Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness

Stefan Becker,Zhen Hou,Michael Engelke,Leo E Wong,Sona Pirkuliyeva,Christian Griesinger,Arshiya Bhatt,Philipp S Erdmann,Jürgen Wienands,Joachim Maier,Jürgen Plitzko

doi:10.1038/s41467-020-14544-1

Abstract

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.

Highlights

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins
The subcellular localization patterns strictly correlated with the ability or disability of SLP65 to trigger mobilization of the second messenger Ca2+ on BCR ligation (Supplementary Fig. 1)
We investigated by confocal fluorescence microscopy, whether granule formation can be recapitulated in vitro using recombinantly expressed SLP65 and Cbl-interacting protein of 85 kDa (CIN85) and synthetic lipid vesicles in different combinations

Summary

Introduction

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. We show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Apart from its C-terminal SH2 domain, SLP65 is an intrinsically disordered protein (IDP)[3] that contains multiple tyrosine phosphorylation sites and proline-rich motifs (PRMs) to interact in a specific manner with SH2 and SH3 domain-containing ligands, respectively[4,5]. To elucidate how exactly the concerted interactions between SLP65, CIN85 and vesicles promote B cell activation, we used a combination of various methods to investigate the structure of these complexes and their functional signaling role in the live B cell. In the live B cell, this results in the formation of intracellular signaling bodies or droplets that are reminiscent of other phase-separated organelles such as stress granules or RNA transport granules[18,19,20]

Methods

Results

Conclusion