B Cells Adapt Their Nuclear Morphology to Organize the Immune Synapse and Facilitate Antigen Extraction

Abstract

Upon interaction with immobilized antigens, B cells form an immune synapse where actin remodeling and re-positioning of the microtubule-organizing center (MTOC) together with lysosomes can facilitate antigen extraction. B cells have restricted cytoplasmic space, mainly occupied by a large nucleus, yet the role of nuclear morphology in the formation of the immune synapse has not been addressed. Here we show that upon activation, B cells re-orientate and adapt the size of their nuclear groove facing the immune synapse, where the MTOC sits, and lysosomes accumulate. Silencing the nuclear envelope proteins Nesprin-1 and Sun-1 impairs nuclear reorientation towards the synapse and leads to defects in actin organization. Consequently, B cells are unable to internalize the BCR after antigen activation. Nesprin-1 and Sun-1-silenced B cells also fail to accumulate the tethering factor Exo70 at the center of the synaptic membrane and display defective lysosome positioning, impairing efficient antigen extraction at the immune synapse. Thus, changes in nuclear morphology and positioning emerge as critical regulatory steps to coordinate B cell activation.

Keywords: B cells; Nesprin-1; Sun-1; actin cytoskeleton; immune synapse; nuclear morphology.

Figure 1

The nucleus reorients towards the B cell immune synapse. (A–D) Representative confocal images of B cells stained for the nucleus (Lamin B, green), actin (phalloidin, red), and microtubules (α-tubulin, magenta). Cells were incubated with antigen-coated beads (white circles) for 0, 30, 60, and 120 min. (B) Scheme depicting method used to measure the orientation of the principal nuclear groove. (C) Percentage of cells with polarized, central, or non-polarized nuclei with respect to the antigen; n≥90. (D) Scheme of nuclear groove rotation in B cells seeded on antigen-coated dishes. (E) Visualization of nucleus rotation (Hoechst, blue) and MTOC (centrin-GFP) in B cells seeded on antigen-coated dishes (filmed every 2 min). (F, G) Representative confocal images and quantification of nuclear groove reorientation in B cells seeded on antigen-coated dishes for indicated times, orientation is indicated between 0-180°. Nuclei (Lamin B): green; lysosomes (LAMP1): magenta; yellow arrowheads: nuclear groove; n≥52 cells. (H) Scheme and quantification of the nuclear groove depth, measuring the height or distance between the point of the nuclear groove farthest from a line traced between the two nuclear lobe apices. All scale bars are 5 µm. Statistical analyses: Kruskal-Wallis with Dunn’s multiple comparisons tests (C, H) and unpaired t-tests (G). *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 2

Nuclear shape is regulated by cytoskeleton stability. (A) Confocal images showing actin cytoskeleton (phalloidin, red) and microtubules (α-tubulin, magenta) strongly associated with the nucleus (Lamin B, green) in resting B cells. Arrowheads: nuclear groove; lobes: yellow; connection between cytoskeleton and nucleus: white. Scale bar 5 µm. (B) Representative confocal images of resting mouse primary B cells; stain colors/arrowheads as indicated as in (A). Primary B cells with a nucleus that occupies almost the entire area of the cell. (C) Diameter of primary B cells and their nuclei and (D) nuclear groove area. (E) Representative confocal images of primary B cells incubated with antigen-coated beads for 60 min. White circles indicate bead position. Arrowheads: nuclear groove. (F) Nuclear orientation, measured as the angle of the nuclear groove. n>55, unpaired t-tests; ****p < 0.0001. (G) Nuclear groove area upon activation. n>55, Mann-Whitney test; ****p < 0.0001; from three independent experiments.

Figure 3

Nesprin-1 and Sun-1 regulate nuclear shape during B cell activation. (A) Nesprin-1 immunoprecipitation (IP) assay to detect LINC complex (Nesprin-Sun) formation in resting or activated B cells for indicated times; data represent three independent experiments. (B) Left: Representative confocal images of nuclear groove slice in control, Nesprin-1-, and Sun-1-silenced B cells under resting conditions. Scale bar: 10 μm. Right: 3D reconstruction images showing whole B cells (total volume of actin and Lamin B); and logical filter applied on actin signal, showing only actin surrounding the nucleus. (C) 3D measurement of the intersections between actin and Lamin B by segmented signals. Quantification of the ratio of intersection of Lamin B and the actin signal divided by the total lamin B signal; n≥48. (D-F) Measurement of nuclear groove rotation in Nesprin-1-, and Sun-1-deficient B cells. Controls and silenced cells were incubated on antigen-coated dishes for indicated times. Scheme depicting the method used to measure nuclear orientation towards the synaptic plane (D), representative confocal images (E), and quantification of complete nuclear lobes rotation (F) between 180° – 0°; n≥60 cells from two independent experiments. (G) Quantification of the nuclear groove depth (height) in control, Nesprin-1-, and Sun-1-silenced B cells under resting and activating conditions. n≥55. Lamin B: green; actin: red in all images. Statistical analyses: Kruskal-Wallis with Dunn’s multiple comparisons tests (C) and two-way ANOVA with Sidak’s multiple comparison tests (F, G); *p < 0.05, **p < 0.01, and ****p < 0.0001.

Figure 4

Nesprin-1 and Sun-1 regulate immune synapse organization. (A) Scheme depicting a mature immune synapse formed by B cells. Arrows indicate nuclear groove space and position of nuclear lobes at the center of the immune synapse. Right side: a confocal image of a B cell seeded on an antigen-coated dish for 30 min, fixed and stained for the nucleus (Hoechst, blue) and actin (phalloidin, red). Nuclear lobules are highlighted with a white line. (B–D) Representative confocal images of the actin signal at the immune synapse of B cells silenced for Nesprin-1 or Sun-1, labeled and activated as in A; quantification of central actin MFI (C) and peripheral actin, which was used to measure immune synapse area, n≥60 (D). (E, F) Confocal images of B cells activated as in A and schemes showing nuclear and lysosome positioning at synaptic center. (E) Upper panel shows cells stained for actin (red) and nucleus (Hoechst in blue, delineated with a white segmented line). Schemes below indicate, the center of mass of the immune synapse (black dot) and the nucleus center of mass (orange dot). Quantification of distance between the nucleus and immune synapse mass center (MC); n>60. (F) Upper panel shows cells stained as in E and stained for lysosomes (LAMP1, green). Schemes below indicate, lysosome positioning respect to the nucleus and the immune synapse. Quantification of lysosome cluster located outside or inside of the perinuclear region, n>60. All scale bars 5 µm. Statistical analyses: Kruskal-Wallis with Dunn’s multiple comparisons tests; *p < 0.05, ****p < 0.0001.

Figure 5

Antigen extraction relies on Nesprin-1 and Sun-1. (A) Epifluorescence images of control or Nesprin-1- and Sun-1-silenced B cells incubated with antigen-coated beads for 0 or 120 min. Cells were stained against OVA (Green), actin (red), and LAMP1 (magenta). White circles indicate bead position. Scale bar 5 μm. (B) Percentage of OVA remaining on antigen-coated beads; n=80, **p = 0.001; ***p = 0.0006; ****p < 0.0001. Means with SEM lines shown. (C) Measurement of LAMP1+ rings surrounding antigen-coated beads; n=80; *p=0.05. (D, E) Confocal images of silenced B cells (as in A) under resting conditions, showing Exo70 (green) with: upper panel, microtubules/MTOC (α-Tub, magenta); and lower panel, actin (red) and nucleus (Hoechst, blue). Quantification of Exo70 distribution (radial profile) from the MTOC. n>30. (F, G) Confocal images of silenced B cells (as in A) activated on antigen-coated dishes for 30 min. Exo70 (green), actin (red) and nucleus (Hoechst, blue). Quantification of Exo70 concentration in central region of immune synapse; n=40, *p < 0.05, **p < 0.01. Statistical analyses: Kruskal-Wallis with Dunn’s multiple comparisons tests (B, C, G), mixed-effects analyses, and Dunnett’s multiple comparisons tests (E).

Figure 6

Model of immune synapse organization controlled by nuclear envelope proteins Nesprin-1 and Sun-1. Upon activation, the immune synapse formed by a B cell and antigen-presenting cell is well organized. The MTOC, Exo70, and lysosomes are recruited to the center of the immune synapse, where antigen-BCR complexes are clustered and internalized. This region is formed by the positioning of the nuclear groove, which orchestrates cytoskeleton remodeling and membrane trafficking. In Nesprin-1- and Sun-1-silenced B cells, connections between actin and the nucleus are lost; the nuclear groove fails to orient towards the antigen contact site, leading to a disorganized immune synapse. This disorganized synapse is characterized by diminished BCR clustering at the center of the synapse and defective Exo70 recruitment, impairing local tethering of lysosomes required to efficiently extract and process immobilized antigens. Further studies are required to elucidate how physical changes in the shape of the nucleus impact immune synapse organization.