| dc.description.abstracteng | The processes of B cell activation and development are stimulated by antigen binding to the B cell receptor. Pre-signaling clusters are present in unstimulated, resting B cells and assemble by tripartite phase separation of two signaling adapter proteins, SLP65 and CIN85, and intracellular VAMP7-positive vesicles. The pre-signaling clusters of the B cell receptor signaling pathway are required for the adaptive immune response. A compromised humoral immune response has been reported in patients where SLP65 or CIN85 malfunction. Also, B cell culture studies show that pre-signaling clusters are required for an intracellular Ca2+ response. Thus, phase separation of these proteins in B cells seems to be functionally relevant.
In order to dissect the thermodynamics of phase separation of SLP65 and CIN85, we determined the affinities of the binding modules, i.e. the proline rich motifs (PRMs) and the SH3 domains individually and modelled it with an appropriate program.
Before this study, the binding preferences and number of relevant binding modules were unknown. Three SH3 domains of each CIN85 monomer that forms a trimer bind to a number of PRMs of SLP65. First, the promiscuous binding of the three SH3 domains of CIN85 to seven potential PRMs of SLP65 was disentangled on the modular level. Therefore, monovalent binding affinities of the individual SH3 domains to the individual PRM peptides were determined. This revealed one particular strong binding motif, PRM4, with dissociation constants of ~200 µM, 6 µM and 35 µM for SH3A, SH3B and SH3C, respectively. The promiscuous interaction further comprises four medium affine binding motifs (PRM1, PRM3, PRM5 and PRM6) with dissociation constants in the range of ~60 µM to 1 mM, and two weakly binding motifs (PRM2 and PRM*) with dissociation constants above 1 mM.
Next, the question was addressed whether PRM4 or SH3B are main drivers for phase separation. Therefore, mutant constructs were designed. Weak (inactivated PRM4) and strong (3xPRM4) binding constructs of SLP65, and a strong binding construct of CIN85-3SH3 (3SH3B) were expressed. Pre-signaling clusters can be reconstituted in vitro with SLP65- and CIN85 protein constructs together with small unilamellar vesicles (SUVs). The critical concentration of phase separation was measured for mixtures in presence or absence of SUVs, and for mixtures with different combinations of mutant constructs. The binding entities 3xPRM4, 3SH3B and SUVs promoted phase separation to a different extent. Comparative analysis showed that phase separation is promoted by SUVs very effectively. The artificially designed strong binding construct 3SH3B showed a similar strong effect. The inactivation of PRM4 by single point mutation increases the critical concentration, indicating that PRM4 has a substantial effect, but to a lesser extent than SUVs. The artificially designed binding entity 3xPRM4 had the smallest effect in promoting phase separation among the mutant constructs designed for strong binding.
Furthermore, we focused on the aspect of the structural arrangement of vesicles inside the condensed phase. The vesicle distribution was visualized in collaboration with Prof. Plitzko’s group by cryo-electron tomography, highlighting that SUVs condense in droplets.
Subsequently, after the detailed characterization of tripartite phase separation in vitro experimentally, an early-stage model was developed to describe the SLP65-CIN85 interaction network beyond the schematic representation. The sticker-spacer lattice modelling program LASSI was phenomenologically parameterized and used to simulate phase separation of the SLP65-CIN85 interaction network. For simulation purposes, SLP65, CIN85-3SH3 and their strong and weak binding mutant constructs shared the same sticker-spacer architecture, but differed in the strength of the interaction terms according to the modular design. Interaction of hexavalent SLP65 (6 x PRMs) with trivalent CIN85 (3 x SH3) constructs were simulated. Initially simulated and experimentally-determined critical concentrations did not agree well. However, the agreement was improved when the energy terms of the C-terminal SH3 module were set to zero in the wildtype-like 3SH3 construct referred to as 2SH3 construct. The validity of this approach is based on experimental evidence ([174] and unpublished results of my colleague Daniel Sieme.)
Finally, the view was shifted towards the biological relevance by correlating the binding affinities to previous ex vivo experiments. We found that the binding affinities determined in vitro are correlated to the partly reconstituted Ca2+ signals in SLP65-/- cells transfected with SLP65-PRM single point mutants.
Moreover, the phase separation properties of mutant constructs were correlated to two readouts, namely the Ca2+ signal capacity and the number of pre-signaling clusters. The wildtype-SLP65 reconstituted cells showed most droplets and highest Ca2+ signal. The abolishment of vesicle interactions showed the most pronounced effect in vitro and in vivo. In vesicle-binding deficient ∆N-SLP65 transformed cells, droplets were absent and the Ca2+ signal was abolished. For SLP65-3xPRM4 reconstituted cells, droplets were still present but reduced, and the Ca2+ signal was decreased, but not abolished. Thus, the SLP65-3xPRM4 reconstituted cells showed the least pronounced effect among the mutant constructs, which is in line with the in vitro observations. The outcomes of the in vitro phase separation assays and the in vivo readouts correlate and indicate that phase separated pre-signal clusters rely on the contribution of individual PRMs. In this work, a comprehensive description of SLP65-CIN85 phase separation is given. | de |