Filopodia Conduct Target Selection in Cortical Neurons Using Differences in Signal Kinetics of a Single Kinase

Abstract

Dendritic filopodia select synaptic partner axons by interviewing the cell surface of potential targets, but how filopodia decipher the complex pattern of adhesive and repulsive molecular cues to find appropriate contacts is unknown. Here, we demonstrate in cortical neurons that a single cue is sufficient for dendritic filopodia to reject or select specific axonal contacts for elaboration as synaptic sites. Super-resolution and live-cell imaging reveals that EphB2 is located in the tips of filopodia and at nascent synaptic sites. Surprisingly, a genetically encoded indicator of EphB kinase activity, unbiased classification, and a photoactivatable EphB2 reveal that simple differences in the kinetics of EphB kinase signaling at the tips of filopodia mediate the choice between retraction and synaptogenesis. This may enable individual filopodia to choose targets based on differences in the activation rate of a single tyrosine kinase, greatly simplifying the process of partner selection and suggesting a general principle.

Keywords: cell signaling; kinase reporter; modeling; neuronal development; optogenetics; photoactivation; synaptogenesis; technology development; transfectable indicator.

Figure 1. EphB receptors are located on the surface of filopodia and colocalize with ephrin-B1, VGLUT1

(A) Combined two-color super-resolution STED and confocal imaging demonstrate that EphB2 in filopodia (green, STED) colocalizes with ephrin-B1 (red, STED) and VGLUT1 (purple, confocal). Neurons were transfected with mNeptune (gray, confocal). Dashed lines show the morphology of transfected neurons. Arrows indicate an example of colocalization of EphB2, ephrin-B1 and VGLUT1 in filopodia. Scale bar = 1 μm. (B) The proportion of EphB2+ and EphB2− filopodia (n = 256). (C) Quantification of EphB2, ephrin-B1 and VGLUT1 colocalization in EphB2+ filopodia (n = 189). (D) Surface staining of EphB2 (green) in DIV7-10 neurons transfected with tdTomato. Arrows indicate EphB2+ filopodia. Dashed lines show the morphology of transfected neurons. Bottom figures show that acid treatment removes surface staining of EphB2. Scale bar = 2 μm. See also Figure S1.

Figure 2. EphB receptors cluster at the tip of moving and stable filopodia, and colocalize with synaptic release sites

(A) Experimental procedure for labeling of presynaptic release sites as in ((Wilhelm et al., 2010), See Methods for details). (B) Representative images show filopodia stable for 30 minutes in neurons transfected with mT2 (Arrows). (C) The same dendrite as in B overlaid with images of EphB2-YFP (yellow) and synaptic release sites (red, labeled with FM4-64). Arrows indicate EphB2-YFP+ filopodial tips colocalized with FM4-64. Dashed lines show the morphology of transfected neurons. Scale bar = 5 μm. (D) Quantification of colocalization (EphB2+: 69 ± 6%, n = 9; EphB2-: 49 ± 6%, n = 9, p = 0.024, t-test). * p < 0.05. Error bars indicate SEM (E) Representative images from 30-minute time-lapse movies of DIV7-10 neurons transfected with EphB2-YFP. Two types of filopodia were identified (Stable and Moving filopodia). Arrows indicate EphB2+ tips across time window. Scale bar = 2 μm. See also Movie S1.

Figure 3. The GPhos indicators selectively report activity of EphB or EphA tyrosine kinases

(A) Design of single- and dual-color GPhos indicators. GPhosEphB and GPhosEphA indicator differ only in the phospho-peptide region (shown in blue). (B) GPhosEphB was immunoprecipitated from HEK293T cells transfected with GPhosEphB indicator and EphB2 or EphA4 receptor and probed for phosphorylation (PY99). Ephrin-B2 treatment resulted in phosphorylation of GPhosEphB only in cells transfected with EphB2. Lower western blots show expression controls. (C) Ratiometric pseudocolor images of single optical sections collected every three minutes from cells transfected with EphB2 and either GPhosEphB (top) or GPhosEphA (bottom). Cells were treated with activated ephrin-B2 as indicated by the orange bar. The pseudocolored lookup table (16 colors) indicates the ratiometric GPhos signal. (D) Quantification of the effects of ephrin-B2 treatment on GPhos signal in HEK293T cells transfected with EphB2 (GPhosEphB: n = 6; GPhosEphA: n = 4, p < 0.0001, two-way ANOVA). * p < 0.05, ** p < 0.01, *** p < 0.001. Western blot shows phosphorylation state of the immunoprecipitated FLAG-EphB2 receptor at 5, 15, and 30 minutes after activation with ephrin-B2. (E) As in C, but ratiometric images of single optical sections were collected when HEK293T cells were transfected with EphA4 and either GPhosEphB or GPhosEphA. GPhosEphA signal increased with ephrin-A1 treatment. (F) Quantification of the effects of ephrin-A treatment (GPhosEphB: n = 10; GPhosEphA: n = 10, p < 0.0001, two-way ANOVA). ** p < 0.01, *** p < 0.001,**** p < 0.0001. Western blot shows phosphorylation state of the immunoprecipitated FLAG-EphA4 receptor at 5, 15, and 30 minutes after activation with ephrin-A1. (G) Ratiometric images of TKI mouse neurons transfected with GPhosEphB indicator and treated with activated ephrin-B2. (H) Quantification of the effects of ephrin-B2 treatment on GPhos signal in transfected neurons (n = 11). (I) Ratiometric images of TKI neurons transfected with GPhosEphB indicator and blocked with 1-NA-PP1 before treatment with activated ephrin-B2. (J) Quantification of the effects of 1-NA-PP1 on ephrin-B treatment (n = 8). The pseudocolor lookup table (fire) indicates the ratiometric GPhos signal in neurons. Scale bars = 5. Error bars indicate SEM in Figures D, F, H, J. See also Figures S2, S3 and Movies S2–S5.

Figure 4. Focal activation of EphB in dendrites by axonal ephrin-B1

(A) An example of persistent contact between axonal mT2-eB1 puncta (cyan) and GPhosEphB (fire) transfected dendrite. Images of RFP channel at the last frame of 15 min movie were shown (pseudocolored, green). The ROI shows the contact between axonal mT2-eB1 puncta and GPhosEphB transfected dendrite (2 min and 15 min). Arrows indicate the colocalization of eB1 puncta (cyan) and persistent GPhos signal (fire). (B) The RFP image (pseudocolored, green) after fixation is shown. Confocal image of EphB2 (red) colocalizes with eB1 puncta (cyan). (C) Quantification of the colocalization of mT2-eB1, EphB2 and GPhos. (D) As in A, an example of persistent contact between axonal mT2-eB1 and GPhosEphB transfected dendrite. Dashed lines show the morphology of axons. Arrows indicate sites of contact. Arrowheads indicate control ROIs used in E. (E) Quantification of GPhos signal in D. (F) Quantification of summary data of GPhos signals in control and mT2-eB1 contacting sites. Data are shown as the average GPhosEphB signal when the mT2-eB1 puncta are in contact with the axon or in control ROIs (arrowheads in D). (Ctrl: 1.00 ± 0.01, n = 19; eB1: 1.23 ± 0.04, n = 19, p ≤0.001, paired t-test). *** p = 0.001. (G) Examples of transient contact between moving axonal mT2-eB1 puncta and GPhosEphB transfected dendrite. Arrows and dashed lines as in D. (H) Quantification of GPhos signal in G. (I) As in F, quantification of GPhos signal in control and mT2-eB1 contacting sites from pooled data (Ctrl: 1.00 ± 0.01, n = 9; eB1: 1.24 ± 0.04, n = 9, p ≤ 0.001, paired t-test). *** p = 0.001. Control ROIs were selected at sites in dendritic shaft without axonal contacts and GPhosEphB signals. Scale bars = 2 μm. Error bars indicate SEM in Figures F and I. See also Figures S4 and Movies S6–S7.

Figure 5. Distinctive patterns of EphB activity in Retracting and Connecting filopodia

(A) An example of retracting filopodia. Axons are labeled with mTuquoise2 and indicated by dashed lines. Ratiometric images (pseudocolored) show transient high GPhosEphB signal (middle panel). Arrowhead indicates focal GPhosEphB signal (ROI). (B) Quantification of GPhosEphB signal in A. (C) GPhosEphB signal in the Retracting filopodia group. Gray lines indicate GPhos signal in individual filopodia and the green line represents mean (n = 9). Time 0 indicates the time filopodia contact a labeled axon (vertical dashed line). The bar indicates the fraction of filopodia in contact with the axons (Black = 100%, White = 0%). Control mean shaft GPhosEphB is indicated by the black line. (D) An example of Connecting filopodia as in A. (E) Quantification of GPhosEphB signal in D. (F) As in C, but the mean GPhosEphB signal in the Connecting filopodia is shown as a red line (n = 12). (G) Illustration of Retracting & Connecting filopodia behavior and EphB signaling. The slope was calculated as the difference between the baseline and the peak value divided by the time from the baseline to the peak. (H) Comparison of the kinetics of average GPhosEphB signal. Retracting group was best fit with a peak model. The Connecting group was best fit with an exponential model. (I) The slope of GPhosEphB signal in Retracting filopodia was significantly sharper than that in Connecting filopodia (Retracting: 0.31 ± 0.04, n = 9; Connecting: 0.17 ± 0.04, n = 12, p = 0.021, Mann Whitney U test) * p < 0.05. Scale bars = 2 μm. Error bars indicate SEM in Figure I. See also Figure S5 and Movies S8–S9.

Figure 6. The dynamics of EphB activity determine filopodial behavior

Slow activation of EphB2 decreases filopodial movement. (A) Dendritic filopodia before and after application of control (FC). (B) Colored lines indicate the distance moved by each filopodium in (A). (C) As in A, but images after application of activated ephrin-B2. (D) As in B. (E) Quantification of GPhosEphB signal in control (Before: 1.3 ± 0.04; After: 1.4 ± 0.09, n = 4, p = 0.358, paired t-test). (F) Quantification of distance moved in control (Before: 0.6 ± 0.06; After: 0.5 ± 0.09, n = 4, p = 0.091, paired t-test). (G) Quantification of GPhosEphB signal in ephrin-B2 treated group (Before: 1.1 ± 0.04; After: 1.5 ± 0.06, n = 4, p ≤ 0.001, paired t-test). ***p ≤ 0.001. (H) Quantification of distance moved in ephrin-B2 group (Before: 0.6 ± 0.04; After: 0.4 ± 0.05, n = 4; p = 0.013, paired t-test). *p < 0.05. (I) Upper: Illustration of the focal application of activated ephrin-B. Lower: Illustration of the EphB signaling induced by exogenous ephrin-B (cyan). (J) An example of Retracting filopodia after ephrin-B treatment (cyan). Arrowheads indicate GPhosEphB signal. (K) An example of Connecting filopodia after ephrin-B treatment as in J. (L) Graph of GPhosEphB in Retracting (green) and Connecting (red) filopodia. Thin lines represent individual filopodial GPhosEphB signals. Means are thick lines. (M) Graph depicting the mean, variance and slope of individual filopodia that have made contact with presynaptic elements (green = Retracting, red = Connecting). Classifying filopodia using a trained linear kernel support vector machine (using held-out data) reveals that Retracting and Connecting filopodia can be correctly identified 91% the time (classification accuracy range across restarts = 83 – 100%). Scale bars = 2 μm. Error bars indicate SEM in Figure E–H. See also Figure S6 and Movies S10–S11.

Figure 7. Fast activation of EphB2 induces filopodial retraction

(A) Design of photoactivatable EphB2. Xfp: fluorescent protein (mCherry or iRFP670). (B) Western blots of HEK293T cells transfected with EphB2-CRY2 variants and treated with activated ephrin-B2 (30 mins) or blue light (440nm, 1 min) (CRY2-cherry: fEphB2-CRY2-mCherry; Oligo-cherry: fEphB2-CRY2-Oligo-mCherry; CRY2hm-cherry: fEphB2-CRY2hm-mCherry; CRY2-iRFP670: fEphB2-CRY2-iRFP670) (C) Western blots of HEK293T cells transfected with wild-type (WT) or kinase-dead (KD) versions of fEphB2-CRY2hm-iRFP670 (fEphB2-CRY2) and treated with ephrin-B2 or blue light. (D) HEK293T cells transfected with WT or KD versions of fEphB2-CRY2 and imaged every 10 seconds. Photostimulation was conducted by a single scan with the 470 nm laser. Arrows indicate filopodia retracting after photostimulation. (E) Quantification of fEphB2-CRY2 transfected filopodia remaining after photostimulation (n = 12, * p = 0.013, ** p = 0.002, *** p ≤ 0.001, paired t-test). (F) Quantification of fEphB2-KD-CRY2 transfected filopodia remaining after photostimulation (n = 13, p > 0.05, paired t-test). (G) Neuron transfected with WT or KD versions of fEphB2-CRY2 and imaged every 10 seconds. Arrows as in D. Dashed lines show the border of photostimulated region. (H) Quantification of filopodia retraction after photostimulation (PHS) (p = 0.025, ANOVA; WT-PHS+: n = 21, vs. WT-PHS-: n = 11, p = 0.019; WT-PHS+: n = 21, vs. KD-PHS: n = 14, p = 0.036, post hoc: Fisher’s LSD test). (I) Quantification of the distance filopodia moved after photostimulation (WT-PHS+: n = 50 vs. WT-PHS-: n = 25, p = 0.025, t-test). Scale bars = 2 μm. Error bars indicate SEM in Figures E, F, H, I. See also Figure S7 and Movies S12–S13.

Figure 8. EphB signaling is elevated in stable filopodia and colocalized with VGLUT1 puncta

(A) An example of Connected filopodia (axonal contact maintained for >30min) contacting an mT2 labeled axon. Dashed lines show the morphology of axons. Arrowheads indicate GPhosEphB signal. (B) Quantification of GPhosEphB signal in A. (C) Quantification of GPhosEphB signal in the pooled data set (ROI: n = 22, indicated by arrowheads; Ctrl: n = 16, p ≤ 0.001, K–S Test). (D) Arrowheads indicate a stable filopodium. Images of RFP channel at the last frame of 30-minute movie and after fixation. (E) Images of GPhosEphB signal in the filopodium in D. (F) The same filopodium as in D and E shown overlaid with VGLUT1 staining (white). Dashed lines show the morphology of the transfected neuron. Arrowheads indicate GPhosEphB signal colocalized with VGLUT1. (G) As in D, arrows point to a stable filopodium. (H) Images of GPhosEphB signal in the filopodium in G. (I) As in F, the filopodium was shown overlaid with staining of VGLUT1. (J) Quantification of GPhosEphB signal in the VGLUT1+ filopodium from D–F. (K) Quantification of GPhosEphB signal in the VGLUT1− filopodium from G–I. (L) Average GPhos signal in VGLUT1+ filopodia was significantly higher than that in VGLUT1− filopodia (VGLUT1+: 1.30 ± 0.05, n = 36; VGLUT−: 1.10 ± 0.04, n = 20, p = 0.005, t-test). Scale bars = 2 μm. ** p < 0.01. Error bars indicate SEM in Figures C and L. See also Figure S8 and Movies S14–S15.