B cell activation from actin rearrangements to cell survival Ph.D. thesis Maus Máté Supervisor: Prof. Sármay Gabriella D.Sc. Biology Doctoral School Immunology Programme Programme leader: Prof. Anna Erdei D.Sc. Eötvös Loránd University, Institute of Biology Department of Immunology Budapest, Hungary 2010.
INTRODUCTION B cells are an essential component of the adaptive immune system. They recognize foreign antigens and mature to antibody secreting plasma cells to mediate humoral immunity. Recognition of the antigen is mediated by their surface antigen receptor the B cell receptor (BCR). Antigen encounter induces the aggregation of BCRs on the B cell surface resulting in the formation of microclusters. This process is a prerequisite of antigen processing and downstream signaling. Microcluster formation requires the reorganization of the actin cytoskeleton on a second timescale with an initial depolymerization and a subsequent repolymerization phase. Regulation of site-specific actin rearrangements is a poorly understood process. B cells have an apoptosis sensitive phenotype, which can be lowered through the BCR signal. Following antigen encounter and microcluster formation a complex network of downstream signaling transmits a signal that can lower the apoptosis sensitiveness of B cells. A major route is the PI3K/Akt survival pathway. 7. M. Maus, D. Medgyesi, G. Sármay.: Gab2 szerepe a B-sejt receptor szignalingban XXXVIII. Magyar Immunológiai Társaság éves gyűlése, Hajduszoboszló, Magyarország, 2007 8. M. Maus, D. Kövesdi, D. Medgyesi, G. Sármay.: Gab2 gather: or how adaptor molecule Gab2 links B cell receptor signal to downstream signaling events? 14 th Symposium on Signals and Signal Processing in the Immune System, Balatonöszöd, Magyarország, 2007 9. M. Maus, N. Szilágyi.: Hypothesis and falsification in complex biological systems, or how can computational biology help guiding experimental biology? FIRST IMMUNOMICS SUMMER SCHOOL, Catania, Olaszország, 2007 10. M. Maus, E. Kiss, J. Matkó, N. Szilágyi, G. Sármay: A calcium szerepe a sejt-morfológia szabályozásában 38 th MEMBRÁN TRANSZPORT KONFERENCIA Sümeg, Magyarország, 2008 1 10
Other publications: 1. M. Maus, K. Paszti., A. Enyedi, D. Medgyesi, E. Kiss, Z. Ács, J. Matkó, N. Szilágyi and G. Sármay: Subcellular distribution of calcium regulates site-specific actin rearrangements Under submission Conference abstract: 1. M. Maus, G. Sármay A Gab adaptorfehérje család a B sejt receptor (BCR) szignalingban XXXVI. Congress of Hungarian Society for Immunology, Szeged, Magyarország, 2004 2. M. Maus, S. Sármay: A Gab adaptorfehérje család B sejtekben XXXVII. Magyar Immunológiai Társaság éves gyűlése, Sopron, Magyarország, 2005 3. M. Maus, S. Sármay: Gab2 adaptor protein and its function in B cells 13 th Symposium on Signals and Signal Processing in the Immune System, Balatonöszöd, Magyarország, 2005 4. M. Maus, D. Medgyesi, G. Sármay.: Gab2, a scaffolding protein regulating the PI3K independent Erk phosphorylation? 1st Joint Meeting of European National Societies of Immunology Under the auspices of EFIS, Paris, France, 2006 5. M. Maus, D. Medgyesi, G. Sármay.: Gab2, a scaffolding protein regulating the PI3K independent Erk phosphorylation? 3 rd INTERNATIONAL IMMUNOMICS CONGRESS, Budapest, Magyarország, 2006 6. M. Maus, D. Medgyesi, G. Sármay.: Gab2, a scaffolding protein regulating the PI3K independent Erk phosphorylation? 3 rd INTERNATIONAL IMMUNOMICS CONGRESS, Budapest, Magyarország, 2006 OBJECTIVES Microcluster formation in B cells is an actin dependent process. It requires actin reorganization with an initial depolymerization phase and a subsequent repolymerization phase. Microclusters form early during B cell activation, thereby regulation of this process needs to be mediated by a very upstream signal. Calcium regulates several actin binding proteins, its subcellular distribution is heterogeneous and its local concentration can vary on a millisecond timescale. These features make calcium a potent regulator of actin rearrangements and thereby microcluster formation.. Broadly speaking, our goal was to test for the existence of a specific interaction between calcium and actin dynamics. In more detail, we aimed to investigate how raising or lowering the cytoplasmic calcium concentration affects the morphology of the cell. How the same effect influenced the structure and density of the actin cytoskeleton. And whether proposed interconnection between calcium and actin dynamics is functional and physiologic. We aimed to decide whether theoretically such an interaction between calcium and actin could explain microcluster formation in B cells. 9 2
When microclusters are formed and the BCR is merged with the signalosomes, a central requirement of B cell activation and BCR induced cell survival is the successful recruitment of PI3K. PI3K activity in the signalosome is necessary for the activation of the PI3K/Akt pathway, for the recruitment of PLCγ and thereby activation of calcium signaling and subsequent NF-κB nuclear translocation. Thereby PI3K is a central player of B cell survival. We have seen that CD19 was suggested to be the main but not exclusive adaptor of PI3K in B cells. Gab family members and especially Gab2 are good candidates to be responsible for the recruitment of PI3K beside CD19. Our goal in this case was to define the role of Gab2 in BCR signaling. We aimed to investigate whether Gab2 is expressed and becomes phosphorylated in B cells. Does Gab2 recruit PI3K and eventually SHP2? Which kinase or kinases are responsible for its phosphorylation? How is Gab2 recruited to the signalosome? Does Gab2 contribute to the PI3K/Akt pathway or eventually to the SHP2/Erk pathway and thereby to cell survival? Publications connected to the thesis: 1. G. Sarmay, A. Angyal, A. Kertész, M. Maus, D. Medgyesi: The multiple function of Grb2 associated binder (Gab) adaptor/scaffolding protein in immune cell signaling Immunol. Lett. 104(1-2):76-82 impact factor: 2,858 2. M. Maus, D. Medgyesi, D. Kövesdi, D.Csuka, G. Koncz, G. Sármay: Grb2 associated binder 2 couples B-cell receptor to cell survival Cell Signal. 2009 Feb;21(2):220-7. Epub 2008 Oct 12. impact factor: 4,305 3
CONCLUSIONS Based on results we could develop a qualitative model for the role of calcium in actin rearrangement. Calcium regulated actin dynamics (CRAD) emphasizes the strong interrelation between calcium and actin dynamics, and says that actin decay occurs at cell sites with high local calcium concentration, while actin polymerization is favored where calcium level is lower as the surrounding. The CRAD model is the very first model for the relationship between calcium and actin dynamics, with both explanatory and predictive power. Based on our results relating the role of Gab2 in B cell receptor signaling, we can say, that beside CD19 and BCAP Gab2 is a third major route for the recruitment of PI3K to the signalosome. The Gab2/PI3K complex contributes to the activity of the PI3K/Akt survival pathway and thereby to B cell survival in death receptor abundant environments. APPLIED METHODS flow cytomertic protocols: apoptotic ratio determination, F-actin measurment, calcium measurment, cell surface molecule measurment (BCR, Fas) microscopy: standard and special confocal microscopy, immunofluorescence, live cell calcium imaging protein analytical methods: immunoprecipitation, western blotting transfection protocols: retroviral transduction, lipofection image processing and data-mining tools: Imaris, median-based search algorithms, mutual information measurment, Neyman s statistics RESULTS 1. Calcium regulated actin dynamics Large rises in the cytoplasmic calcium concentration induce the loss of cell structure and morphology. Raising the cytoplasmic calcium level induces F-actin decay, while lowering calcium concentration induces saturation like F-actin growth. Ionomycin induces the collapse of the actin cytoskeleton in living HELA cells. Calcium oscillations induce cortical actin oscillations with maximum peaks of actin, where calcium has its minimums in mcherry-actin expressing A20 cells. BCR internalization is speeded up in EGTA treated A20 cells. 7 4
Akt phosphorylation is selectively enhanced in EGTA treated A20 cells. T cells form a ring like actin structure at the cell-cell contact zone during immunological synapse formation. The mitochondria translocates to the synapse in a ring like structure. NCX is enriched at the IS-site. T cells maintain at the synapse a ring like calcium low area. 2. Gab2 in the BCR signaling Gab2 mutants lacking the PH-domain do not alter neither Akt nor Erk phosphorylation. A20 cells overexpressing WT Gab2 show a lower apoptotic rate when exposed to anti-fas antibody JO2 than control and PH-domain deficient Gab2 expressing cells. A20 cells overexpressing WT Gab2 show a lower apoptotic rate when exposed to anti-igg and subsequently to anti Fas antibody JO2 than control and PH-domain deficient Gab2 expressing cells. Gab2 is expressed in B cells. Gab2 becomes phosphorylated upon BCR engagement and recruits PI3K and SHP2. PI3K recruitment can only partially be inhibited by LY294002. SHP2 recruitment can fully be inhibited by LY294002. Gab2 in Lyn KO B cells cannot recruit PI3K upon BCR engagement. Gab2-SHP2 complex is formed in Lyn KO B cells. Inhibition of Syk blocks SHP2 recruitment to the Gab2 complex. Inhibition of Syk does not alter PI3K recruitment to the Gab2 complex. Gab2 is coimmunoprecipitated with Igβ in resting B cells, and with Igα in activated B cells Gab2 overexpression enhances Akt phosphorylation but does not alter Erk phosphorylation. 5 6