptides. performed following our published protocol (6). Briefly, prelabeled J558L-B1- 8-IgM cells were imaged by TIRFM with a 640-nm laser at an output power of Photochemistry. Caged peptides in PBS buffer (pH 7.4, containing 10 mM DTT) × (1 mg/mL, 20–50 μL) were placed in a 1.5-mL Eppendorf tube on ice. Irradi- 10 mW at the objective lens in the epi-fluorescence mode. A subregion of 100 ation was performed at 365 nm using the Omnicure S1500 (EXFO Photonic 100 pixels of the available area of the electron-multiplying CCD chip was used to Solutions Inc.) with a light intensity of 18 mW/cm2. RP-HPLC analysis was achieveanexposuretimeof30ms/frame,thetimeresolutionofwhichwas used to determine the photolysis kinetics. Peptides were quantified based found to be sufficient to reliably track the single molecule BCRs as reported (6, on the HPLC standard curves of the caged and noncaged derivatives. The 31). Single molecule tracking of BCR molecules was analyzed as described in our amount of each peptide was normalized to the amount of the internal previous study (6, 31). MSD and short-range diffusion coefficients for each BCR standard (Benzamide). molecule trajectories were calculated from positional coordinates and plotted as CDP. ELISA. We measured the binding capacity of anti-NP IgG to different NP peptides by ELISA. Each peptide (20 μg/mL) was photoactivated for the in- Subpopulation Analysis of Molecule Motion. BCR or MHC-I molecules in each dicated amount of time and was then diluted to 10 or 5 μg/mL Theses NP temporal segment are grouped into a fast population and a slow population, peptides were then coated on maxisorb plates (Nunc) by an overnight in- achieved by mathematically fitting the distribution of the instant diffusion

cubation at 4 °C. The plates were further blocked with 0.3% (wt/vol) gelatin coefficient (D0) by a mixture model of two components. The fitting begins by in PBS buffer (2 h at 37 °C), followed by the addition of anti-NP antibodies. transforming D0 to its logarithm, x = logðD0Þ. For the slow population, the After an incubation at 37 °C for 1 h, 1:10,000 diluted HRP-conjugated goat PDF, noted by ps, is assumed to be Gaussian anti-mouse IgG was used to detect NP antibody. After washing, the sub- " # strate solution, composed of 0.325% orthophenylenediamine dihydro- − μ 2 μ σ = ffiffiffiffiffiffi1 − ðx Þ chloride (OPD; Sigma) and 0.085% H O in 0.3 M Tris-citrate buffer, pH 6.0, psð , , xÞ p exp . 2 2 2πσ 2σ2 was added. After incubation at room temperature for 10 min in the dark, the reaction was stopped with 2.5 M H2SO4 and the plates were measured For a molecule exhibiting 2D Brownian motion of average diffusion co- using the ELISA plate reader (Bio-Rad) at 490 nm. 2 efficient D, D0 follows χ distribution of 2°. Hence the PDF pB is   Photoactivation of Caged-NP and Molecule Imaging by TIRFM. The behaviors of expðxÞ expðxÞ p ðD, xÞ = exp − . NP-specific B cells before and after the photoactivation of caged-NP were B D D examined by TIRFM imaging. Caged-NP peptides were first tethered to acid- treated coverslips for 30 min at 37 °C. Caged-NP peptides can also be captured The fast population is modeled by a mixture of groups of Brownian mole- to the coverslips presenting PLBs. In this case, we first prepared Ni-NTA– cules. Considering the complexity of cell conditions, Brownian molecules will containing PLBs following our published protocol (6, 18, 30, 43). Briefly, we follow χ2 distributions of parameter D slightly different to each other. To mixed 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; Avanti Polar Lipids) and formulate this, the PDF of the fast population pf equals to pB blurred by a 1,2-dioleoyl-sn-glycero-3-[N(5-amino-1-carboxypentyl) iminodiacetic acid]-succinyl Gaussian function

E566 | www.pnas.org/cgi/doi/10.1073/pnas.1517612113 Wang et al. Downloaded by guest on October 6, 2021 Z∞ ! showing distinct distances to the boundaries of the centralized cell body of PNAS PLUS 2 ffiffiffiffiffiffi1 t B cells in TIRFM images. pf ðD, σs, xÞ = pBðD, x − tÞ p exp − dt. 2πσ 2σ2 −∞ s S Pretreatment of B Cells with Pharmaceutical Inhibitors. B cells were pretreated The log likelihood function Lðμ, σ, D, σs, qÞ on molecule set fx1, x2, ..., xng is with 0.2 μM cytochalasin D for 10 min at 37 °C to block actin polymerization or were pretreated with 1 μM jasplakinolide for 45 min at 37 °C to block actin X∞ μ Lðμ, σ, D, σ , qÞ = log½qp ðD, σ , x Þ + ð1 − qÞ p ðμ, σ, x Þ, depolymerization (6). B cells were pretreated with 50 M of the Src family s f s i s i μ i=1 tyrosine kinase inhibitor PP2 for 10 min at room temperature or with 50 M Syk inhibitor piceatannol for 10 min at room temperature (6). Where q is the proportion of the fast population. Parameters are estimated by maximizing the log likelihood function, solved by the matlab function fminsearch. ACKNOWLEDGMENTS. We thank Dr. Susan K. Pierce (National Institute of Allergy and Infectious Diseases, National Institutes of Health), Dr. Klaus Rajewsky Quantification of BCR Microclusters and B-Cell Probing Behaviors. The math- (Immune Regulation and Cancer, Max-Delbrück-Center for Molecular Medicine), ematical quantification of BCR microclusters for precise FI and position in- and Dr. Mark Shlomchik (Yale University) for generously providing experimental formation was performed following our published protocol by a MatLab- materials. This work was supported by funds from National Science Foundation based 2D Gaussian analysis algorithm (6, 43). This function was used to China Grants 81422020, 81361120384, 21532004, and 31270913, Ministry of Science and Technology of China Grants 2014CB542500, 2013CB932800, and quantify each of the 2D FI profiles for some critical parameters of each 2014AA020527, the Specialized Research Fund for the Doctoral Program microcluster, such as position (xc, yc) and integrated FI (I). B-cell probing of Higher Education Grants 20130002110059 and 20120002130004, One- behaviors were quantified by counting the number of membrane pseudo- Thousand-Youth-Talents Program Grant 2069999-3 of the Chinese Cen- pods/protrusions per cell per TIRFM image. In this report, the membrane tral Government, and Tsinghua University Initiative Scientific Research Program protrusions were defined as the puncta-structured membrane protrusions Grant 20131089279.

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