Mitchell, J. W. | bioRxiv Science

bioRxiv2022

Coordination of Pickpocket ion channel delivery and dendrite growth in Drosophila sensory neurons

Sensory neurons enable an organism to perceive external stimuli, which is essential for survival. The sensory capacity of a neuron depends on the elaboration of its dendritic arbor and the delivery of sensory ion channels to the dendritic membrane. However, it is not well understood how ion channels are trafficked to sensory dendrites and whether their delivery is coordinated with dendrite growth. We investigated the trafficking of the DEG/ENaC/ASIC ion channel Pickpocket (Ppk) in peripheral sensory neurons in fruit fly larvae. We used CRISPR-Cas9 genome engineering to tag endogenous Ppk1 and visualize it live, including monitoring Ppk1 membrane localization via a novel secreted split-GFP approach. Strikingly, Ppk1 is present throughout the membrane of actively growing dendrites, and Ppk1 density scales in proportion to the dendritic membrane, even when dynein-mediated transport to dendrites is disrupted. Our data suggest that Ppk1 is integral to the membrane of growing dendrites and implicate the recycling endosome GTPase Rab11 in the forward trafficking of Ppk1 to dendrites. Together, our results suggest that Ppk channel transport is coordinated with dendrite morphogenesis, thus ensuring proper ion channel levels and distribution in sensory dendrites. Author SummaryPeripheral sensory neurons are essential for an organism to interact with its environment. Neurons are composed of signal-receiving dendrites and a signal-sending axon. Ion channels distributed throughout sensory dendrites transduce external stimuli into chemical signals, however the mechanisms that localize ion channels to sensory dendrites are not well understood. Both the composition of ion channels in the dendrites and the structure of a sensory neurons dendritic arbor are important for how it functions to sense external stimuli. Using live imaging and genomic engineering, we have discovered that the localization of a sensory ion channel, Pickpocket, in fruit fly sensory neurons is coordinated with growth of the dendritic arbor and that Pickpocket levels scale in proportion to dendrite length, even when transport to dendrites is disrupted. We also developed a novel genetically encoded approach to visualize the membrane expression of proteins in a living organism utilizing the split-GFP system. We found that both the secretory and endosomal networks mediate the forward trafficking of Pickpocket during neuronal morphogenesis, thus coordinating membrane growth with ion channel delivery. Our findings reveal that actively growing sensory dendrites are equipped with the ion channels needed for sensing external stimuli.

neuroscience↗

bioRxiv2021

Tubulin Acetylation Promotes Penetrative Capacity of Cells Undergoing Radial Intercalation

The post-translational modification of tubulin provides a wide diversity of differential functions to microtubule networks. Here we address the role of tubulin acetylation on the penetrative capacity of cells undergoing radial intercalation in the skin of Xenopus embryos. Radial intercalation is the process by which cells move apically and penetrate the epithelial barrier via inserting into the outer epithelium. As such there are two opposing forces that regulate the ability of cells to intercalate: the restrictive forces of the epithelial barrier versus the penetrative forces of the intercalating cell. By positively and negatively modulating tubulin acetylation specifically in the intercalating cells, the timing of intercalation can be altered such that cells with more acetylated microtubules penetrate the epithelium faster. Moreover, the Xenopus epithelium is a complex array of variable types of vertices and we find that intercalating cells preferentially penetrate at higher order "rosette" vertices as opposed to the more prevalent tricellular vertices. We observed differential timing in the ability of cells to penetrate different types of vertices, indicating lower order vertices represent more restrictive sites of insertion. Interestingly, we are able to shift the accessibility of early intercalating cells towards the more restrictive tricellular junctions by modulating the level of tubulin acetylation and the subsequent penetrative capacity of intercalating cells. Overall our data implicate tubulin acetylation in driving tissue penetration of intercalating cells.

cell biology↗