mouse anti nesprin 1  (Developmental Studies Hybridoma Bank)

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2021.801164

Figure Lengend Snippet: 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.

Article Snippet: For Western blot, the following antibodies were used: rabbit anti-Sun-2 (Abcam #124916, 1:100), mouse IgG1 anti-Nesprin1 (Invitrogen #MANNES1A (7A12), 1:500), rat anti- α- tubulin (Genetex, #GTX76511), 1:100), rabbit anti- γ-Tubulin (Abcam, #Ab11317, 1:1,000), mouse anti-actin (cloneC4, ImmunO, #691001), mouse Nesprin 1 mouse (invitrogen, Ma5-18077, 1:500) rabbit anti-Histone 3 (Abcam #ab1791).

Techniques: Activation Assay, Immunoprecipitation, Incubation

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2021.801164

Figure Lengend Snippet: 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.

Article Snippet: For Western blot, the following antibodies were used: rabbit anti-Sun-2 (Abcam #124916, 1:100), mouse IgG1 anti-Nesprin1 (Invitrogen #MANNES1A (7A12), 1:500), rat anti- α- tubulin (Genetex, #GTX76511), 1:100), rabbit anti- γ-Tubulin (Abcam, #Ab11317, 1:1,000), mouse anti-actin (cloneC4, ImmunO, #691001), mouse Nesprin 1 mouse (invitrogen, Ma5-18077, 1:500) rabbit anti-Histone 3 (Abcam #ab1791).

Techniques: Staining, Labeling

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2021.801164

Figure Lengend Snippet: 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) .

Article Snippet: For Western blot, the following antibodies were used: rabbit anti-Sun-2 (Abcam #124916, 1:100), mouse IgG1 anti-Nesprin1 (Invitrogen #MANNES1A (7A12), 1:500), rat anti- α- tubulin (Genetex, #GTX76511), 1:100), rabbit anti- γ-Tubulin (Abcam, #Ab11317, 1:1,000), mouse anti-actin (cloneC4, ImmunO, #691001), mouse Nesprin 1 mouse (invitrogen, Ma5-18077, 1:500) rabbit anti-Histone 3 (Abcam #ab1791).

Techniques: Incubation, Staining, Concentration Assay

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2021.801164

Figure Lengend Snippet: 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.

Article Snippet: For Western blot, the following antibodies were used: rabbit anti-Sun-2 (Abcam #124916, 1:100), mouse IgG1 anti-Nesprin1 (Invitrogen #MANNES1A (7A12), 1:500), rat anti- α- tubulin (Genetex, #GTX76511), 1:100), rabbit anti- γ-Tubulin (Abcam, #Ab11317, 1:1,000), mouse anti-actin (cloneC4, ImmunO, #691001), mouse Nesprin 1 mouse (invitrogen, Ma5-18077, 1:500) rabbit anti-Histone 3 (Abcam #ab1791).

Techniques: Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Nuclear Envelope Alterations in Myotonic Dystrophy Type 1 Patient-Derived Fibroblasts

doi: 10.3390/ijms23010522

Figure Lengend Snippet: Primary antibodies used to detect the multiple proteins analyzed by Western blotting and immunocytochemistry.

Article Snippet: Mouse monoclonal anti-nesprin-1 (MANNES1E 8C3) , Developmental Studies Hybridoma Bank , WB—0.4 μg/mL ICC—1.5 μg/mL.

Techniques: Western Blot, Immunocytochemistry