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Fig. 6. F-actin-rich protrusion from the central domain of growth cones are detected by 3D SIM. (A,B) SIM images of Xenopus spinal neuron growth cones on laminin (A) or thin gelatin (B), fixed and stained for F-actin with Alexa-568 phalloidin. Note the cross-linked actin meshwork within the peripheral veil and the F-actin-rich foci within the growth cone central domain (arrowhead in A and red box in B). (C) 2.5× zoom of the boxed region in B. (D) Orthogonal view (y-z) through the F-actin-rich foci shown in C. Note this F-actin foci spans from the basal (Ba) substratum to the apical (Ap) membrane where it protrudes from the growth cone surface (arrowhead). (E,E′) SIM images of an iPSC-derived human forebrain neuron growth cone on laminin, fixed and stained for F-actin with Alexa-568 phalloidin (E) and immunolabeled for βIII-tubulin (E′). Note the F-actin-rich foci located within the growth cone central domain (arrowhead). (E′) Merged image of F-actin (magenta) and βIII-tubulin (green) labeling. (F-F′) Orthogonal view (x-z) images through the F-actin-rich foci shown in E-E′. Note how MTs track along F-actin within the apical protrusion (arrowhead). (G-G′) 3D SIM images of a Xenopus spinal neuron growth cone in a collagen-I gel stained for F-actin (G) with phalloidin and immunolabeled for Cttn (G′). Note in the merge image (G′) F-actin-rich foci (purple) within the C-domain colocalize with Cttn (green, arrowheads in G′). (H) Orthogonal view of a triple-labeled growth cone (Cttn, green; F-actin, red; βI+II tubulin, blue) in a collagen-I gel showing apical (white arrowheads) and basal protrusions (red arrowhead). (I-K) 3D SIM images of a peripheral Rohon-Beard growth cone in the skin immunolabeled for NCAM (green) and Cttn (red) and viewed at three orientations. Cttn-containing NCAM puncta in the C-domain viewed in x-y (I, solid arrowhead) associates with an apical protrusion viewed as a 90° rotation along the x-axis (J, solid arrowhead). A second prominent apical protrusion (open arrowheads) extends ∼10 µm toward the peripheral skin as seen in a 90° rotation along the y-axis (J,K). Scale bars: 5 µm (A,B,E,G,I(x,y),J(z),K(z)); 1 µm (C,D); 2 µm (F,H). |
