Abstract
Neurite formation relies on finely-tuned control of the cytoskeleton. Here we identified a novel protein-protein interaction between the ion and metabolite channel protein Pannexin 1 (Panx1) and collapsin response mediator protein 2 (Crmp2), a positive regulator of microtubule polymerization and stabilization. Panx1 and Crmp2 co-precipitated from both Neuro-2a (N2a) cells and mouse ventricular zone (VZ) tissue. In vitro binding assays between purified proteins revealed the interaction occurs directly between the Panx1 C-terminus (Panx1 CT) and Crmp2. Because Crmp2 is a well-established microtubule-stabilizing protein, and we previously observed a marked increase in neurite formation following treatment with the Panx1 blocker, probenecid, in N2a cells and VZ neural precursor cells (NPCs), we investigated the impact of probenecid on the Panx1-Crmp2 interaction. Probenecid treatment significantly disrupted the Panx1-Crmp2 interaction by both immunoprecipitation (IP) and proximity ligation analysis, without altering overall Crmp2 protein expression levels. In the presence of probenecid, Crmp2 was concentrated at the distal ends of growing neurites. Moreover, probenecid treatment increased tubulin polymerization and microtubule stability in N2a cells. These results reveal that probenecid disrupts a novel interaction between Panx1 and the microtubule stabilizer, Crmp2, and also increases microtubule stability.
Highlights
Pannexin 1 (Panx1) was first discovered about 20 years ago (Panchin et al, 2000) and forms large-pore membrane channels that mediate passage of ions and metabolites
Based on a substantial body of evidence that has established it as a microtubule-associated protein that plays a critical role in neurite stability, we decided to focus our attention on collapsin response mediator protein 2 (Crmp2) (Dpysl2; Figure 1A)
We found that the Panx1 C-terminus (Panx1CT) enriched with purified Crmp2-GST bound to glutathione-agarose beads, but not to control GST
Summary
Pannexin 1 (Panx1) was first discovered about 20 years ago (Panchin et al, 2000) and forms large-pore membrane channels that mediate passage of ions and metabolites (reviewed in Chiu et al, 2018; Whyte-Fagundes and Zoidl, 2018). Using a combination of models, mouse neuroblastoma Neuro-2a (N2a) cells and VZ NPCs, we made several key observations suggesting Panx is a negative regulator of neurite formation (Wicki-Stordeur and Swayne, 2013). Work from another group supported these findings, demonstrating that disruption of Panx (block or KO) increased axonal caliber and growth rate in dorsal root ganglion neurons (Horton et al, 2017). Together these data from others and us suggest that Panx is a negative regulator of neurite extension and/or stability, the underlying mechanism has not yet been resolved
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