Figure 1.
Bin2 is mainly expressed in hematopoietic cells and leucocyte-enriched tissues. A
: Expression of Bin2 in rat tissues. Cell homogenates from the indicated tissues were resolved by SDS-PAGE and immunoblotted with our polyclonal anti-Bin2 antibody (BACT). B: Endogenous expression of Bin2 in different leucocytic cell lines. An anti-calnexin antibody was used as a loading control. Molecular weight markers (Broad Range, Promega) are indicated.
Figure 2.
The structure of the hBin2 BAR domain. A
: Ribbon representation of the dimeric crystal structure found in the asymmetric unit. Subunit A is depicted in green and B in purple. Kinks in helix 2 (K1 and K2) and helix 3 (K3) are indicated. B: The different conformation of the BAR ends. Ribbon representation of the dimer superimposed on itself by a 180° rotation, so that subunit A is superimposed on B and vice versa. The central region, Cα atoms from amino acids 38–131 and 197–238 were used for the superimposition. The dimer and its rotated version are shown in dark and light colours respectively, green and purple as in (A). A view of the concave face is show on the left and the “end-on” view on the right. C: Comparison of hBin2 and dAmph BAR structures. On the top: ribbon representation of the superposed monomers. hBin2 subunit A in green and dAmph in yellow. In the middle and bottom: superimposition of the BAR dimers showing the slightly smaller curvature of Bin2 defined by the concave surface. Arrows indicate the regions that contribute to the differences in curvature. A side view is shown in the middle and a view of the concave surface on the bottom. D: Molecular surface representation of hBin2 BAR dimeric crystal structure showing the dimerization interface. Subunit A is depicted in transparent green and subunit B is depicted in pink. Residues V81 and S214 located on the surface of protomer B are highlighted in violet.
Table 1.
Data collection, phasing and refinement statistics.
Figure 3.
In vitro binding of hBin2 N-BAR to membranes.
A: hBin2 N-BAR binds more tightly to membranes that dAmph, despite the high sequence homology. Coomassie-stained gels of cosedimentation assays of hBin2 and dAmph N-BARs, with Folch liposomes. S, supernatant and P, pellet. B: hBin2 tubulates liposomes in a similar fashion to dAmph N-BAR. Electron micrographs of Folch liposomes tubulated by hBin2 (12–15 nm diameter) or dAmph N-BARs (∼12 nm diameter). C: Molecular surface representation of modelled N-terminal amphipathic helices. In blue, positive charges, in red, negative charges and in green, hydrophobic residues. D: Bin2 H0 helix is essential for membrane binding and bulky hydrophobic residues of Bin2 H0 helix contribute to the enhanced affinity to membranes compared with dAmph. Sequence alignment of hBin2 and dAmph H0 helix. Bulky hydrophobic amino acids are highlighted in red. Liposome cosedimentation assay as performed in (A) with hBin2 N-BAR amphipathic helix-mutants: F13A,F21A and H0 deletion mutant (Δ31). E: Bin2 has no specificity for phosphoinositides (PIPs) but prefers membranes enriched in PIPs. Lipid cosedimentation assays with synthetic liposomes (70% PC, 30% PS, 10% cholesterol, 1% PIPs).
Figure 4.
Bin2 localizes at podosomes of adherent leucocytes and is an adaptor for SH3-domain containing proteins. A
: Epifluorescence images of endogenous Bin2, stained with a polyclonal anti-hBin2 (BACT) antibody, in rat mast cells (RBL·2H3 cells). Transiently overexpressed Bin2-EGFP in mast cells (live cell imaging), human B cells (fixed 721.221 cells), and mouse macrophages (BAC1·2F5 cells, live cell imaging). Podosome like structures are highlighted by the white circle. Boxed areas show enlarged regions. Scale bars, 5µm. B: Bin2 and podosome markers: snapshots of living RBL cells transiently expressing rBin2-EGFP and LifeAct-mCherry or cortactin-mCherry. Box on the right shows fixed RBL·2H3 cells expressing rBin2-EGFP (left) stained with an anti-Vinculin Ab (middle). Small panels show maximized views of highlighted areas (white boxes). Scale bars, 5µm. C: A dimeric N-BAR domain is required for Bin2 targeting to podosomes. Snapshots of living rBRAP-siRNA treated RBL·2H3 cells transiently expressing different rBin2-EGFP siRNA-insensitive mutants: full-length protein, the N-BAR only (1–238), which also localizes at these adhesive structures, the BAR domain (32–238), which goes to lysosomes (the image shows colocalization with lysotracker (red), the C-terminal end (239-end), which is cytosolic and the full-length dimerization mutant (V81R, S214E), mutations destabilize dimer formation and the protein becomes cytosolic. Scale bars, 5 µm. D: Bin2 immunoprecipitations (IPs) from human B cells (721.221) (left) or rat mast cells (RBL·2H3) (right) using our BACT Ab shows binding with PIX and Git2 proteins. In control experiments (labelled as C) these proteins are missing. E: LC-MS/MS data for the bands indicated in (D). F: Bin2 directly interacts with α-PIX. In vitro pull-down assays using GST-hBin2-CT versus a GST (control) with purified human α-PIX SH3 domain. Lanes 1–6 show the GST pull-downs. The amount of α-PIX increases over these samples and only binds to GST-Bin2-CT (red box). Lane 7, broad range molecular markers, BioRad. Molecular weights (kDa) are indicated. Lane 8 GST-hBin2-CT or GST alone and lane 9 α-PIX alone. Proteins were detected by Coomassie stain.
Figure 5.
Bin2 regulates cell movement. A
: Bin2 is at the leading edge of moving cells: (A) Snapshots of a migratory macrophage (mouse BAC1·2F5 cell line, left) and a B cell (human 721.221 cell line, right) overexpressing Bin2-EGFP. Arrows indicate the direction cells are moving. Scale bars, 5 µm. B: Polarization of hBin2-EGFP to the leading edge of a B cell (human 721.221 cell line) that is migrating towards a neighbouring NK cell (human YT cell line). Phase contrast image (left) and epipluorescence micrograph (right). Scale bars, 5 µm. C: Transwell migration assay with monocytes (human U937 cell line). Top: Western blot showing the depletion of Bin2 when cells are treated with siRNA6 and siRNA8 (Dharmacon). Calnexin was used as a loading control. Bottom: Bin2-depleted monocytes (siRNA6 and siRNA8) show a decreased motility when compared with untransfected cells (UTC) or cells transfected with a control siRNA. Cell migration was performed under stimulating (addition of 60 nM RANTES, left) and non-stimulating (right) conditions. An inhibition of cell movement (addition of 5 µM calphostin, a PKC inhibitor) was also performed as a control. Data are the mean ± SD.
Figure 6.
Bin2 is located at the phagocytic cup of macrophages and regulates phagocytosis. A
: Epifluorescence micrographs of fixed macrophages (mouse BAC1·2F5 cell line) transiently overexpressiong Bin2-EGFP and LifeAct-mCherry. Cells were incubated with 0.9 µm latex beads for 3 min. before fixation to be able to visualize phagocytic cups (white stars). B: Confocal micrographs of two different rat alveolar macrophages (RAM) over-expressing Bin2-EGFP. Cells were incubated with alexa-labelled immune-complexed ovalbumin (pink) for 10 minutes at 37°C. Nuclei stained with DAPI (blue). Bin2 is enriched at the phagocytic cup. C: Left: western blot of RAM lysates from wild type (WT) and 48h-rBin2 siRNA transfected cells developed with polyclonal anti-hBin2 (BACT) and anti-actin (loading control) antibodies. Molecular weight markers (Broad Range, Promega) are indicated. Right: phagocytosis assay (expressed as phagocytic index (geometric mean fluorescence of positive cells)) performed with RAM cells at 4° (surface binding without internalization) and 37°C (internalization). Cells were incubated with alexa-labelled ovalbumin for 45 min before analysis by flow cytometry. Uptake from cells overexpressing rBin2-EGFP (Bin2) and rBin2 N-BAR-EGFP (N-BAR) and Bin2-depleted cells (Bin2 siRNA) where compared with control experiment (EGFP). Overexpression of a siRNA-insensitive protein on Bin2 depleted cells rescues the phenotype (Rescue). A siRNA control was also performed (siRNA control). Data are the mean ± SD. Significance was calculated using the Student’s t test (* = p<0.05). D: Confocal micrographs of RAM cells over-expressing Bin2-EGFP (Bin2, green, left) or control (EGFP, green, right). Cells were incubated with alexa-labelled immune-complexed ovalbumin (pink) for 120 minutes 37°C. Nuclei stained with DAPI (blue).