Dynamics and composition of signaling droplets reveal an impact of cellular transport during B cell activation

by Nadia Paglilla
Doctoral thesis
Date of Examination:2024-12-17
Date of issue:2025-03-13
Advisor:Dr. Michael Engelke
Referee:Dr. Michael Engelke
Referee:Prof. Dr. Henning Urlaub
Persistent Address: http://dx.doi.org/10.53846/goediss-11139

 

 

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Abstract

English

B cell activation during immune responses relies on the signaling network downstream the B cell antigen receptor (BCR). BCRs engaged by an antigen recruit a plethora of signal transducers forming BCR signalosomes, which results in the diversification of various signaling cascades. Nevertheless, the details underlying the translocation of these multiple signaling effectors to the sites of BCR activation have remained elusive. The scaffold SH2 domain-containing leukocyte protein of 65 kDa (SLP65) plays an essential role in orchestrating BCR signaling, being thereby crucial for B cell development and antibody production. Previous evidence collected by our working group indicates that the compartmentalization of SLP65 with its constitutive interaction partner CIN85 and vesicles in phase-separated droplets is a requirement for efficient B cell activation. In line with this, BCR signaling effectors assembled in droplets might rely on the cellular vesicle transport machinery for their translocation to the sites of BCR activation. This thesis aimed at characterizing SLP65/CIN85 signaling droplets in order to shed light on the mechanisms by which they support B cell activation. I established a system to monitor the droplet dynamics in real time, revealing a de novo formation of droplets, which navigate unidirectionally as whole entities to engaged BCRs. I set up proximity labelling methods, which coupled to mass spectrometry-based proteomics allowed for the identification of the protein composition of the B cell signaling droplets, constituting the “Vesicleome”. The Vesicleome comprises BCR signal transducers, as well as vesicle traffic and cytoskeleton regulatory proteins, highlighting an influence of intracellular transport processes in the BCR signaling network. I addressed the function of the Vesicleome components ARAP1 and ASAP1 in B cells, showing that they regulate BCR signaling by modulating the actin cytoskeleton and the BCR-induced translocation of signaling effectors, respectively. My thesis provides first evidence that B cell phase-separated droplets are specialized compartments endowed with a signaling purpose, which provide the BCRs with functionally diverse effectors to integrate a range of signaling cascades and cellular responses. This work brings together signal transduction and cellular transport processes, thereby extending our current understanding of B cell activation.
Keywords: B cells; Droplets; Cellular transport; Signaling