Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella

Abstract

Sorting maturing neurons into distinct layers is critical for brain development, with disruptions leading to neurological disorders and pediatric cancers. Lamination coordinates where, when, and how cells interact, facilitating events that direct migrating neurons to their destined positions within emerging neural networks and control the wiring of connections in functional circuits. While the role of adhesion molecule expression and presentation in driving adhesive recognition during neuronal migration along glial fibers is recognized, the mechanisms by which the spatial arrangement of these molecules on the cell surface dictates adhesive specificity and translates contact-based external cues into intracellular responses like polarization and cytoskeletal organization remain largely unexplored. We used the cerebellar granule neuron (CGN) system to demonstrate that JAM-C receptor cis-binding on the same cell and trans-binding to neighboring cells controls the recruitment of the Pard3 polarity protein and drebrin microtubule-actin crosslinker at CGN to glial adhesion sites, complementing previous studies that showed Pard3 controls JAM-C exocytic surface presentation. Leveraging advanced imaging techniques, specific probes for cell recognition, and analytical methods to dissect adhesion dynamics, our findings reveal: 1) JAM-C cis or trans mutants result in reduced adhesion formation between CGNs and cerebellar glia, 2) these mutants exhibit delayed recruitment of Pard3 at the adhesion sites, and 3) CGNs with JAM-C mutations experience postponed sorting and entry into the cerebellar molecular layer (ML). By developing a conditional system to image adhesion components from two different cells simultaneously, we made it possible to investigate the dynamics of cell recognition on both sides of neuron-glial contacts and the subsequent recruitment of proteins required for CGN migration. This system and an approach that calculates local correlation based on convolution kernels at the cell adhesions site revealed that CGN to CGN JAM recognition preferentially recruits higher levels of Pard3 and drebrin than CGN to glia JAM recognition. The long latency time of CGNs in the inner external germinal layer (EGL) can be attributed to the combined strength of CGN-CGN contacts and the less efficient Pard3 recruitment by CGN-BG contacts, acting as gatekeepers to ML entry. As CGNs eventually transition to glia binding for radial migration, our research demonstrates that establishing permissive JAM-recognition sites on glia via cis and trans interactions of CGN JAM-C serves as a critical temporal checkpoint for sorting at the EGL to ML boundary. This mechanism integrates intrinsic and extrinsic cellular signals, facilitating heterotypic cell sorting into the ML and dictating the precise spatial organization within the cerebellar architecture.