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

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.

The Eukaryotic Linear Motif resource: 2022 release

“…SLiM-mediated interactions typically contribute to receptor clustering and the formation of the associated, membrane-proximal signalling networks (so called signalosomes), such as the synaptic densities of excitatory ( 57 , 58 ) and inhibitory synapses ( 59 ), presynaptic active zones ( 60 ), T- and B-cell receptor signalosomes ( 61 , 62 ), the nephrin-associated signalling network specific for kidney podocytes ( 63 ), ABC transporter-linked condensates ( 64 ) and tip-link densities of stereocilia and microvilli of inner ear hair cells and intestinal enterocytes, respectively ( 65 ). SLiMs also play central roles in the assembly of several nuclear bodies, such as SPOP/DAXX bodies ( 66 ), Promyelocytic leukaemia nuclear bodies ( 67 ), nuclear splicing speckles ( 68 ), heterochromatin ( 69 ) and transcription regulatory condensates ( 70 ), as well as cytoplasmic phase-separated condensates of diverse functions, like yeast P-bodies ( 71 ), Balbiani bodies ( 72 ) and miRISC complexes ( 55 ), among others.…”

“…PxpxPR motifs within the B-cell linker protein (BLNK, also called SLP-65) belong to the newly annotated LIG_SH3_CIN85_PxpxPR_1 motif class and mediate multivalent interactions with CIN85 trimers, forming the extended molecular scaffold underlying phase-separated B-cell receptor signalling clusters ( 62 ).…”

Biomolecular phase separation through the lens of sodium‐23 NMR

“…NMR spectroscopy is a powerful technique allowing experimental access to biomolecular structure and dynamics at a broad range of time and length scales in different environments, including the liquid‐ and gel‐like biomolecular condensates 8,9 . Several recent studies have utilized high‐resolution NMR to monitor the structural dynamics of proteins and other biomolecules within condensed phases 10–13 . Recently, natural abundance 17 O relaxation of water molecules has been utilized as a reliable proxy to report the internal fluidity of phase‐separated biological hydrogels and the ordering of water molecules across lipid nanodiscs 14,15 .…”

Comparative roles of charge, π , and hydrophobic interactions in sequence-dependent phase separation of intrinsically disordered proteins

“…LLPS underpins formation of a variety of biomolecular condensates (10), including intracellular bodies, such as nucleoli and stress granules, that are often referred to as membraneless organelles (4,11), and precursor of extracellular materials as in the case of sandcastle worm adhesive (12) and elastin in vertebrate tissues (8). These dynamic, phase-separated condensates perform versatile functions, as underscored by their recently elucidated roles in synapse formation and plasticity (7,13), organization of chromatin (14), regulation of translation (15,16), B cell response (17), and autophagosome formation (18). The pace of discovery in this very active area of research has been accelerating (19)(20)(21)(22)(23)(24)(25)(26)(27)(28).…”