A colleague of mine, a former postoc in the lab, Chris Deppmann, has just published his first paper as an independent investigator at UVA. I’m really glad to see this story finally come out as this was one of his first projects during his postdoc and now and then I can still see some of his random reagents and constructs used in this project floating around in the lab freezer.
The paper is about NGF, which is the field I’m in right now too. This study aims to tackle a central question of the NGF biology: how can retrograde NGF/TrkA signal have a long-lasting activity and downstream effect, unlike other growth factor-receptor tyrosine kinase system such as EGF/EGFR?
Part of the answer might be…..coronin. Coronin has been implicated in the survival of mycobacteria in macrophages via lysosome escaping. Similarly here in neurons, retrogradely transported NGF endosomes in cell bodies containing coronin are less frequently and more slowly fused with lysosomes. The problem here is that, they use lysotracker as a lysosome marker. At least in my hands, and from other people, this is not a reliable way of labeling lysosome in cultures for a prolonged time. Since lysotracker is in the cells for over 10hrs in most of their experiments, I doubt the identity of the labeled vesicles.
An interesting point in this part of the story is that, it appears, from both their ICC and functional study, that the association of TrkA endosome with coronin happens only in cell bodies but not in axons and growth cones. While this is consistent with the macrophagy system and might make more sense since lysosomes are highly aggregated in cell bodies, it does (almost) rule out a protective role of coronin in axons during the transport process. And I think it’s still an open question that whether there’s an active mechanism to allow TrkA endosomes to evade degradation in axons.
Next they raised a really novel role of coronin in the recycling and transcytosis of TrkA in cell bodies. They first showed that Rab11 association with TrkA requires coronin.then, using a recycling assay developed by Francis Lee’s group, which basically can specifically label a receptor that’s inserted into plasma membrane, internalized and then recycled back, they showed coronin is necessary for the transcytosis of TrkA. The problem here is that, they can’t distinguish membrane-bound TrkA versus the ones that undergo endocytosis again. Stripping off the fluorescent signal for membrane TrkA, in my mind, is the way to go.
They conclude that coronin sustains NGF/TrkA signal by preventing lysosomal fusion on one hand and by promoting TrkA recycling on the other hand. In this way, there’s less degradation and at the same time there is more receptor recycling. But they never got a way to separate the two situations. It’s natural to think that, if you block one route, in order to have some kind of equilibrium, there’s gotta be more in the other route. In this case, increasing recycling could be a consequence of shutting down degradation, or vice versa. And their Fig4a can’t help here because the number of TrkA punctae quantified here, is again, a combination of both membrane and cytosolic TrkA. One way to address this is to do the stripping assay in addition to the recycling assay so that you can tell if there’s indeed more recycling. Another control experiment one can do is to block recycling (I don’t have a great small molecule inhibitor on top of my head, but certainly a dominant negative Rab11 should do) and to see if there’s more degradation and vice versa.
But the recycling hypothesis is certainly an attractive way of sustaining signal. If no recycling, all the receptors are routed to the degradation pathway. Now you have a recycling mechanism that allows a portion of the receptors to go back to plasma membrane. This basically buys a round of endocytosis-worth of time for them to signal, and depending on how long they are internalized again, the signal can be prolonged for minutes and hours. As a result, the retrogradely transmitted signal is prolonged by converting part of it to a more local signal eminent from the cell body, which is released with some delay. The killer question here though is whether those recylced receptors can bring more signal. again, that’s an open question.