Article Figures & Data
Figures
- Figure 1.
Expression of N-cadherin-GFP in Rohon-Beard neurons. a, A maximum intensity projection of a two-photon image stack showing expression of soluble GFP in a Rohon-Beard neuron of a ∼24-hr-old zebrafish embryo. GFP fluorescence is both continuous and bright within the neuronal processes, effectively labeling even thin filopodia. B, A maximum intensity projection of a two-photon image stack showing expression of an Ncad-GFP fusion protein in a Rohon-Beard neurons of a ∼30 hr old zebrafish embryo. In contrast to soluble GFP, Ncad-GFP shows highly punctate staining within the longitudinal axons, as well as the peripheral sensory arbor. In addition, there is a faint labeling of the cell body plasma membrane, but very little background labeling in the axons. Scale bar, 8 μm.
- Figure 2.
Synaptic localization of zebrafish Ncad-GFP in cultured rat hippocampal neurons. a, Image of a cultured hippocampal neuron transfected with Ncad-GFP (green) and immunolabeled with an antibody against rat PSD-95 (red). When expressed in hippocampal neurons, zebrafish Ncad-GFP exhibits a punctate distribution as is seen in zebrafish embryos in vivo and by previous immunolabeling studies in vitro. b, A higher magnification of the boxed area in a, showing the punctate distribution of the Ncad-GFP. c, The same area showing staining for rat PSD-95. d, The same area showing antibody labeling for PSD-95. h, An overlay showing extensive colocalization of Ncad-GFP (green) with PSD-95 (red). Quantitation of the data reveals that there is 74% colocalization (n = 212 Ncad-GFP puncta) between the fusion protein and the synaptic marker. e, A confocal image of an axon from a lateral-line ganglion neuron expressing Ncad-GFP, showing dim, diffuse labeling along the axon, except for a large punctum. f, The same segment of axon immunostained with an antibody against the synaptic vesicle protein, SV2. g, An overlay of the Ncad-GFP and SV2 showing that the Ncad-GFP colocalizes with a large SV2 punctum. Scale bar: (in g) e-g, 5 μm; h-j, 11 μm. h, A two-photon image of a segment of an Ncad-YFP-labeled Rohon-Beard axon in a live embryo. i, The same area labeled with VAMP-CFP. j, Overlay of the Ncad-YFP and VAMP-CFP signals, showing colocalization of the N-cadherin with accumulations of VAMP.
- Figure 3.
Ncad-GFP labels discrete-punctate transport organelles. Shown are images taken from an in vivo time-lapse sequence, revealing the movement of discrete-punctate transport organelles in a Rohon-Beard axon. Axons exhibit both stable and highly dynamic fluorescent puncta, with very little background staining of the axons. The discrete-punctate transport organelles move in either the anterograde or retrograde directions and reach velocities of up to 34 μm/min. The arrowheads follow one such transport organelle. Scale bar, 10 μm.
- Figure 4.
Ncad-GFP labels tubulovesicular transport organelles. Shown are images taken from an in vivo time-lapse sequence, revealing the anterograde transport of a tubulovesicular organelle. In contrast to the discrete-punctate transport organelles, the tubulovesicular structures stain an extended portion of the axon, in some cases up to ∼20 μm. This extended accumulation of fluorescence moves en masse within the axon, predominantly in the anterograde direction. Scale bars, 13 μm.
- Figure 5.
Formation of a stable Ncad-GFP punctum. a, In addition to the mobile fluorescent puncta present in the developing Rohon-Beard axons, there are also stable puncta. The stable accumulations (arrowhead), as shown in this time-lapse sequence, form soon after the departure of the migrating axonal growth cone. Scale bar, 10 μm. b, Cumulative probability of stable punctum formation. The formation of multiple stable puncta were characterized relative to the axonal growth cone. The fraction of puncta formed was plotted as a function of time after initial growth cone arrival. The plot shows that nearly all stable Ncad-GFP accumulations form within 1 hr after arrival of the growth cone, with an average time to formation of 32 min.
- Figure 6.
Formation of a stable VAMP-GFP punctum. a, VAMP-GFP is a well characterized marker of synaptic vesicle clusters. Shown is a time-lapse sequence revealing the formation of a stable accumulation of zebrafish VAMP-GFP (arrowhead) in the wake of a migrating growth cone, similar to what we have shown for Ncad-GFP. b, A plot of cumulative probability for VAMP-GFP accumulation, similar to that shown in Figure 5b. The time course of VAMP-GFP accumulation (gray trace, gray triangles) was essentially the same as for Ncad-GFP (black trace, black circles), indicating that they accumulate with similar kinetics.
- Figure 7.
Localization of N-cadherin mutants expressed in Rohon-Beard axons. a, Punctate distribution of full-length Ncad-GFP in a Rohon-Beard axon. b, Two examples of Rohon-Beard axons labeled with NcadΔCP-GFP. Like the full-length N-cadherin, the deletion mutant appears as discreet puncta along the axon, with relatively little background labeling of the plasma membrane along the axon shaft. c, Two examples of Rohon-Beard axons expressing the NcadΔEC-GFP deletion mutant. In contrast to both Ncad-GFP and NcadΔCP-GFP, there is diffuse, uniform labeling of the plasma membrane in the growth cone and axon. In addition, NcadΔEC-GFP does not accumulate in stable, discrete puncta. Scale bar, 15 μm. d, The time course of NcadΔCP-GFP accumulation is similar to that found for both Ncad-GFP and VAMP-GFP, although it appears to stabilize slightly more quickly, with an average time-to-formation of 25 min. Scale bar, 10 μm.
Additional Files
Supplemental data
Files in this Data Supplement:
In this issue
