Figure 1.
Morphological characterization of PC12 varicones and growth cones.
(A–C) Examples of differentiated PC12 cells observed using Differential Interference Contrast (DIC). A large varicosity at the tip of many neurites is apparent (“v” in A and C) while other terminals resemble neuronal growth cones (“gc” in A and B). (D–G) Fluorescence images of several PC12 cell neurite terminals labeled with a β-III-tubulin antibody and phalloidin to visualize tubulin and actin. While some terminals display a morphology similar to neuronal growth cones, including a tubulin-rich central domain (c) surrounded by an actin-rich peripheral domain (p) as shown in D, others have a large varicosity (E–G) and “growth cone” that can be collapsed (E) or more clearly visible (F–G). In some neurites, both the varicosity (v) and the growth cone (gc) appear as a connected structure (connected arrows in E). Scale bar A = 20 µm, B–G = 10 µm.
Figure 2.
Immunocytochemical characterization of PC12 terminals I: growth cone markers.
(A–I) Fluorescence images of hippocampal neuron growth cones, PC12 growth cones and PC12 varicones, immunolabeled with growth cone markers (white or green) and co-labeled with phalloidin (actin, red). Arp3 (A–C), Cortactin (D–F) and GAP-43 (G–I) localize to the actin-rich regions of the growth cones, including the actin-rich region associated with varicones (C, F, I), but not to the varicosity associated with varicones (white arrow). Scale bar = 10 µm.
Figure 3.
Immunocytochemical characterization of PC12 terminals II: varicosity markers.
(A–I) Fluorescence images of hippocampal neuron growth cones, PC12 growth cones and PC12 varicones, immunolabeled with non-growth cone markers (white/green) and co-labeled with phalloidin (actin, red). Calpain (A–C) is a protease enriched at the neurite shaft in neurons (A) but not so in PC12 cells (B–C), where it localizes almost exclusively to varicosities (white arrow). Synaptophysin (D–F) is a presynaptic marker that is barely detected in young growing neurites in hippocampal neurons (D) but enriched at PC12 varicosities (arrow in F). Scale bar = 10 µm.
Figure 4.
Calpain-2 subcellular localization in PC12 cells.
(A–C) Double immunolabeling of calpain-2 (red) and actin (phalloidin, green) in a differentiated PC12 cell. Calpain-2 (B) localizes almost exclusively to the varicosity contained in the varicone (A). In a Z-projection across the varicone (C), actin staining is detected around the varicosity, while calpain-2 fills the space within. Scale bar = 10 µm.
Figure 5.
Onset of PC12 process outgrowth.
(A–C) Double immunolabeling of calpain-2 (white or red) and actin (phalloidin, green) in PC12 cells at different stages of differentiation. (A) Calpain-2 labeling in the initial stages of process outgrowth of PC12 cells concentrates around and along the initial buds (arrowheads). (B) Some neurites don't have visible accumulations of calpain-2 (empty arrowheads), and they invariably lack a varicosity (as determined by morphology). (C) Some neurites develop a clear varicone (arrowhead) and calpain-2 labeling concentrates at the varicosity. (D) An example of PC12 cell showing multiple processes with a variety of morphologies. Calpain staining uncovers the varicosity contained in the PC12 terminals, including those in which the presence of a varicosity was not easy to determine by morphology, such as those of triangular shape (asterisk). An incipient process is also highlighted by the presence of calpain-2 puncta (arrowhead in D). Scale bar = 10 µm.
Figure 6.
Misidentification of varicones in the literature.
Histogram showing the frequencies of growth cones and varicones in our cultures (top bar) and in the images published in the literature (second bar), including the notation used in those articles (third), the central topic of the article (fourth) and the frequency of errors in the identification of growth cones (always correctly identified) or varicones (usually regarded as a growth cone) in the literature (lower bar).
Figure 7.
PC12 terminal morphology include the genuine growth cone (red, left) and two examples of varicones containing a varicosity (green) and a more or less apparent growth cone (green/red or green). Both terminals are characterized by the expression of different set of markers (those shown in this article are in bold).