Structurally distinct endocytic pathways for B cell receptors in B lymphocytes

Abstract

B lymphocytes play a critical role in adaptive immunity. Upon antigen binding, B cell receptors (BCR) cluster on the plasma membrane and are internalized by endocytosis. In this process, B cells capture diverse antigens in various contexts and concentrations. However, it is unclear whether the mechanism of BCR endocytosis changes in response to these factors. Here, we studied the mechanism of soluble antigen-induced BCR clustering and internalization in a cultured human B cell line using correlative super resolution fluorescence and platinum replica electron microscopy. First, by visualizing nanoscale BCR clusters, we provide direct evidence that BCR cluster size increases with F(ab’)2 concentration. Next, we show that the physical mechanism of internalization switches in response to BCR cluster size. At low concentrations of antigen, B cells internalize small BCR clusters by classical clathrin-mediated endocytosis. At high antigen concentrations, when clusters size increases beyond the size of a single clathrin coated pit, B cells retrieve receptor clusters using large invaginations of the plasma membrane capped with clathrin. At these sites, we observed early and sustained recruitment of actin and an actin polymerizing protein FCHSD2. We further show that actin recruitment is required for the efficient generation of these novel endocytic carriers and for their capture into the cytosol. We propose that in B cells, the mechanism of endocytosis switches to accommodate large receptor clusters formed when cells encounter high concentrations of soluble antigen. This mechanism is regulated by the organization and dynamics of the cortical actin cytoskeleton.

Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles

“…A positive membrane curvature may thus facilitate clathrin-coated pit generation, because artificially generated positive membrane curvatures are preferred for CCS generation ( Zhao et al., 2017 ). Likewise, in immune B cells and macrophages, clathrin has been shown to form on large invaginations of the plasma membrane ( Roberts et al., 2020 ). We propose that plasma membrane invaginations are a major platform for clathrin-coated pit formation by providing positive curvature, where unique proteins, lipids, tensions, adhesion energies, phases, or cytoskeletal structures may enhance clathrin lattice growth and development.…”

“…This finding suggests that two seemingly independent forms of endocytosis, clathrin-mediated endocytosis, and bulk endocytosis, work together to enhance endocytic capacity. Such a collaboration is of broad importance, given that clathrin-mediated endocytosis is essential for the life of most eukaryotic cells ( Mettlen et al., 2018 ), and bulk endocytosis is a major form of endocytosis in secretory cells and beyond ( Clayton et al., 2008 ; Kononenko et al., 2014 ; Richards et al., 2000 ; Roberts et al., 2020 ; Watanabe et al., 2013 ; Wu et al., 2014a ; Wu and Wu, 2007 ).…”

Bidirectional feedback between BCR signaling and actin cytoskeletal dynamics

“…FCH and double SH3 domain-containing protein 2 (FCHSD2), a protein that generates F-actin at sites of clathrin-mediated endocytosis, selectively co-localizes with relatively larger nanoscale BCR-Ag clusters, generating F-actin around them, which enhances their endocytosis. In contrast, relatively smaller nanoscale BCR clusters (induced by lowering the concentration of soluble antigen) were only associated with classical clathrin-mediated endocytic pits [93]. The BCR can internalize antigen through clathrin independent pathways (Fig.…”

The Ins and Outs of Antigen Uptake in B cells

“…This mechanically demanding process requires actin polymerization which may be mediated by interaction between clathrin light chain and its actin-binding partner Hip1R ( 16 , 19 ). The specific clathrin adaptors, redundancy, stoichiometry and dynamics may be altered depending on antigen concentration ( 20 ).…”