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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). |
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Fig. 8. Motoneuron growth cones in the spinal cord extend invadosome-like protrusions toward the peripheral myotome, which are necessary for proper axon extension into the periphery. (A,B) Maximum z-series projected images of a whole-mount embryo (lateral view, anterior left) with descending motoneuron growth cones on the ventral fascicle labeled with GFP by using targeted blastomere injection. This embryo was immunolabeled for βI+II tubulin (A) and GFP (B). (C) Merged image of motoneurons labeled for tubulin (red) and GFP (green) showing a robust protrusion that extends toward the notochord (n) from the central domain of the lead growth cone. Note that a MT has polymerized into this invadosome-like protrusion (A, arrowhead) that extends diagonally away from the spinal cord, as seen in an x-z view (C, inset). (D) Maximum z-series projected image (inverted contrast) of a whole-mount embryo (lateral view, anterior left) immunolabeled with the Znp-1 antibody. Note several peripheral axons and fine protrusions extend from motoneurons (arrows). (E) Magnified image from the boxed region in D shows a terminal motoneuron growth cone with many fine protrusions. (F) x-z view resampled along the dashed line in E shows an invadosome-like protrusion that extends deep into the lateral tissue (arrow). (G,H) Maximum z-series projected images of whole-mount embryos (lateral view, anterior left) expressing GFP (G) or δPX-Tks5-GFP (H) in motoneurons. Embryos were immunolabeled for βI+II tubulin (red) and GFP (green). Note that several peripheral GFP-expressing motoneuron axons with growth cones (G, arrowheads) have exited the spinal cord en route to the peripheral myotome, whereas motoneurons expressing δPX-Tks5-GFP remain within the spinal cord (H, arrows). (I) The percentage of tubulin-positive peripheral motoneuron axons that express δPX-Tks5-GFP is significantly less than that expressing GFP in control embryos (see Materials and Methods). *P<0.05, Fisher's exact test. The n values for numbers of peripheral axons (tubulin-positive, GFP-positive), image z stacks and embryos, respectively, are 64, 14, 28, 7 for control and 140, 16, 32, 8 for experimental conditions. Scale bars: 10 µm (A-C); 30 µm (D,G,H). |
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Fig. S8. Model illustrating F-actin rich foci as growth cones
invadosomes. A. SIM image of a Xenopus spinal neuron growth cone on
LN, fixed and stained for F-actin (green) with Alexa-568 phalloidin and
immunolabeled for βI/II-tubulin (magenta). Note the F-actin rich foci within the
growth cone C-domain (arrows). B. Illustration of a cross-sectioned growth
cone responding to environmental cues by remodeling the ECM with F-actin
foci that become protruding invadosomes. Note how invadosomal F-actin
orients perpendicular to the planar F-actin network within the growth cone. C.
Magnified schematic view of a single cross-sectioned growth cone
invadosome. Growth cones first establish adhesions with the ECM through
the interaction of integrins, Src and adhesion proteins such as paxillin and
FAK. Once localized to adhesion sites, Src may phosphorylate Tks5, as well
as proteins implicated in lipid raft formation. Phosphorylated Tks5 targets to
lipid rafts and initiates actin polymerization through cortactin, N-WASP, the
Arp2/3 complex and Ena/Vasp proteins. This network of branched actin
assembles F-actin rich columns that span the width of the growth cone and
extend protrusions orthogonal to the plane of outgrowth. Membrane bound
and secreted proteases at the tips of invadosomes begin to degrade the ECM,
allowing the actin column to transition into a 3-dimensional membrane
protrusion. Invadosomal protrusions, formed of both branched and
unbranched actin filaments, are stabilized by proteins such as α-actinin. In
mature invadosomes, MTs polymerize into the protrusion providing increased
stability and the delivery of vesicular cargo, such as proteases and guidance
cue receptors. Growth cone invadosomes may represent a novel mechanism
for growth cones to respond to environmental cues by remodeling surrounding
tissues with 3-dimensional projections during axon guidance. |
