Transcript 12mb ppt - UCLA.edu

• M. Carrie Miceli
• May 4, 2005
• TCR Signal Transduction and Membrane
Dynamics
• Reading
• Huppa, J. B., Davis, M. M. T-cell-antigen
recognition and the immunological synapse. Nat
Rev Immunol 3: 973-83 (2003)
• Behind the scenes of anergy: a tale of 3 E3s,
Davis and Ben-Neriah, Nature Immunogy
March 2004 5:238
• "In the context of immune responses, the most critical feature of the
cell is its surface. The surface of a cell is not smooth and flat, like a
ball bearing; it is more like a microscopic garden tended in darkness,
bathed by warm salty fluid, a rounded and shaggy convex landscape
with cellular vegetation waving like seaweed above the cell membrane.
This vegetation represents the aboveground tendrils of proteins and
other molecules, which are anchored in the membrane. These are the
eyes, the ears, the taste buds, the nerve endings of the cell. At any
given time, the surface of a typical cell may be studded with thousands
of different tendrils. But perhaps the hardest concept to bear in mind,
sometimes even for scientists, is that this moist, shaggy landscape is
dynamic. It is constantly changing. New vegetation shoots up and old
vegetation collapses as in in a time-lapse film. In real time, these
proteins bloom and shrivel, unfurl and fold up, in the course of hours,
sometimes minutes, depending on what's going on in the immediate
neighborhood, because cells are exquisitely sensitive, and constantly
reacting, to their local ecology. Certain receptors, like perennial
flowers, grow on the surface, so permanent and unchanging a fixture
of the cellular landscape that they can in essence serve as a reliable
molecular landmark, or fingerprint, that reveals the identity of the cell
itself. Indeed, they are known as the clusters of differentiation...."
•
From "A Commotion in the Blood: Life, Death and the Immune System" by Stephen S.
Hall. Henry Holt, 1997 ISBN 0-8050-5841-9
TH1 T cells clones
Allen group and Germain
group both publish in cell
Agonists processively
phosphorylate zeta to the
pp21 (pp23) isoform; ZAP
70 is associated and
phosphorylated (activated)
Antagonists/partial
agonists induce pp18 (21)
isoform and then stall;
ZAP-70 associates but
doesn’t get phosphorylated
(activated)
Hb agonist
Gln, ser 79
pa/antag
ppZAP70
Anti-pY western total cell lysates
ppz
big
small
Is the TCR must be dimerized/multimerized and undergo
a conformational change to signal?
Alam…..Travers PJ. "Qualitative and quantitative differences
in T cell receptor binding of agonist and antagonist ligands.
Immunity, 1999 Feb, 10(2):227-37."
• Boniface JJ……..Davis MM. Initiation of signal
transduction through the T cell receptor requires the
peptide multivalent engagement of MHC ligands.
Immunity, 1998 Oct, 9(4):459-66.
– Using recombinant MHC/peptides, monovalent or multimerized
demonstrate that MHC/peptide monomers are not sufficient
• Single peptide MHC is enough on the surface of an APC
(davis,april NI 2004)
• Role for partial agonists/antagonists helping?
• Role for costimulators/adhesion molecules
• Contraints imposed by mimbrane micoenvironment
Kinetic proofreading models of T cell activation; it takes time
The engagement of peptide MHC does not immediately lead to TCR triggering
because a series of phosphorylation steps followed by recruitment and
activation of ZAP70 need to be performed, a process that requires time. If the
duration of the interaction is sufficient, the phosphorylation and docking events
will proceed until the fully active complex is assembled and can transmit a
signal to downstream pathways. In contrast a premature dissociation before the
process has been completed will lead to the formation of inactive intermediates
that by sequestering substrates inhibit activating by agonists resulting in TCR
antagonism. Alternatively, intermediates generated might deliver an
intermediate or negative signal , thus explaining partial agonism and
antagonism.
Increases Fidelity
Allows for discrimination of small differences in off rates/affinity.
TCR z Chain Undergoes a Series of Ordered
Phosphorylation Events Upon TCR Ligation
Antagonists
p18 z
Agonists
pp21 z
pp23 z
ZAP-70 recruitment,
But no activation
T cell inactivation
ZAP-70 recruitment,
Complete activation
T cell activation
Lat functions as an adapter to link TCR to multiple downstream
pathways ( Annual review of Immunology 2002, Samelson)
LAT is multiply phosphorylated, different tyrosines adapt to
different signaling pathways…is this a site of control? Could
degree of processive phoshorylation control functional outcome?
2) Naïve T cell activation requires
sustained and continual receptor
engagement: 6-12 hours of APC:T cell
contact.
• Premature distruption of an agonist
signal-> aborted signal. Lanzavecchia
• Whereas the earliest TCR signals (z chain
phosphorylation), ZAP-70 activation
happens within seconds to minutes of
receptor engagegment , sustained
engagement is required to for T cell
activation.
• 3) The TCR is serially engaged. Also
Lanzavecchia The finding that TCR
engagement results in its internalization in
an antigen dose and time dependent
fashion provided investigators with a
readout as to the number of TCR that had
been engaged. Using TCR internalization
as a readout of TCR engagegment and by
accurately labeling and counting the
number of receptors that were internalized
it was determined that few (100 per APC)
agonistic peptide-MHC complexes engage
and trigger a much larger number of
TCRs (2000-18,000).
• Andrey S. Shaw and
Michael L. Dustin .
Making the T Cell
Receptor Go the Distance:
A Topological View of T
Cell Activation Immunity
1997 6: 361-369
• It needs a space
•
•
•
•
•
Topologic model of T cell activation; it needs a space
Emphasizes the requirements for the rearrangement of
membrane proteins at the area of contact between the T cell
and APC in addition to TCR engagement for successful T
cell activation.
Liganded engagement of short similarly sized molecules
are proposed to concentrate at the activation cap while
taller heavily glycosylated are proposed to relocate outside
the area of contact
The actin cytoskeleton is proposed to play a role in
affecting this molecular reorganization.
Such interactions are proposed to increase the local 2D
affinity by concentrating T cell activation
molecules/transducers within the contact cap and increasing
local membrane rigidity and signal procession.
Coincident with recruitment of T cell accessory molecule
and the exclusion of CD45 is the recruitment of associated
intracelluar tyrosine kinase activity and the exclusion of
phosphatase activity.
The T Cell:APC contact site is organized into the cSMAC and
pSMAC (Monks CR et al, Nature 1998 395:82-86)
The immune synapse partitions into
cSMACs and pSMACS
• Supramolecular activation complexes
• Central cSMAC
–
–
–
–
–
–
TCR/CD3
Pep/MHC
CD28/CD80
PKC theta
Lck/Fyn
Lipid rafts???
• Peripheral SMAC
– LFA-1 (ICAM-1)
– Talin
• Even further out CD45 -D-SMAC (distal) and
CD43
The Immunological Synapse: A Molecular Machine
Controlling T Cell Activation Science 1999 July 9; 285: 221-227.
Arash Grakoui, 1 Shannon K. Bromley, 1 Cenk Sumen, 2 Mark M. Davis, 2 Andrey S. Shaw, 1 Paul M.
Allen, 1 Michael L. Dustin 1*
The specialized junction between a T lymphocyte and an antigen-presenting cell,
the immunological synapse, consists of a central cluster of T cell receptors
surrounded by a ring of adhesion molecules. Immunological synapse formation
is now shown to be an active and dynamic mechanism that allows T cells to
distinguish potential antigenic ligands. Initially, T cell receptor ligands were
engaged in an outermost ring of the nascent synapse. Transport of these
complexes into the central cluster was dependent on T cell receptor-ligand
interaction kinetics. Finally, formation of a stable central cluster at the heart of
the synapse was a determinative event for T cell proliferation.
Quicktime movie= synapse live
TCR transgenic T cells plated on lipid bilayer reconstituted
with fluorescent MHC/peptide and ICAM-1 (LFA-1 ligand)
30 min
post antigen
Agonist peptide
induces SMACs
Antagonist peptide
doesn’t induce SMACs
proliferation
dose
response
Figure 2. Immunological synapse formation and MHC-peptide dose. 2B4 T cells on Oregon green Ek-GPI loaded with different peptides and
Cy5 ICAM-1-GPI at 200 molecules per square micrometer. Images show accumulated MHC-peptide (green) and ICAM-1 (red). (A) to (E)
Ek(MCC88-103) agonist at 80 molecules per square micrometer. (A) Bright-field image; (B) IRM image; (C) Ek only (green); (D) ICAM-1
only (red); (E) Ek and ICAM-1 overlay. (F) Ek(MCC88-103) agonist at 0.6 molecules per square micrometer Ek plus ICAM-1 overlay. (G)
Ek(MCC88-103 K99A) null at 80 molecules per square micrometer Ek plus ICAM-1 overlay. (H) Dose-response for Ek(MCC88-103)
agonist for T cell proliferation on bead-supported bilayers (41, 42). (I) Dose-response for Ek(MCC88-103) agonist for cluster formation at 30
min. The asterisk indicates no immunological synapse formation. Original image elements = 0.018 µm2. Data are representative of two
Wulfing and Davis Dec 1998 Science
• Peripheral T cells from TCR transgenic loaded
with Ca+ sensitive dye.
• APC pulsed with agonist antigen
• Beads coated with strepavidin (T cells are
biotinylated)
• Within 4 minutes of CA+ flux get
repolarization of membrane toward the T
cell:APC interface.
• Beads are dragged the interface as a result of
reversal of polarity.
• Requires presence of B7 and/or ICAM on APCs
(ligands for CD28 and LFA respectively).
• The sequel: requires actin:myosin motor
• Synapse
Live Quicktime move
•TCRs intially engaged at edges
of contact site and then cluster centrally
• How is a synapse constructed?
•Wulfing movie
•Reversal of polarity
•Synaptice membrane recruitment
•Does it function in signal transduction?
T Lymphocyte Costimulation Mediated by Reorganization of Membrane Microdomains Antonella Viola, Susanne Schroeder, Yoichi
Sakakibara, and Antonio Lanzavecchia Science 1999 January 29; 283: 680-682.
Figure 3. CD28-induced raft redistribution to the site of TCR engagement. (A to C) Resting T cells were stained with FITC-CTx and
analyzed by confocal microscopy. (A) Unstimulated resting T cells. (B) T cells stimulated for 20 min with beads coated with anti-CD3
alone (10 µg/ml) and (C) stimulated for 20 min with beads coated with anti-CD3 (10 µg/ml) plus anti-CD28. Representative data are
from one of six experiments. (D and E) Redistribution of rafts induced by costimulation. (D) Surface staining by FITC-CTx at various
times after stimulation of T cells with beads coated with anti-CD3 alone () or anti-CD3 plus anti-CD28 (). () Unstimulated control
cells. (E) Surface (filled bars) and total (open bars) FITC-CTx staining in live and permeabilized cells, respectively.
Lipid Rafts/Membrane Microdomains

“Lipid Rafts" are composed primarily of sphingolipids (including the
glycosphingolipid GM1) and cholesterol and float as laterally associated units
within in an otherwise glycerophospholipid-rich plasma membrane.

Enriched in GPI-linked proteins (the long and saturated acyl chains of their
carboxyterminal GPI-lipid modification exhibit typical features of lipid raft
components).

Enriched in doubly acylated Src family members with fully saturated fatty acids
including Lck and Fyn.

Enriched in other signal transduction molecules including Ras, heterotrimeric
G proteins, nitric oxide synthase, LAT, phosphatidylinositol-(4,5)-biphosphate and
sphingomyelin.

Enriched in actin, actin binding proteins and Rho, Rac, Cdc42 implicated in
cytoskeletal communication.

Specifically exclude CD43 and CD45 and contribute to the partitioning of
phosphatase and kinase activities within T cell membranes.
CD48
L
A
T
Lck
CD48 is Expressed on the Surface of T cells and is a Ligand for CD2
APC Surface
Class II
MHC
 
TCR
T Cell Surface
 
CD2
CD48
 
zz
GPI
lck
CD48/ TCR Costimulation
(Moran and Miceli, Immunity 1998)
• CD48/TCR coengagement enhances TCR z tyrosine
phosphorylation and cytoskeletal association
– requires intact lipid rafts
– requires the actin cytoskeleton
• CD48/TCR coengagement induces F-actin redistribution
and early morphological changes
• CD48/TCR costimulation enhances TCR induced IL-2
production
Therefore:
– Costimulators can enhance TCR signals by recruiting
lipid rafts to the contact site.
– Lipid rafts function to integrate TCR engagement and
cytoskeletal reorganization at the contact cap.
CD43
CD43
TCR/CD48
stimulation
CD45
CD48
CD45
CD48
TCR
CD48
Lck
Lck
Lck
CD48
TCR
Fyn
Lck
Fyn
G-actin
F-actin
F-actin
Modified topological model which included contributions of lipid rafts
Costimulators (CD48 and CD28) function to organize the contact cap through
reorganization of membrane microdomains.
Detergent insoluble glycoclipid membranes (DIGs) are a
biochemical approximation of lipid rafts
Lipid Raft Fractionation
Sucrose
5%
Lysis:
non-ionic
detergent
(1% Brij-58)
Cell lysate in
40% sucrose
200,000g
centrifugation
Lipid Rafts/
DIGS
30%
Non-Rafts
Membrane Compartmentation Is Required for Efficient T Cell
Activation, Xavier….Seed; Immunity, June 1998
A) western blots demonstrating membrane microdomain localization
of various proteins. B) Anti-phosphotyrosine analysis of proteins in
the cytoplasmic membrane and DIG (raft) fractions of Jurkat cells
before and after treatment with anti-TCR antibody
Xavier et
al
Immunity
1998
TCR
antigen
receptor
complex
localizes to
DIG/Raft
Compartment
following
TCR
activation
Anti-CD3 mediated redistribution of Signaling molecules
•Zhang…. Samelson LAT
Palmitoylation: Its
Essential Role in
Membrane Microdomain
Targeting and Tyrosine
Phosphorylation during T
Cell Activation Immunity
1998 9: 239
Wt Lat partitions within
lipid rafts (GEMS),
C26/29A; C26A and C29A
palmitoylation mutants
don’t. Mutants can’t be
tryosine phosphorylated.
Without LAT phosphorylation, no T cell activation
The Lck SH3 Domain is Required for
Polarized Lipid Raft Clustering at the TCR Contact Site
Patel, Moran, Low and Miceli, JI 166(2):754, 2001
neo
CD3
neo
CD3 + CD48
F505
CD3
F505
CD3 + CD48
LckF505 second site SH3 mutation
does not affect:
AF/F505
CD3 +CD48
YLDY/F505
CD3 + CD48
Induction of protein tyrosine
phosphorylation
apoptosis
does affect:
Sustenance of protein tyrosine phosphorylation
Raft clustering at the TCR contact
IL-2 production
Costimulator shortening of TCR engagement requirements for IL-2
A Two Signal Model for T Cell Activation Reliant on
Distinctly Regulated Raft-Mediated Signals One and Two
Synaptic raft clustering requires: Lck SH3 Patel et al JI 2001;
MAGUK scaffold Dlgh1 Round et al J Exp Med 2005; WASP
Dupre et al Immunity, 2002
Cbl-b ablation lifts the requirement for CD28 in activation and
enhances synaptic raft/TCR clustering through WASP
Krawczyk…Penninger Immunity 2000
Immunity, Vol 17, 809-822, December 2002
Dynamics of p56lck Translocation to the T Cell
Immunological Synapse following Agonist and
Antagonist Stimulation
Lauren I. Richie Ehrlich1, Peter J.R. Ebert1, Matthew F.
Krummel , Arthur Weiss, and Mark M. Davis
•
Lck-GFP Translocates to the Immunological Synapse following Agonist
but Not Antagonist Stimulation in D10 T Cells (from intracellular
vesicles)
•
To observe lck translocation to the immunological synapse, lck-GFP D10 cells were imaged as
they interacted with CA-pulsed CH27 B cells. The T cells were loaded with the calcium indicator
dye fura-2 before imaging. Agonist-pulsed CH27 B cells were added to the T cells, and images
were acquired for 15 min at 15 s time intervals. At each interval, four images were acquired: first, a
differential interference contrast (DIC) image, second and third 340 nm and 380 nm images for
calcium analysis, and fourth a 20 µm z stack of GFP images acquired at 1 µm intervals. From this
z stack, we generated 3-dimensional (3-D) reconstructions of lck-GFP that were rotated to view the
immunological synapse en face. In addition, we followed a central horizontal GFP slice of the cell
over time.
Staging and resetting
T cell activation in
3 min
SMACs
Benjamin A.
Freiberg…. Abraham
8 min
Kupfer Nature
Immunology 3, 911 917 2002
CD45 green 23 min
Lck red
hang together early
(3 minutes); by 8
minutes start to
segregate. CD45
outside both SMACs
Green TCR; blue CD45; red talin
at 23 minutes
The Immunological Synapse--a Multitasking System P. Anton van der
Merwe and Simon J. Davis Science 2002 February 22; 295: 1479-1480.
What is the purpose
of the immune
synapse?
A machine for
modulating signals
-TCR or
-costimulators?
A mechanism for
polarized secretion
A mechanism for
internalization,ligand
counting and TCR
desensitization
Current Opinion
in Pharmacology
August 2004, Pages
415-422
Rangachari and
Penninger
Figure 1. Simplified overview of signals provided by TcR, Cbl-b and CD28. Binding of the TcR to antigen/MHC results in recruitment of Src
family kinases such as Lck, followed by recruitment of ZAP-70. ZAP-70 subsequently phosphorylates the membrane-associated adaptor molecule
LAT, which forms a complex with the adaptor SLP-76 and the guanine nucleotide-exchange factor Vav1. Calcium mobilization, IL-2 production
and SMAC formation depend, in part, on Vav1, which is negatively regulated by the E3 ubiquitin ligase Cbl-b. Costimulation through CD28
represses Cbl-b, permitting activation of Vav1 and the lipid kinase PI3K. This allows Vav1-mediated activation of a molecular complex that
induces cytoskeletal changes leading to SMAC formation. Further, Vav1 and PI3K co-operate to localize PKC to the SMAC, where it acts
upstream of PLC1 to drive calcium flux and activation of IL-2 transcription factors such as AP-1, NF-B and NF-AT. The Vav1 homologues Vav2
and Vav3 may have overlapping functions with Vav1. Integration of TcR and/or CD28 signals might have several outcomes. Appropriate ones
include tolerance to self-Ag and immune response to pathogen; the decision between tolerance and response is influenced by the function of Cblb. However, in situations of immune dysregulation, autoimmune attack against self-tissue can proceed. Recent studies suggest that GRAIL and
Itch might promote T cell tolerance
Anergizing conditions upregulate E3 ligases (some sustained NFAT without AP1)
Figure 4. Upregulation of E3 ligases in T cells subjected to sustained Ca2+ signaling.
(a) Upregulation of Itch, Cbl-b and Tsg101 in anergic T cells. D5 cells were left resting (-) or were
stimulated (+) with ionomycin (Iono), CsA or both. Cell extracts were evaluated for Itch, Tsg101, Cbl-b
and Nedd4 by immunoblotting, and relative protein expression was quantified (below lanes). (b) D5 cells
were left untreated or were stimulated with ionomycin (Iono) or ionomycin plus CsA for 10 h, and
expression of Itch, Cblb, Rnf128 (GRAIL) and Plcg1 mRNA was evaluated by real-time RT-PCR, normalized
to amounts of mRNA encoding the ribosomal protein L32. Data represent the average s.d. of the ratio of
mRNA expression in ionomycin-treated or ionomycin and CsA-treated to that in untreated cells..
Restimulation of anergized cells induces targeted proteolysis PLC PKCq,
RasGAP degradation (ubiquitination and lysosomal targeting) and induces
anergy/unresponsiveness
c) Cells were pretreated for 16 h with ionomycin and restimulated for 1 h with
anti-CD3, anti-CD3 plus anti-CD28, ionomycin, or PMA plus ionomycin. A)
pretreated wth iono w or w/out CSA and then restimulated
(a,b) Primary TH1 cells from 2B4 TCR transgenic mice were left untreated (top rows; control) or were
pretreated with ionomycin (bottom rows), then were incubated for 40 min on planar phospholipid bilayers
containing Oregon green-labeled I-EK-agonist MCC peptide complexes and indocarbocyanine-labeled
ICAM-1. The distribution of ICAM-1 (red) and I-Ek-MCC (green) molecules in T cell-bilayer contact zones
was captured at different times. The gray panels in a are interference reflection microscopy images in
which cell-bilayer contacts appear as dark areas. We have obtained similar results in more than four
independent experiments. (c) Involvement of PLC-1 in synapse stability. Mature T cell synapses were
allowed to form, then weak or strong PLC- inhibitors were added. Right, percentage of cells with mature
synapses relative to the same cells before the addition of inhibitors.
Calcineurin
imposes T cell
unresponsiven
ess through
targeted
proteolysis of
signaling
proteins
Heissmeyer…
Anjana Rao Nature
Immunology 5, 255265
Figure 5. Ionom
ycin-anergized
T cells show
decreased
stability of the
immunological
synapse.
Nature Immunology 5, 238 - 240 (2004)
Matti Davis & Yinon Ben-Neriah
Figure 1. A model for destabilization of the T cell synapse due to anergy-induced ubiquitination:
'Unauthorized' APC engagement of TCR in the absence of costimulation induces NFAT-dependent
transcription of 'anergy genes', including those encoding several E3s (Cbl-b, Itch and GRAIL).
After subsequent engagement with an 'authorized' APC, the upregulated E3s are activated, targeting SMAC
components (PLC-1 and PKC-q) to lysosomal degradation. This might be achieved in three steps: SMAC
component recruitment to the endosome mediated by Cbl-b, CIN85 or endophilin; subsequent ubiquitination of
SMAC and endocytic components by Itch and/or GRAIL in the endosome; and trafficking of the ubiquitinated
proteins into the lysosome, where the SMAC proteins could be degraded. Protein degradation will eliminate
recycling of PLC-1 and PKC- back to the synapse and thus may shorten the lifespan of the synapse. In addition,
Cbl-b may compromise SMAC through inhibition of the WASP-Arp2/3 actin polymerization pathway by negative
modification of the WASP activator PI3K and by elimination of PKC- q, a negative regulator of WASP inhibitor WIP.
Only after restimulation and E3s synaptic recruitment does “defect’ present
THE SYNAPSE AT DIFFERENT STAGES
OF T CELL DEVELOPMENT MAY DIFFER
Lck (and likley raft) distribution differs thoughout
development of antigen specific CD8 T cells
Bachman…Viola J exp Med
Eric Hailman, W. Richard Burack, Andrey S. Shaw, Michael L. Dustin, and Paul M. Allen
Immature CD4+CD8+ Thymocytes Form a Multifocal Immunological Synapse with Sustained
Tyrosine Phosphorylation
Immunity 2002 16: 839-848.
The Immunological Synapse of DP Thymocytes
(A)
DP thymocytes from 3A9 H-2b RAG-1-/- mice were
incubated on lipid bilayers containing fluorescently
labeled ICAM-1 (red) and I-Ak-HEL (green).
(B)
(B) DP thymocytes from N3.L2 or 3A9, H-2b RAG-1-/mice were incubated on lipid bilayers containing
fluorescently labeled ICAM-1 (red) and I-Ek loaded with
agonist peptide (for N3.L2) or I-Ak with covalently
linked agonist peptide (I-Ak-HEL, for 3A9). Similar
patterning, with multiple areas of exclusion (holes) in
the ICAM-1 fluorescence pattern, was seen in >90% of
adherent cells in 15 separate experiments.
(C) 3A9 H-2b RAG-1-/- thymocytes were incubated on lipid
bilayers containing fluorescently labeled ICAM-1 (red)
and I-Ak-HEL (green). The same cell is shown at three
time points, with intervals of 2 min between images.
Balamuth, F….
Bottomly, K Immunity Nov. 2001Distinct patterns
of membrane microdomain partitioning in Th1
and Th2 cells
• Th1s cluster rafts at synapse, TH2 don’t
• Dsiruption of lipid rafts affects TH1, but not
TH2
• Th1 induce SMACs (TCR/LFA-1); TH2 dont
Figure 4. CTL Secretory Lysosomes Insert between
the Signaling Molecule Patch and the Adhesion Ring
Lck (red) and either CD11a (green) and cathepsin D (blue, [A–
C]), or granzyme A (green) and actin (blue, [D–G]).
Copyright © 2004 Cell Press.
Immunity, Vol 15, 751-761, November 2001
The Immunological Synapse of CTL Contains a Secretory
Domain and Membrane Bridges
Jane C. Stinchcombe , Giovanna Bossi , Sarah Booth , and Gillian M.
Griffiths
•
CTL Secretory Lysosomes Insert between the Signaling Molecule Patch and the Adhesion Ring
•
CTL-P815 target cell conjugates stained with antibodies against Lck (red) and either CD11a (green)
and cathepsin D (blue, [A–C]), or granzyme A (green) and actin (blue, [D–G]). CTL and P815 target
cells are shown on the right and left, respectively, in (A), (B), and (E–G). Three separate conjugates
are shown (A–C, D–F, and G).
•
(A), (E), and (G) are single confocal sections, (B) and (F) are projected confocal sections taken 0.4
É m apart through the sample (shown as 3D image reconstructions in Supplemental Movies S2 and
S3), and (C) and (D) are z axis image reconstructions shown in the plane of the contact site. The lytic
granules insert to the side of the signaling protein marker (A, B, E, and F) within the adhesion ring (A
and B). (C) and (D) clearly show that the signaling and secretory molecules are distributed into two
distinct domains within the adhesion ring (green in [C] and shown by the black ring lacking signal in
[D]) at the contact site. Note, especially in (B) and (F), that different stages of granule polarization
and secretion are happening simultaneously with some granules already inserted between the
signaling patch and adhesion ring while others are still polarizing.
•
(G) A single confocal section demonstrating granule content protein (green) appearing on the target
cell (left) side of the CTL (right) membrane defined by the Lck (red) signal. The scale bars represent
10 É m (A, B, E, and F) and 4 É m (G).
Remaining Questions
•What controls alternate synapse assembly/raft clustering in thymocytes vs naïve T
cells?
•What controls raft clustering and transducer partitioning
throughout T cell development
PreTCR constitutively raft associated
naïve T cells have a significant fraction of their rafts
contained in intracellular vesicles, whereas as
activated effectors release them to the surface
TH1 are more raft dependent than TH2?
•Can engagement of costimulators/accesory molecules function
to modify arrangement within rafts/SMACs?
•How does synapse assembly influence costiimulator signaling?
•How are effectors specifically routed to the synapse?
•How does macromolecular assembly translate into alternate
signals (or is this just about receptor internalization
or directed secretion)?
•Relationship betwee rats microdaomins and SMACS
•More than one flavor of lipid raft microdoamain?
Alternate organizers of membrane microdoains (sugar packing)