Harder, Thomas

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Frontiers in Immunology

The T cell plasma membrane lipid bilayer stages TCR-proximal signaling events

Thomas Harder

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IntroductIon
The T cell plasma membrane lipid bilayer 
attracts the attention of immunologists as 
fundamental two-dimensional platform 
for molecular networks which mediate 
T cell antigen receptor (TCR) activation 
responses. Plasma membrane lipids criti-
cally influence formation and activity of 
signaling networks linked to the T cell 
plasma membrane. This opinion arti-
cle outlines recent advances in under-
standing functional implications of their 
organization into highly dynamic raft lipid 
nanoassemblies which can be triggered to 
coalesce into ordered raft membrane phases 
and then serve as functional platforms for 
multimolecular TCR signaling machineries. 
Raft lipid dynamics are also incorporated 
into models of initiating TCR signal trans-
duction. Hence, including the biochemical 
and biophysical complexity of the plasma 
membrane lipid bilayer is critical for sys-
tematic characterization of TCR-proximal 
signaling functions.
FunctIonal plasma membrane 
organIzatIon FollowIng t cell 
actIvatIon
T cell activities are induced by TCR signals 
which are triggered by its interaction with a 
cognate peptide MHC (pMHC) ligand pre-
sented on the surface of an antigen-present-
ing cell (APC) or target cell. TCR-proximal 
signals are passed on from plasma mem-
brane-associated signaling TCR microclus-
ters to cytoplasmic and nuclear activities 
which mediate downstream immunological 
T cell effector functions.
A highly organized immunological syn-
apse (IS) between the T cell and the cognate 
cell is marked by segregation of functionally 
distinctive supramolecular activation clus-
ters (SMACs) into domains of the T cell 
plasma membrane (Huppa and Davis, 2003). 
Spatial organization of TCR activation in 
the contact region of the T cell with T cell-
activating membrane surface was followed 
by video microscopy. These studies noted 
on-going TCR-proximal signaling in TCR 
microclusters which form in the periphery 
of the IS and are then transported centrip-
etally. At the central SMAC TCR-proximal 
signaling is attenuated and TCR signaling 
complexes are tagged for endocytosis into 
the degradative lysosomal pathway (Campi 
et al., 2005; Varma et al., 2006).
Dynamic TCR signaling clusters in the 
plasma membrane were initially imaged by 
confocal microscopy of Jurkat T cells plated 
on glass coverslips coated with αCD3 TCR-
activating antibody (Bunnell et al., 2002). In 
later studies, interactions of plasma mem-
brane-anchored signaling marker proteins 
with antibody-induced TCR signaling clus-
ters were assessed by tracking translational 
trajectories of single protein molecules and 
measuring their diffusional retardation at 
TCR signaling domains. These studies 
highlighted signaling-induced protein/
protein interactions as important drivers 
of TCR plasma membrane signaling clus-
ters formation, while raft lipid-dependent 
interactions were not observed (Douglass 
and Vale, 2005). A related approach was 
used to determine diffusion coefficients of 
inner leaflet raft-anchored proteins relative 
to TCR signaling clusters. In these studies 
diffusion properties of membrane-linked 
marker proteins did indicate contributions 
of raft lipid- and actin-dependent interac-
tions to diffusional retardation of cyto-
plasmic leaflet membrane raft-anchored 
proteins in the vicinity of TCR signaling 
clusters (Ike et al., 2003).
nanometer-scale raFt lIpId 
connectIvIty and coalescence oF 
condensed, ordered raFt 
membrane phase at tcr sIgnalIng 
domaIns
Insight into highly dynamic, nanometer-
scale sphingolipid/cholesterol raft assemblies 
in the plasma membrane of unstimulated 
cells was achieved by approaches which 
circumvent the diffraction-limit in resolv-
ing power of conventional lens-based light 
microscopy (Hell, 2009). Using these tech-
nologies it was possible to track dynamic 
nanoscale interactions of raft lipids in the 
plasma membrane (Eggeling et al., 2009). 
New strategies in correlation spectroscopy 
were used by He, Marguet and colleagues 
to show that phosphatidylinositol-3,4,5-
triphosphate (PIP
3
)-dependent Akt/PKB 
activation at the plasma membrane is con-
trolled by nanoscale raft lipid-interactions 
in the cytoplasmic leaflet of the lipid bilayer 
(Lasserre et al., 2008).
Physical structure of TCR plasma mem-
brane domains following receptor activa-
tion was monitored using fluorescent 
probes Laurdan and di-4-ANEPPDHQ 
which report on relative packing density 
of lipid membranes. These dyes revealed 
condensation of the plasma membrane 
at the IS which depended on TCR sign-
aling and intact actin cytoskeleton (Gaus 
et al., 2005; Owen et al., 2010). Dense 
packing of these T cell plasma membrane 
sites indicated coalescence of ordered raft 
lipid domains at sites of TCR activation. 
In support of this notion, TCR signaling-
dependent membrane condensation was 
perturbed by incorporation of ordered 
membrane phase-disrupting lipid com-
pounds; 7-ketocholesterol and polyun-
saturated fatty acid (PUFA), into T cell 
membranes (Rentero et al., 2008; Zech 
et al., 2009). Coalescence and stabilization 
of ordered bilayer phases at TCR signaling 
plasma membrane domains is mediated 
by a vast molecular network of signaling 
proteins which was recently character-
ized by proteomic analyses (De Wet et al., 
2011). Interestingly, disruption of plasma 
membrane condensation at TCR signal-
ing sites correlated with reduced tyrosine 
phosphorylation in TCR signaling clusters 
and impeded assembly of TCR signaling 
protein complexes in the plasma mem-
brane (Rentero et al., 2008), indicating 
The T cell plasma membrane lipid bilayer stages TCR-proximal 
signaling events
Thomas Harder*
35510 Butzbach, Germany
*Correspondence: tharder@faciendum.org
www.frontiersin.org March 2012 | Volume 3 | Article 50 | 1
OpiniOn Article
published: 20 March 2012
doi: 10.3389/fimmu.2012.00050
contributions of both, ordered-phase lipid 
membrane and signaling protein networks, 
to the generation of TCR signaling clusters.
New mass spectrometric technology 
was developed which quantitatively charts 
molecular lipid composition of complex 
membranes. Mammalian cell membranes 
are made up of cholesterol, and thousands 
of glycerophospholipid- and sphingolipid-
species which differ in their polar head-
groups, their fatty acid-positions, -lengths, 
and -saturation (Shevchenko and Simons, 
2010). Charting the lipidome of isolated 
TCR signaling plasma membrane domains 
revealed that physical condensation of TCR 
signaling plasma membrane was indeed 
mirrored in its lipid chemistry. The TCR 
signaling lipidome accumulated molecular 
raft lipids; cholesterol, saturated phosphati-
dylcholine (PC) species, and sphingolipids 
(Zech et al., 2009). As interesting additional 
feature, the raft lipidome at TCR activation 
sites accumulates inner leaflet glycerophos-
pholipid phosphatidylserine (PS) (Zech 
et al., 2009). A correlation between densely 
packed raft lipid domains and accumula-
tion of phosphatylserine was observed in 
ordered raft lipid membranes of other sys-
tems (Lorizate et al., 2009; Fairn et al., 2011).
Influence of PS on the structure of 
TCR/CD3 receptor complex was studied 
by in vitro analyses. These studies showed 
that a peptide corresponding to the cyto-
plasmic portion of TCR ζ-chain interacts 
with liposomes composed of negatively 
charged glycerophospholipids like PS and 
then adopts helical structure and is refrac-
tory to tyrosine phosphorylation (Aivazian 
and Stern, 2000). Similarly it was found that 
CD3ε cytoplasmic region specifically inter-
acts with PS via basic amino acid residues in 
the CD3ε cytoplasmic protein segment (Xu 
et al., 2008). It was proposed that this inter-
action represents a safety switch to avoid 
erroneous TCR/CD3 tyrosine phosphoryla-
tion under resting T cell conditions (Kuhns 
and Davis, 2008). This proposal was subject 
to discussion following reports that tyros-
ine phosphorylation of CD3ε cytoplasmic 
domain did not increase when these basic 
residues were mutated (Fernandes et al., 
2010; Gagnon et al., 2010).
A role of PS in generation of signaling pro-
tein membrane networks at TCR triggering 
sites was suggested by reconstructing LAT-
anchored TCR lipid/signaling protein net-
work in vitro: tyrosine phosphorylated LAT 
was recombinantly expressed as membrane-
anchored variant in insect cells and inserted 
into liposomes. Nucleation of cooperative 
multimolecular signaling protein networks 
around these phosphotyrosineLAT-proteoli-
posomes effectively occurred on membranes 
which were composed of PS- but not of 
PC-lipid. This indicated the fundamental 
importance of the lipid bilayer platform 
and its composition for the nucleation of 
LAT-anchored TCR downstream signaling 
complexes (Sangani et al., 2009).
ImplIcatIon In tcr trIggerIng 
mechanIsms
In vitro reconstitution is indeed a power-
ful approach to reduce the complexity of 
collective systems to isolated features. This 
has also been performed with great success 
in model lipid membranes which feature 
cholesterol-dependent segregation of liquid 
ordered (l
o
) and liquid disordered (l
d
) phases 
(Ahmed et al., 1997). Immiscibility of arti-
ficial l
o
- and l
d
-phases in model membranes 
recapitulates (but cannot be equated with 
Kaiser et al., 2009) segregation of ordered 
raft- and disordered non-raft phases in cell 
plasma membranes. It was shown that in iso-
lated cell plasma membrane vesicles at 37°C 
connectivity of raft lipids does not suffice 
to segregate bulk lipid membrane phases. 
However, segregation of ordered and dis-
ordered phases in cell plasma membrane 
vesicles could be induced by choleratoxin 
B subunit (CTB)-mediated crosslinking 
of raft GM1 ganglioside lipid component 
(Lingwood et al., 2008). These results suggest 
that resting cell plasma membranes are held 
below a critical threshold above which raft 
lipid connectivity causes bulk phase separa-
tion at physiological conditions. Segregation 
of ordered and disordered phases can then 
be triggered by passing this threshold with 
relatively small shifts in membrane status, 
for example by pentavalent crosslinking of 
GM1 using CTB. Hence, it can be envisioned 
that ligand-induced alterations of TCR con-
figuration in the plasma membrane bilayer 
locally induces ordering of its lipid envi-
ronment. This causes coupling of ligand-
engaged TCR to membrane-anchored Lck 
Src-family kinase which prefers an ordered 
lipid bilayer environment (Janes et al., 1999; 
Ike et al., 2003). Increased accessibility of 
Lck kinase to ligand-engaged TCR com-
plex would then cause ITAM phosphoryla-
tion and receptor activation. Indeed, it was 
recently shown that a significant fraction of 
Lck kinase in T cells is in its active form prior 
to TCR activation and will phosphorylate its 
ITAM substrate in the TCR/CD3 complex 
once it gains access (Nika et al., 2010).
outlook
Induction of ordered raft phase by ligand 
engagement has been put forward as cen-
tral element of models describing ini-
tial TCR triggering (van der Merwe and 
Dushek, 2011). Testing this experimentally 
poses the challenge of comparing structure 
of TCR lipid bilayer environment prior to 
and post ligand engagement and moni-
toring how ligand engagement causes raft 
phase coalescence in the immediate, pos-
sibly nanometer range TCR environment. 
This needs to be experimentally discrimi-
nated from segregation of micrometer 
scale ordered raft phase shown to be a 
consequence of TCR signaling at T cell 
activation domains.
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Received: 28 November 2011; accepted: 29 February 2012; 
published online: 20 March 2012.
Citation: Harder T (2012) The T cell plasma membrane 
lipid bilayer stages TCR-proximal signaling events. Front. 
Immun. 3:50. doi: 10.3389/fimmu.2012.00050
This article was submitted to Frontiers in T Cell Biology, a 
specialty of Frontiers in Immunology.
Copyright © 2012 Harder. This is an open-access article 
distributed under the terms of the Creative Commons 
Attribution Non Commercial License, which permits 
non-commercial use, distribution, and reproduction in 
other forums, provided the original authors and source 
are credited.
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www.frontiersin.org March 2012 | Volume 3 | Article 50 | 3
Harder Raft dynamics in TCR triggering


The T Cell Plasma Membrane Lipid Bilayer Stages TCR-proximal Signaling Events

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The T Cell Plasma Membrane Lipid Bilayer Stages TCR-proximal Signaling Events

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