DENDRITIC ENDOSOMAL SORTING OF NEWLY SYNTHESIZED AXONAL MEMBRANE PROTEINS

The accurate sorting and delivery of newly synthesized axonal membrane proteins from the somatodendritic compartment remain poorly understood. Increasing evidence suggests that axonal proteins initially enter dendrites, where dendritic endosomes function as sorting hubs that redirect them to the axon via transcytosis. Using proximity-dependent labeling with TurboID coupled to mass spectrometry, we identified 172 axonal proteins enriched in dendritic endosomes. From this dataset, we selected six axonal membrane proteins for detailed analysis of their localization and trafficking pathways: voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1), contactin-1 (CNTN1), ephrin type-A receptor 5 (EPHA5), N-cadherin (NCAD), receptor-type tyrosine-protein phosphatase S (PTPRS), and teneurin-2 (TENM2).
Immunocytochemistry in cortical neurons revealed that all six proteins localize to both dendritic and axonal compartments. Colocalization with transferrin receptor-positive endosomes further supported that these cargos are sorted to the axon through dendritic endosomes. Spatiotemporally resolved imaging showed that after exiting the endoplasmic reticulum, these proteins first traffic to dendrites and are subsequently transported to the axon, supporting axonal membrane delivery via transcytosis.
In parallel, we are investigating the molecular machinery required for axonal protein sorting within dendritic endosomes at distinct stages of trafficking. By synchronizing cargo release from the biosynthetic pathway and performing time-resolved affinity purification-mass spectrometry, we aim to define the dynamics and components of this process. Ultimately, our goal is to identify axonal proteins that transit through dendritic endosomes and the machinery responsible for their sorting. Understanding these mechanisms will provide critical insight into pathways essential for neuronal function and maintenance under physiological and disease conditions.