Unraveling details of CIN85/CD2AP assistance to SLP65-mediated B cell activation
Arshiya Bhatt
Doctoral thesis
Date of Examination:
2019-09-17
Date of issue:
2020-06-18
Advisor:
Wienands, Jürgen Prof. Dr.
Referee:
Wienands, Jürgen Prof. Dr.
Referee:
Schwappach, Blanche Prof. Dr.
Persistent Address: http://hdl.handle.net/21.11130/00-1735-0000-0005-13DF-B
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Doctoral thesis
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Abstract
English
The hallmark of adaptive immunity is the production of specific antibodies. This process is initiated upon the ligation of the B cell antigen receptor (BCR) which activates multiple intracellular signaling cascades through the phosphorylation of the B cell ‘master regulator’: SLP65. Our lab found that in order to function, SLP65 interacts constitutively – in a stimulation independent manner, with molecules of the CIN85/CD2AP-family of adaptor proteins, harbouring multiple protein interaction domains. Mouse models with B-cell specific CIN85 deletion and patients with a germline deletion in CIN85 encoding gene have shown that CIN85 is mandatory for B cell activation and the subsequent B cell responses. The molecular basis of the mechanism driving this, however, needed further elucidation. The primary cells pose a limitation to the scope of genetic and mechanistic studies due to the scarce availability of the reserve. I addressed these aspects through the generation of CIN85-deficient B cells from the established B cell lines, followed by subsequent deletion of CD2AP. The cellular system thus created was used to conduct genetic and biochemical functional assays to assess how these proteins work. I observed that CIN85 significantly impacts the Ca2+ flux upon BCR-ligation. Moreover, CIN85 could additionally affect an upstream NF-κB pathway signaling element called PKCβ. I also used the abovementioned CIN85/CD2AP-double deficient cells to investigate further binding partners of SLP65 employing SILAC-based mass spectrometry to look for possible functional redundancies. In our collaboration with Prof. C. Griesinger’s group, we found that CIN85 and SLP65 undergo phase-separation into supramolecular-clusters, representing the more recent and exciting intracellular compartments of membrane-free, mini-organelles, that orchestrate signaling in B cells. Due to their capacity to interact with multiple signaling elements, droplet signalosomes could be the modus operandi for CIN85/CD2AP-family of adaptor proteins for scaffolding of relevant signaling proteins.
Keywords: BCR signaling; B cell activation; SLP65; CIN85; CD2AP
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GGNB - Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [824]
GGNB - Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences