Presented by Jeremy Smirnov
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.
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.
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)
Cell Spreading
(Phase contrast and Scanning EM microscopy)
Cell Spreading
Cell Spreading
Cell Spreading
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)
Rap-dependent Spreading
Rap-dependent Spreading
Rap-dependent Spreading
Rap-Dependent Spreading (Other cell lines)
Evaluate the amounts of Rap-GTP
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.
Rap activation required for pSMAC
F-Actin-rich cup formation
 “Cups” are formed in response to particulate BCR ligands.
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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
Cup formation
Cup formation
Cup formation
Cup formation
Cup formation (A20)
Cup formation (Splenic)
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.
Signaling
Signaling (ERK)
Signaling (Akt)
Signaling
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.