Article Figures & Data
Figures
- Figure 1.
Expression of ephrinBs and EphB receptors in mixed hippocampal cultures. The low-density 14 DIV primary hippocampal cultures were stimulated for 15 min with the indicated preclustered ectodomain ephrin/Eph–Fc fusion proteins or as a control with preclustered control Fc before fixation. After permeabilization, the cultures were stained with antibodies against the Fc portion (A–H). Specific binding of the Fc fusion proteins leads to the appearance of characteristic clusters (as in B). Glial cells and neurons were identified by their typical non-neuronal like (large cytoplasm, compact morphology; A′–D′) and neuronal-like (multiple neurites; E′–H′) morphologies by bright-field microscopy, respectively. The non-neuronal/glial-like morphology was correlated with GFAP-positive cells (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Scale bars, 10 μm. I, Quantification of ephrin and Eph expression. Three to four images for each condition were analyzed using MetaMorph software (supplemental Materials and Methods, available at www.jneurosci.org as supplemental material). The background (Fc) staining was subtracted from each image, and only round unmerged clusters were counted.
- Figure 2.
Release of EphB2-containing vesicles from 16-d-old neurons. A, B, High-density 14 DIV primary hippocampal cultures were transfected with expression constructs coding for fluorescently tagged EphB2–YFP receptors. Two days later, transfected neurons were imaged by time-lapse microscopy at 1 frame/min. Large images show merge of phase contrast in red and the EphB2–YFP fluorescence of transfected neurites (and its growth cone; A) in green. Dashed boxes indicate the imaged area in the bottom row. Glial cells (asterisk) are indicated by lighter areas surrounded by dashed lines. Darker areas indicate glia-free area (diamond). Scale bars, 20 μm. A1–A4, B1–B6, Fluorescent images from the dashed box of the large images. Time points of imaging are indicated in top right corners. A1–A4, Within 6 min, a fluorescent cluster is moving away from the middle filopodium-like protrusion toward a neighboring cell, most likely a glial cell in close contact with the neuron (fluorescent cluster indicated by arrow) (supplemental movie S1, available at www.jneurosci.org as supplemental material). B1–B6, Within 8 min, a fluorescent cluster is moving away from a small neuronal filopodium toward a neighboring cell, most likely a glial cell (fluorescent cluster indicated by arrow) (supplemental movie S2, available at www.jneurosci.org as supplemental material). C, Comparison of efficiency of release of fluorescent clusters generated by different fusion proteins. Primary hippocampal high-density cultures were transfected at 13–15 DIV with expression constructs coding for either EphB2–YFP, YFP–ephrinB1, YFP–ephrinB2, GFP-tagged TrkB.T1 receptor (GFP–TrkB.T1), or a membrane-tagged (myristoylated) form of YFP (memYFP). Time-lapse microscopy was done as above. Each movie was analyzed for trans-endocytosis events and counted as positive (yes), negative (no), or ambiguous (maybe). Movies were counted as positive when a fluorescent cluster moved away from a dendrite or dendritic filopodium, or as negative when a fluorescent cluster did not move at all or moved back and forth or alongside dendrites (data not shown). The numbers of time-lapse recordings used for the analysis are indicated in the bars. Clusters covered an area of 4.9 ± 2.8 pixels2 (mean ± SD) corresponding to 1.1 ± 0.6 μm2.
- Figure 3.
Inhibition of EphB2–YFP release by soluble Fc proteins and implication of astrocytes. A–G, Hippocampal cultures were established and imaged as in Figure 2. A–C, After 35 min of imaging, unclustered Fc, ephrinB1–Fc, or EphB2–Fc were applied to the cultures. Representative frames of the analyzed movies before and after Fc protein (A) or ephrinB1–Fc (B) application. Time points in minutes are indicated in the bottom corners. Gray arrows indicate the position at which trans-endocytosis events are just about to happen, and white arrows indicate released EphB2 clusters. A, Supplemental movie S3 (available at www.jneurosci.org as supplemental material); B, supplemental movie S4 (available at www.jneurosci.org as supplemental material). C, Quantification of trans-endocytosis events before and after bath application of the indicated proteins. Data are expressed as mean ± SEM (N.S., not significant; **p < 0.0001, Student’s t test). D–G, Association of a GFAP-positive astrocyte with pinched-off EphB2 clusters in low-density cultures. D, Merge of the phase-contrast image (red) and the YFP fluorescence (green) (similar to Fig. 2). Scale bar, 10 μm. E, Bright-field image of the cell depicted in D. F, The same cell immunostained with anti-GFAP antibodies (GFAP). Dashed lines in the images outline the astrocyte. G, Fluorescent time-lapse images depicting a filopodium-like protrusion (arrow) growing on top of the glial cell and showing the detachment of a fluorescent cluster. Time points of imaging are indicated in the top right corners (supplemental movie S5, available at www.jneurosci.org as supplemental material).
- Figure 4.
EphB2 and ephrinB clusters are endocytosed into astrocytes at sites of cell-to-cell contact. A, B, Mature recipient astrocytes were cultured on glass coverslips and stimulated with HeLa cells (green) transiently transfected to express either EphB2–YFP (A) or YFP–ephrinB1 (B). After 1 h, cells were fixed and imaged for YFP fluorescence. Note the green fluorescent puncta in the cytosol of the astrocyte that is in contact with EphB2 (A) or ephrinB1 (B)-expressing HeLa cells. The insets depict YFP fluorescence only. C, D, Parallel astrocyte/HeLa cultures were immunostained for EphB2 (anti-FLAG) or ephrinB1 (anti-HA) on the surface and for the endocytic marker EEA1 after the cells had been permeabilized (supplemental Materials and Methods, available at www.jneurosci.org as supplemental material). Note the presence of EphB2-positive puncta inside the astrocyte (green arrows in Cb) colocalizing with EEA1 (white arrows in Ce).
Additional Files
Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental material
- supplemental material - Supplementary Figure 1: GFAP-positive cells in dissociated 14 DIV hippocampal cultures. 14 DIV primary hippocampal low-density cultures were fixed, permeabilized and immunostained with antibodies against the glial fibrillary acidic protein (GFAP) to reveal the morphology of astrocytes in hippocampal cultures (GFAP, right column, A�-B�). The corresponding bright-field images are shown on the left (phase, A-B). Scale bars = 10 �m.
- supplemental material - Supplementary Figure 2: Expression of ephrinBs and EphB receptors in 14 DIV hippocampal cultures. 14 DIV primary hippocampal low-density cultures were stimulated for 15 min with preclustered unfused Fc protein (negative control), ephrinB2-Fc, EphB1-Fc or EphB4-Fc, fixed and stained as described in Figure 1. Scale bars = 10 �m.
- supplemental material - Supplementary Figure 3: Expression of ephrinAs and EphA receptors in 14 DIV hippocampal cultures. 14 DIV primary hippocampal low-density cultures were stimulated for 15 min with preclustered unfused Fc protein (negative control), or the indicated ephrinA-Fc, and EphA-Fc fusion proteins, fixed and stained as described in Figure 1. Scale bars = 10 �m.
- supplemental material - Supplementary Figure 4: Comparison of endogenous and exogenous EphB2 expression. 14 DIV hippocampal high-density cultures were transfected with expression constructs encoding either EphB2-YFP (A-D, I-L) or YFP (E-H). Two days later, cultures were stimulated with either preclustered ephrinB1-Fc (A-H) or as a control with preclustered Fc (I-L) for 15 min, fixed and stained as described in Figure 1. Left row shows merge of YFP fluorescence of transfected neurites in red and the ephrinB1-Fc immunostaining in green. YFP fluorescence depicts exogenous EphB2 expression (C), whereas ephrinB2-Fc stains both endogenous EphBs and exogenous EphB2 (A,B). Right row shows phase contrast. Scale bars = 10 �m.
- supplemental material - Supplemental movie 1
- supplemental material - Supplemental movie 2
- supplemental material - Supplemental movie 3
- supplemental material - Supplemental movie 4
- supplemental material - Supplemental movie 5
