Segmentation of Membrane Protein Motion in the Axon Initial Segment

Abstract

The axon initial segment (AIS) is a structure rich in specific cytoskeletal molecules that play important roles in the concentration of ion-channels that are required for action-potential generation. The establishment of a postulated diffusion barrier to the lateral exchange of membrane molecules in the AIS correlates with the enrichment of specific cytoskeletal molecules at this structure during development.Recently, a repetitive pattern of actin, spectrin and ankyrin forming ring-like structures perpendicular to the direction of axonal propagation has been discovered, that is interconnected via spectrin tetramers. This structure may finally provide the long sought direct physical correlate to the diffusion barrier at the AIS.Here, we perform repeated high-throughput single-molecule tracking on individual live primary hippocampal neurons during AIS development (DIV 3 - 10). We furthermore analyze the lateral mobility of lipid-anchored and transmembrane molecules with microsecond tracking at a resolution of few nanometers via interferometric scattering (iSCAT). Finally, we correlate the lateral motion of membrane molecules to the organization of the AIS cytoskeleton.We find that the lateral motion of membrane molecules becomes reduced in the AIS during development and that this reduction correlates with cytoskeletal organization into ring-like structures. The lateral motion of membrane molecules in the AIS plasma membrane is locally confined to awithin a repetitive pattern of 190 nm spaced segments along the AIS axis, consistent with the observed spacing of the cytoskeletal rings.Our data provide mechanistic insight into the diffusion barrier function in of the AIS.