NrCAM Coupling to the Cytoskeleton Depends on Multiple Protein Domains and Partitioning into Lipid Rafts

Abstract

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-␤-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.

A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

“…Although the concept of membrane microdomains and their potential roles in cellular processes have been widely studied in yeast and mammalian systems (Simons and Toomre, 2000;Garcia et al, 2003;Parton, 2003;Falk et al, 2004;Lafont et al, 2004;Salaün et al, 2004;Hansen et al, 2005 ;Lajoie and Nabi, 2010;Lingwood and Simons, 2010;Simons and Gerl, 2010), current knowledge on the composition and roles of such membrane microdomains remains sparse in plant cells (Bhat et al, 2005).…”

Characterization of Lipid Rafts from Medicago truncatula Root Plasma Membranes: A Proteomic Study Reveals the Presence of a Raft-Associated Redox System

“…These membrane domains were termed lipid rafts by Simons and Ikonen in the late 1990s, a name which rapidly raised their popularity both in mammalian and yeast (Saccharomyces cerevisiae) biology (Rajendran and Simons, 2005). Lipid rafts have been proposed to play biological roles in signal transduction (Simons and Toomre, 2000), exocytosis (Salaü n et al, 2004), endocytosis (Parton and Richards, 2003), apoptosis (Garcia et al, 2003), and actin cytoskeleton organization (Wickstrom et al, 2003;Falk et al, 2004). In addition, lipid rafts appear to be used for toxin and virus cellular entry (Rosenberger et al, 2000;Lafont et al, 2004) and for viral budding (Brugger et al, 2006).…”

Definition of Arabidopsis Sterol-rich Membrane Microdomains by Differential Treatment with Methyl-β-cyclodextrin and Quantitative Proteomics

“…In mammalian cells, lipid rafts have been shown to play a role in many different events: endocytosis via caveolae (9), virus budding or pathogen entry (10), regulation of exocytosis (11), actin cytoskeleton organization (12), and apoptosis (13). Cholesterol-dependent segregation of lipid raft proteins from non-raft proteins was visualized in mammalian cells and is consistent with the view that raft domains in the plasma membrane of cells are usually small and highly dispersed, but their size can be modulated by oligomerization of raft components (2).…”