The turning point

[Introduction|Part 2|Discussion]

Tojima et al (2007) find that the growth cone's response to attractive guidance cues requires asymmetrical vesicle transport and exocytosis. They cultured dorsal root ganglion (DRG) cells from embryonic chicks, and produced localized elevations in calcium ion concentration on one side of the growth cone by photolytic release of the caged calcium ion compound DMNT-EDTA. In cells cultured on a substrate of cell adhesion molecule L1, this causes calcium-induced calcium release (CICR), and elicits a turning response in the direction of the calcium signal. In cells cultured on laminin, there is no CICR, and the growth cone turns away from the calcium signal.

The movements of vesicles were observed by exposing isolated DRG cells to the fluorescent lipophilic dye FM1-43. In cells grown on L1, but not in cells grown on laminin, vesicles were seen to be transported from the central to the peripheral domain of the growth cone in response to the calcium signal.

Vesicle Transport occurred in the direction of the calcium signal, within 1 minute of televation of calcium concentration, and transfecting cells with an alpha-tubulin-GFP fusion construct showed that vesicle transport preceded any changes in the cytoskeleton.

Transport was abolished when neurons were preloaded with the selective calcium ion chelator BAPTA-AM, or when they were treated with paclitaxel or nocodazole, which stabilize and depolymerize microtubules respectively.

A fusion protein called pHVenus-VAMP2 was then generated. This protein fluoresces when a vesicle is exposed to the high pH of exracellular solution during exocytosis. In pHVenus-VAMP2-transfected cells grown on L1, but not on laminin, pHVenus-VAMP2 fluorescence increased in response to elevated calcium concentration or membrane depolarization. This increase in fluorescence was observed only on the same side of the growth cone as the calcium signal, and was abolished with addition of BAPTA-AM or tetanus toxin, which blocks VAMP2-mediated exocytosis.

Together, these findings show that attractive, but not repulsive, guidance cues elicit transport of vesicles and clathrin-mediated exocytosis. Vesicles are transported asymmetrically, in the direction of the calcium signal, along pre-existing, and not newly-formed, microtubules. This occurs before any visible turning response of the growth cone.

By contrast, Piper et al find that growth cone collapse requires localized protein synthesis and endocytosis. They cultured Xenopus retinal neurons in Slit2-conditioned medium. They first added radioactively-labelled leucine to the culture medium, and observed that the radioactively-labelled amino acids were incorporated into newly synthesized proteins, confirming that proteins are synthesized during growth cone collapse.

They found that collapse was blocked by the translation inhibitors anisomycin and cyclohexamide, but not by the transcriptional inhibitor α-amanitin. Translation inhibitors also blocked collapse of isolated growth cones, confirming that the protein synthesis required for collapse takes place in the growth cone and not in the cell body or axon. Collapse was also blocked by phenylarsine oxide, a non-specific endocytosis inhibitor, and monodansyl cadaverine, which specifically blocks clathrin-mediated endocytosis.

Using immunofluorescence, they then showed that Slit2 causes an increase in the phosphorylated (active) form translation initiation factor 4E (elF4E) and the phosphorylated (inactive) form of its inhibitor, elF4EBP-1-P. Slit2 also increased levels of the actin depolymerizing protein cofilin-1, but this increase was blocked when translation inhibitors were added to the culture medium.

Piper et al also identified some of the components of the signalling pathways activated by Slit2. When the protein kinases p38, MEK 1 and MEK2 were inhibited in collapse assays, protein synthesis (as determined by 3H-leucine incorporation) was abolished and collapse did not occur. Inhibition of mammalian target of rapamycin (mTOR), a mediator of cap-dependent translation, also abolished protein synthesis.

Thus, it appears that Slit2 activates at least 2 different MAP kinases, which leads to phosphorylation (and inactivation) of elF4EBP-1-P; as a consequence, elF4E is also phosphorylated, so that it can initiate translation of its target mRNAs. Cofilin-1 mRNA is translated, leading to actin depolymerization and then to growth cone collapse.