Peles, E. | bioRxiv Science

bioRxiv2023

Antibody-directed extracellular proximity biotinylation reveals Contactin-1 regulates axo-axonic innervation of axon initial segments.

Axon initial segment (AIS) cell surface proteins mediate key biological processes in neurons including action potential initiation and axo-axonic synapse formation. However, few AIS cell surface proteins have been identified. Here, we used antibody-directed proximity biotinylation to define the cell surface proteins in close proximity to the AIS cell adhesion molecule Neurofascin. To determine the distributions of the identified proteins, we used CRISPR-mediated genome editing for insertion of epitope tags in the endogenous proteins. We found Contactin-1 (Cntn1) among the previously unknown AIS proteins we identified. Cntn1 is enriched at the AIS through interactions with Neurofascin and NrCAM. We further show that Cntn1 contributes to assembly of the AIS-extracellular matrix, and is required for AIS axo-axonic innervation by inhibitory basket cells in the cerebellum and inhibitory chandelier cells in the cortex.

neuroscience↗

bioRxiv2022

Coiling of cellular protrusions around extracellular fibers

Protrusions at the leading-edge of a cell play an important role in sensing the extracellular cues, during cellular spreading and motility. Recent studies provided indications that these protrusions wrap (coil) around the extra-cellular fibers. The details of this coiling process, and the mechanisms that drive it, are not well understood. We present a combined theoretical and experimental study of the coiling of cellular protrusions on fibers of different geometry. Our theoretical model describes membrane protrusions that are produced by curved membrane proteins that recruit the protrusive forces of actin polymerization, and identifies the role of bending and adhesion energies in orienting the leading-edges of the protrusions along the azimuthal (coiling) direction. Our model predicts that the cells leading-edge coils on round fibers, but the coiling ceases for a fiber of elliptical (flat) cross-section. These predictions are verified by 3D visualization and quantitation of coiling on suspended fibers using Dual-View light-sheet microscopy (diSPIM). Overall, we provide a theoretical framework supported by high spatiotemporal resolution experiments capable of resolving coiling of cellular protrusions around extracellular fibers of varying diameters. Significance StatementCells adhere and migrate in environments that are composed of fibrous structures, such as the thin filaments of the extracellular matrix, or the wider axons and dendrites of neurons. In recent experiments, cells have been observed to form leading edge protrusions on such surfaces, that seem to coil around the extracellular fibers. However, the mechanism responsible for the formation of such coiling protrusions is not understood. Here, we provide a combined experimental and theoretical approach to explain the emergence of coiling protrusions. Our model is based on the self-organization of curved proteins that recruit actin polymerization at the leading edge of the cell, when spreading over an adhesive fiber.

biophysics↗