1. Presented by Jeremy Smirnov

2. BCR and LFA-1 Induce B Cell Spreading Cell Spreading - a change in morphology that allows the B cell to “search” for antigens in the interstitial fluid surrounding the cell or on the surface of APCs. BCR - B Cell Receptor. Activates Rap1 and Rap2 (RAP GTPase), which regulate actin dynamics. LFA-1 – Lymphocyte function-associated antigen-1. Located on the surface of B Cells. It binds ICAM-1 on the surface of the APC and functions as an adhesion molecule.

3. Importance of Research B cells are key components of the immune system. Understanding the processes that regulate their functions gives us a better understanding of the immune system. This project set out to discover the effect of Rap GTPases on the activation of B cells – specifically, the steps that trigger activation upon recognition of the antigen.

4. Cell Spreading Plate B cells on surfaces coated with antibodies specific to BCR and LFA-1. This “captures” and immobilizes the cell and spreading can be observed. Spreading characterized by elongation, presence of membrane extensions, and cell length > 1.5x width A20 B Cell Lymphoma cells also spread when plated on ICAM-1 (LFA-1 ligand). NO spreading observed with CD40 or FcγRIIB plating. Cytoskeleton facilitates spreading – no spreading observed following treatment with latrunculin A or cytochalasin D (depolymerize actin), or PP2 (kinase inhibitor for Src – a kinase instrumental in actin cytoskeleton regulation)

5. Cell Spreading (Phase contrast and Scanning EM microscopy)

6. Cell Spreading

9. Spreading Depends on Rap Activation Expressing RapGAPII converts Rap into its inactive form (GDP). This is more effective than single gene knockdowns - there are several different Rap types and they are all encoded by separate genes – functionally redundant. RapGAPII takes care of them all! Rap activation can also be blocked by expression of Rap1N17 – a form of Rap1 that interferes with other Rap types. (Inhibited spreading, but not to the same extent as the first method)

10. Rap-dependent Spreading

13. Rap-Dependent Spreading (Other cell lines)

14. Evaluate the amounts of Rap-GTP

15. Rap Activation Required for pSMAC formation B cell recognizes antigen and forms the immunological synapse (IS). Ag-bound BCRs are then clustered into cSMAC (central supramolecular activation cluster). Reason: amount of antigen can be insufficient for activation, so clustering creates a central, concentrated area that will lead to activation of the B cell. LFA-1 binds ICAM-1 and is organized into a pSMAC (peripheral) around the cSMAC – this increases the contact area – stabilizes the immunological synapse.

16. Rap activation required for pSMAC

17. F-Actin-rich cup formation “Cups” are formed in response to particulate BCR ligands. Actin is stained with rhodamine-phalloidin (for red appearance), nuclei are stained with DAPI (blue appearance). Coated beads are used as a particulate Ag model. BCR induces tyrosine phosphorylation at the contact site, RapL (binds active Rap) relocalizes to the contact site as well  Rap-GTP must be there! No cup formation for CD40 and LFA-1 coatings. Latrunculin A prevents formation of cups (no data shown!) Rap activation is required for the cytoskeletal reorganization that facilitates cup formation

18. Cup formation

22. Cup formation (A20)

23. Cup formation (Splenic)

24. Rap activation is important for BCR signaling pathways Intracellular staining used to quantify pTyr (phosphorylated form) – no big differences with or without active Rap. Conclusion – Tyr phosphorylation is NOT dependent on Rap activation ERK (extracellular signal-regulated kinase) and Akt (also known as protein kinase B – PKB) phosphorylation IS dependent on Rap activation. This is perhaps due to an inability to form cups – treatment with LatA also produces decreased ERK and Akt phosphorylation.

25. Signaling

26. Signaling (ERK)

27. Signaling (Akt)

28. Signaling

29. Conclusions Rap GTPases are important in regulation of cytoskeleton dynamics of B cells. This is important in cell activation. Rap activation is needed for successful cell spreading, formation is cups, and BCR signaling pathways that affect cell activation.