* The Section on Lymphocyte Signaling, The Unit of Organelle Biology, Cell Biology and Metabolism Branch, National
Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
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Abstract |
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The nonreceptor protein tyrosine kinase
ZAP-70 is a critical enzyme required for successful T
lymphocyte activation. After antigenic stimulation,
ZAP-70 rapidly associates with T cell receptor (TCR)
subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the
TCR chain expressed as a chimeric protein (TT
and
T
), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using
ZAP-70 and T
fused to green fluorescent protein
(ZAP-70 GFP and T
-GFP, respectively). Time-lapse
imaging using confocal microscopy indicated that the
activation-induced redistribution of ZAP-70 to the
plasma membrane, after a delayed onset, is of long duration. The presence of the TCR
chain is critical for
the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed.
Binding specificity to TT
was indicated using mutant
ZAP-70 GFPs and a truncated
chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. T
-
GFP is relatively immobile, while peripherally located
ZAP-70 in stimulated cells is less mobile than cytosolic
ZAP-70 in unstimulated cells, a phenotype confirmed
by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided
new insights into the TCR
-ZAP-70 interaction and
will be a powerful tool for continuing studies of lymphocyte activation.
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Introduction |
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ENGAGEMENT of the T cell receptor (TCR)1 by antigenic ligand, in the form of a short linear peptide
bound in the cleft of a major histocompatibility
complex (MHC) class I or II molecule, is the critical binding event leading to T cell activation (Babbitt et al., 1985;
Townsend et al., 1986
; Bentley and Mariuzza, 1996
; Garcia
et al., 1996
). The TCR is comprised of multiple integral membrane proteins (Jorgensen et al., 1992
; Weiss, 1993
;
Weissman, 1994
) and serves to initiate intracellular signaling, leading to new gene expression, protein synthesis, induction of multiple effector functions, and clonal expansion (Samelson and Klausner, 1992
; Weiss and Littman,
1994
). The
heterodimer binds to the antigen-MHC
ligand, and the CD3
,
,
, and TCR
chains translate this
event into biochemical signals within the cell (Cantrell, 1996
; Garcia et al., 1996
; Wange and Samelson, 1996
; Qian
and Weiss, 1997
). Since none of these molecules contains
any intrinsic enzymatic activity, they recruit and bind signaling proteins via their conserved immunoreceptor tyrosine-based activation motifs (ITAMs), which are present
as a single copy in each of the CD3 chains and in triplicate
in TCR
(Reth, 1989
; Weiss and Littman, 1994
; Wange
and Samelson, 1996
). The CD3 and TCR
chains are phosphorylated on the tyrosines within their ITAMs within
seconds of TCR engagement by the Src kinases Lck and
Fyn (Iwashima et al., 1994
; van Oers et al., 1996
; Sloan-Lancaster and Samelson, 1998
). The phospho-ITAMs are
then able to bind SH2 domain-containing proteins, allowing a multiprotein complex to form under the membrane,
which includes enzymes and adaptors responsible for triggering the various intracellular signaling pathways for successful T cell activation (Weiss and Littman, 1994
; Wange
and Samelson, 1996
).
ZAP-70, a nonreceptor protein tyrosine kinase expressed exclusively in T cells, thymocytes, and natural
killer cells, is a critical enzyme in early T cell signaling
(Chan et al., 1992; Wange et al., 1992
; Arpaia et al., 1994
;
Chan et al., 1994
; Elder et al., 1994
; Negishi et al., 1995
).
After binding via its tandem SH2 domains to the two
phosphotyrosines of an individual ITAM during TCR engagement (Wange et al., 1993
), ZAP-70 is phosphorylated by Lck and/or Fyn and is thus activated (Iwashima et al.,
1994
; Wange et al., 1995a
; Kong et al., 1996
). Subsequently, these kinases phosphorylate other specific substrates, resulting in the activation of the various intracellular signaling pathways required for T cell function.
Although the absolute requirement of functional ZAP-70
for T cell activation has been clearly demonstrated both biochemically and genetically (Wange et al., 1995b
; Qian
et al., 1996
; Williams et al., 1998
), few studies have examined its intracellular localization and how this is affected
by cellular stimulation. The primary structure predicts that
ZAP-70 is a cytosolic protein (Chan et al., 1992
), and biochemical data have shown that it rapidly translocates to
the TCR upon activation (Wange et al., 1992
; Chan et al.,
1991
). We have recently developed a cellular approach to
examine the location and movement of ZAP-70 in single
cells over real time, using a chimera of ZAP-70 fused to
the green fluorescent protein (GFP) and time-lapse imaging confocal microscopy (Sloan-Lancaster et al., 1997
).
Our initial study revealed that ZAP-70 GFP was present
not only throughout the cytosol but also in the nucleus, in
both transiently transfected COS 7 cells and ZAP-70-deficient T cells stably reconstituted with the chimera. In COS
7 cells, ZAP-70 GFP rapidly moved from the cytosol to the
cell surface in response to pharmacological stimulation.
This was surprising since COS 7 cells do not express any
TCR chains or other molecules known to contain ITAMs.
We reasoned that another membrane-associated protein,
which becomes tyrosine phosphorylated upon cellular
stimulation, was able to bind ZAP-70 in order for this
translocation and apparent binding to occur (Sloan-Lancaster et al., 1997
).
Since the current model of T cell activation dictates that
ZAP-70 is bound and concentrated at the region of activated TCR via a specific interaction with the phosphorylated ITAMs of TCR subunits, we wanted to refine our experimental system to study this association. This would
enable us not only to assess the real time binding kinetics,
but also to demonstrate the fine specificity of the molecular interaction in individual living cells. In addition, we
wanted to measure the mobility of ZAP-70 in the different
intracellular compartments to understand the mechanisms
of retention at the plasma membrane. Here we report the
stimulation-dependent translocation of ZAP-70 to the cell
surface in HeLa cells is dependent on expression of a chimeric TCR chain. We describe the kinetics of this interaction and show that ZAP-70 translocation is enhanced by
coexpressing active Lck. Moreover, we provide evidence
that relocated ZAP-70 is specifically bound to the chimeric
chain, with properties that correspond precisely
with the data generated biochemically (Wange et al., 1993
;
Koyasu et al., 1994
). Using photobleaching techniques, we
have revealed the highly mobile and freely diffusible nature of cytosolic and nuclear ZAP-70 and its conversion to
a more static state accompanying its translocation to the
cell periphery. Cell surface-located ZAP-70 is more diffusible than TCR
, a transmembrane protein, a phenotype
confirmed by calculating the diffusion constants for the individual proteins, which indicated that peripheral ZAP-70 diffuses 20-fold faster than TCR
. Such observations suggest that the interaction between ZAP-70 and TCR
upon
cellular stimulation is dynamic.
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Materials and Methods |
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Cells, Antibodies, and Reagents
HeLa cells were grown in complete D10 medium (DME containing 10%
FBS, 2 mM glutamine, and 50 µg/ml gentamicin). All stably transfected
lines were cultured in complete D10 medium supplemented with 1 mg/ml
geneticin (G418; GIBCO BRL, Gaithersburg, MD) for maintenance of
transgene expression. H/TT, H/T
, and H/T
truncated (H/T
trunc)
cells stably expressed the appropriate fusion protein as determined by frequent FACS® and immunoprecipitation analyses.
mAbs used include anti-IL-2 receptor chain, 33B3.1 (Immunotech,
Inc., Westbrook, ME), for FACS® analysis; rabbit anti-ZAP-70 antiserum
(Wange et al., 1995a
); mouse anti-human
-tubulin (Sigma Chemical Co.,
St. Louis, MO); rhodamine-coupled goat anti-mouse IgG; and fluorescein-coupled goat anti-rat IgG (KPL, Inc., Gaithersburg, MD).
Plasmids, Constructs, and Transfection
The generation of pSXSR-Lck F505, pEGFP/ZAP-70, and pEGFP/
kin.neg. (with deleted kinase domain) ZAP-70 have been described previously (Wange et al., 1995a
; Sloan-Lancaster et al., 1997
). pEGFP/double
SH2 was made by ligating the NheI/XmnI fragment from pEGFP/ZAP-70, containing both SH2 domains and both interdomains, to the NheI/
SmaI-digested pEGFP-N1 vector. For construction of pEGFP/ZAP-70
kin.dom. (expressing the kinase domain alone), the NheI/XmnI fragment
was removed from pEGFP/ZAP-70, and the vector was religated using
the oligos 5' CTA GCA CCG GTG GAT CCT CTA GAA TGA AGC 3'
and 5' GCT TCA TTC TAG AGG ATC CAC CGG TG 3'. For pEGFP/
SH2(C) + kin.dom., the NheI/KpnI fragment of pEGFP/ZAP-70 was removed, and the vector religated using the oligos 5' CTA GCG ATA TCA
TGC CAG ACC CCG CGG CGC ACC TGC CCT GGT AC 3' and 5'
CAG GGC AGG TGC GCC GCG GGG TCT GGC ATG ATA TCG 3'.
pEGFP/SH2(N) + kin.dom. was made in two steps. First, an intermediate
vector, ZAP 1 + 2, encoding the kinase domain alone with an inserted
KpnI site, was derived by annealing the NheI/XmnI-digested pEGFP/
ZAP-70 to the oligos 5' CTA GCG ATA TCT GCA GGG TAC CTC
GAG AAG C 3' and 5' GCT TCT CGA GGT ACC CTG CAG ATA
TCG 3'. The NheI/KpnI fragment of pEGFP/ZAP-70, encoding the NH2-terminal SH2 and interdomain 1, was then ligated to the NheI/KpnI-cut
ZAP 1 + 2. pEGFP/T
was constructed as follows: the EcoRI/BamHI
fragment from pXSSR
/T
(Letourneur and Klausner, 1991
) was ligated to EcoRI/BamHI-digested pEGFP-N1 vector to create the intermediate plasmid pEGFP/T
/BamHI. A PCR fragment from the BamHI
site of T
was created with an introduced COOH-terminal AgeI site, using the oligos 5' GCA GGG ATC CAG AGA TGG GAG GC 3' and 5'
GAC GAC CGG TGA GCG AGG GGC CAG GGT CTG 3'. Then
BamHI/AgeI-digested PCR product and BamHI/AgeI-digested pEGFP/
T
/BamH1 vector were ligated together to produce the final construct,
pEGFP/T
. The construction of the TT
, T
, and T
trunc chimeras
have been described elsewhere (Letourneur and Klausner, 1991
). All
three contain the extracellular domain of the human IL-2 receptor
chain
and the intracellular domain of TCR
. TT
includes the transmembrane region of the IL-2 receptor
chain, while T
contains the transmembrane
region of TCR
. T
trunc is a shortened form of the latter chimera, terminated after TCR
amino acid residue 65, and thus lacks all three ITAMs
(Letourneur and Klausner, 1991
). HeLa cells, or their stably transfected
counterparts, were electroporated using 15 µg of each DNA construct at
250 V and 500 µF using a Gene Pulser (Bio-Rad Labs., Hercules, CA) and used 20-24 h after transfection.
Immunofluorescence Staining
HeLa cells were grown overnight on sterile glass coverslips (10-mm diameter, No. 1 thickness). Cells, untreated or pretreated with nocodazole (33 µM, 30 min incubation at 4°C, followed by 30 min at 37°C), were then fixed in 3.7% paraformaldehyde in PBS for 30 min at room temperature, washed (three times) in PBS containing 10% fetal bovine serum (PBS/ FBS), permeabilized using 0.1% Triton X-100 in PBS for 4 min at room temperature, washed (three times), and incubated for 45 min in PBS/FBS for preblocking. Cells were then incubated with a mouse anti-human tubulin Ab in PBS/FBS for 45 min at room temperature, washed, and incubated with rhodamine-coupled goat anti-mouse IgG for 45 min, followed by washing with PBS (three times). The coverslips were then mounted onto glass slides using Fluoromount G (Southern Biotechnology Associates, Inc., Birmingham, AL) and viewed using the 568-nm laser line of a confocal laser scanning microscope (model LSM 410; Carl Zeiss, Inc., Thornwood, NY) with a 100× planapochromat oil immersion objective (NA 1.4) and optics for rhodamine.
Fluorescence Microscopy, Time-Lapse Imaging, and Image Processing
Transfected cells were grown overnight in coverglass chambers (LabTek,
Naperville, IL) in complete D10 medium. For time-lapse imaging experiments, the slides were mounted on a custom-made platform (of a confocal
laser scanning microscope; Yona Microscope and Instrument Co., Rockville, MD) equipped with a triple line Kr/Ar laser, a 100× 1.4 NA Planapochromat oil immersion objective, a 25× 0.8 NA Neofluar immersion
corrected objective, and a temperature-controlled stage. Time-lapse sequences were recorded with macros programmed with the Zeiss LSM
software package that allow autofocusing on the coverslip surface in reflection mode before taking confocal fluorescence images. The media was
replaced by PBS supplemented with magnesium and calcium salts before
the start of imaging. In Fig. 4 d, cells were treated with nocodazole as
above before beginning the time-lapse imaging. Two images of each cell
were taken before addition of the pervanadate (PV) stimulant directly to
the chambered coverglass, and subsequent images were taken at 30-s intervals thereafter until 15 min after stimulation, as previously described
(Sloan-Lancaster et al., 1997).
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Photobleaching Experiments
Fluorescence loss in photobleaching (FLIP) experiments were performed
at room temperature on a custom-made stage of a confocal microscope
(model LSM 410; Carl Zeiss, Inc.) using the 63× objective and the 488-nm
line of a 400-mW Kr/Ar laser, which delivered 0.9 mW power (Cole et al.,
1996). In brief, HeLa cells expressing ZAP-70 GFP alone, ZAP-70 GFP
together with Lck F505, or T
GFP were left untreated or stimulated
with PV for 12 min before beginning the FLIP experiment, as indicated in
figure legends. A small rectangular region defined by the boxed area was
repeatedly illuminated with the laser at 100% power, 100% transmission.
Between each intense illumination, the entire field of view was imaged at
low-power laser light (20% power, 1% transmission) to assess the extent of loss of fluorescence outside the box as a consequence of photobleaching within the box. The time lapse between images was ~25 s. The possibility that regions on the edge of the illuminated box are progressively bleached by light leakage during FLIP was ruled out by repeating FLIP
identically on fixed cells, which showed bleaching only in the area exposed
to illumination. Furthermore, there was no significant photobleaching
while imaging the recovering cell since control cells in the field did not
lose any significant fluorescence intensity during the time followed.
Fluorescence recovery after photobleaching (FRAP; Edidin, 1994) was
performed at room temperature on a confocal microscope (model LSM
410; Carl Zeiss, Inc.) essentially as described (Ellenberg et al., 1997
). For
the qualitative D measurements shown in Fig. 7, cells were stimulated for
12 min before commencement of photobleaching. The width of the rectangular regions of interest used were 2 µm (T
GFP, some ZAP-70 GFP)
or 4 µm (ZAP-70 GFP). Fluorescence within the strip was measured at
low laser power (20% power, 1% transmission) before the bleach (prebleach intensity) and then photobleached with full laser power (100%
power, 100% transmission) for 0.218 s (T
GFP) or 0.436 s (ZAP-70
GFP) (which effectively reduced the fluorescence to background levels in
fixed material). Recovery was followed after 2 s with low laser power at
2-s intervals for 200 s (T
GFP) or 1-s intervals for 50 s (ZAP-70 GFP)
and then at 10-s intervals until the recovered fluorescence intensity within
the strip had reached a plateau. Zero of time t, taken as the midpoint of
the bleach, was 2.399 s for T
GFP and 2.513 s for ZAP-70 GFP. Numerical simulations were used to determine D using the prebleach intensity of
entire cells (to assess the effects of geometry and nonuniform fluorescence
density) and compared with experimentally derived D values, as described
(Ellenberg et al., 1997
; Sciaky et al., 1997
).
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Results |
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Redistribution of ZAP-70 to the Plasma Membrane in HeLa Cells Requires Both Cellular Activation and Coexpression of a TCR Chain
In a previous report, we made use of a ZAP-70 GFP chimera to study the intracellular location of this protein tyrosine kinase, and how it changed in response to cellular
stimulation, using time-lapse imaging (Sloan-Lancaster
et al., 1997). This approach revealed the very rapid redistribution of cytosolic ZAP-70 to the cell surface, with significant membrane accumulation detected as early as 1 min after stimulation. The phenotype was enhanced when
an active form of Lck was coexpressed, which itself induced some ZAP-70 translocation. These results were
somewhat surprising since the COS 7 cells, in which the
chimeric ZAP-70 GFP was expressed, do not contain any
TCR chains. We were therefore curious to determine whether the introduction of a TCR chain in this experimental system had any observable effect on the redistribution of ZAP-70.
To compare results in the presence or absence of a TCR
chain, we made use of the HeLa cell line, which lacks any
TCR chains, and its transfected derivative, H/TT, which
stably expresses a chimeric form of TCR
comprised of
the extracellular and transmembrane portions of the human IL-2 receptor
chain, Tac, fused to the entire intracellular region of TCR
(Letourneur and Klausner, 1991
).
The fusion protein is successfully expressed on the cell surface as an integral membrane protein independent of any
other TCR component and provides an experimental system in which the contribution of the tandem ITAMs of
TCR
can be examined apart from the CD3 molecules
(Letourneur and Klausner, 1991
). In non-T cells, cross-linking TT
at the cell surface does not induce cellular activation. Thus, to stimulate the cells, we used the pharmacological agent pervanadate (PV). PV inhibits intracellular
phosphatases, thereby creating a steady state in which tyrosine residues are phosphorylated normally but not dephosphorylated and which is used as a surrogate for antigen or anti-TCR cross-linking (O'Shea et al., 1992
; Secrist
et al., 1993
). Both HeLa and H/TT
cells were transfected
with ZAP-70 GFP, and time-lapse imaging was used to
monitor the movement of the chimeric fluorescent molecule in response to pharmacological stimulation. Two images were taken before PV addition, with subsequent images taken at 30-s intervals thereafter.
Little if any ZAP-70 redistributed to the plasma membrane in HeLa cells with PV stimulation (Fig. 1, top row),
while significant membrane accumulation was evident as
early as 2 min after stimulation in COS 7 cells (Sloan-Lancaster et al., 1997). The lack of a similar phenotype in
HeLa cells suggests that no phosphotyrosine- or ITAM-containing proteins capable of binding ZAP-70 are expressed in these cells. Thus, in the absence of a TCR chain,
pharmacological stimulation had little effect on ZAP-70
redistribution in HeLa cells. However, when H/TT
cells
were stimulated with PV, ZAP-70 dramatically redistributed to the cell surface in all cells examined (Fig. 1, second
row). Unlike its rapid accumulation to the plasma membrane in COS 7, there was a significant delay in H/TT
, with
little detectable redistribution until 8-10 min after stimulation. At this time, movement to the plasma membrane,
accompanied by cytosolic clearing, continued steadily
around each cell until ~15 min after stimulation, when
ZAP-70 was uniformly distributed over the inner surface.
Results from a semiquantitative analysis of individual cells
confirmed this phenotype, in which mean fluorescence intensity in a region of interest (ROI) over the center of
each cell (cytosol and plasma membrane) was compared
with an ROI at the edge (plasma membrane). These data
indicated that there was, on average, a 1.1-fold increase in
the surface to cytosolic fluorescence ratio 4 min after stimulation, which increased to 1.5-fold by 8 min and to 3.1-fold by 15 min after PV addition. The uniform distribution around the cell surface was consistent with ZAP-70 binding specifically to the chimeric TT
molecule, which is localized throughout the plasma membrane under these
conditions (data not shown).
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Lck F505 Enhances ZAP-70 GFP Movement to the Cell Surface
The current model of physiological early T cell signaling
suggests that ZAP-70 binds to the ITAMs of TCR only
after they have been phosphorylated by Lck and/or Fyn
(Iwashima et al., 1994
; Wange and Samelson, 1996
; Qian
and Weiss, 1997
). Our earlier studies in COS 7 suggested
that expression of active Lck followed by PV stimulation
enhances the redistribution of ZAP-70 to the cell surface
over that induced by PV alone (Sloan-Lancaster et al., 1997
). Thus, we performed time-lapse imaging in cells coexpressing the constitutively active Lck F505 to determine
if this kinase influenced the recruitment of ZAP-70 and its
binding to TCR
. In HeLa cells, the coexpression of Lck
F505 had no apparent effect on the location of ZAP-70 before or after PV stimulation, since ZAP-70 was not detected at the plasma membrane at any time point (Fig. 1,
third row). This further confirmed the lack of other ZAP-70-binding proteins in HeLa cells, and thus the specificity of the interaction with the ITAMs in H/TT
. With the expression of the chimeric TCR
chain, Lck F505 enhanced
the redistribution of ZAP-70 to the cell surface, and presumably its binding to TT
(Fig. 1, bottom row). The time-lapse imaging results suggested that Lck F505 kinetically
enhanced the accumulation of ZAP-70 at the cell surface.
ZAP-70 accumulation was detected at the plasma membrane as early as 4-5 min after stimulation, reaching a plateau by 8 min (Fig. 1, bottom row). This was determined in
several cells by comparing fluorescence intensities within
ROIs over the center of the cell (cytoplasm and plasma
membrane) and at the edge (plasma membrane only) with
time. These data showed an increase in fluorescence intensity at the plasma membrane at a much earlier time after
stimulation in cells coexpressing Lck F505. However, there was no apparent quantitative enhancement by Lck
F505, since surface fluorescence increased threefold compared with cytosolic levels whether or not Lck F505 was
coexpressed.
The Specificity of ZAP-70-TT Interaction
The above experiments indicated that, in the HeLa system, the specific molecular interaction of ZAP-70 and TT
in response to cellular stimulation could be monitored at
the single cell level. We next used this assay system to assess the basis of this interaction. To do so, we constructed
various chimeras consisting of mutant ZAP-70 with GFP
and used them to determine the contribution of individual
protein domains in the translocation and binding of the kinase to TT
(Fig. 2). Lck F505-expressing H/TT
cells were cotransfected with the indicated ZAP-70 GFP mutant, and cells were monitored before and after addition of
PV. Accumulation of ZAP-70 at the cell surface was monitored in response to PV stimulation in cells expressing
wild-type ZAP-70 (Fig. 2 a, unstimulated, and b, 15 min
PV). A similar pattern of redistribution was observed using a kinase-dead form of ZAP-70 (Fig. 2 c, unstimulated,
and d, 15 min PV, and data not shown), and an analysis of
multiple cells in several experiments indicated that the kinetics and amount of wild-type and kinase-dead ZAP-70
translocated to the cell surface did not differ significantly
(data not shown). These data confirm that the kinase activity of ZAP-70 is not necessary for its binding to TCR
(Wange et al., 1993
; Hatada et al., 1995
).
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We next assessed the roles of the individual protein domains of ZAP-70 in its binding to TCR. While a mutant
ZAP-70 GFP, containing the tandem SH2 domains without the kinase domain, redistributed to the cell surface
with kinetics indistinguishable from those of the entire
molecule (Fig. 2 e, 15 min PV, and data not shown), neither SH2 domain expressed alone with the kinase domain
moved to the plasma membrane (Fig. 2 g, NH2-terminal
SH2, and h, COOH-terminal SH2, 15 min PV). Moreover,
the kinase domain by itself could not bind to TT
(Fig. 2 f,
15 min PV). These data agree with biochemical evidence
showing that the tandem SH2 domains of ZAP-70, but not the kinase domain, are absolutely required for a stable
interaction with any individual phosphorylated ITAM
(Wange et al., 1993
; Iwashima et al., 1994
; Koyasu et al.,
1994
).
A similar in vivo analysis of the molecular properties of
required to bind to ZAP-70 was also undertaken. For
this, two additional HeLa cell lines, which stably express
distinct forms of the
chimera, were used. The first expresses T
, which differs from TT
in that its transmembrane domain is derived from TCR
instead of from Tac.
This ensures that T
is expressed as a disulfide-linked homodimer on the cell surface. The second expresses a
shorter form of T
, called T
trunc, terminated after
amino acid 65 and resulting in a homodimer with no
ITAMs (Letourneur and Klausner, 1991
). A schematic of
the structures of these
chimeras is shown in Fig. 3 A. We
tested the ability of these
chimeras to acquire phosphotyrosine, since only phospho-ITAMs can bind ZAP-70 biochemically (Bu et al., 1995
; Isakov et al., 1995
). The
chimeras were examined for phosphotyrosine content before
or after PV stimulation of the respective cell lines. As expected, only H/TT
and H/T
, but not HeLa or H/T
trunc, displayed a phosphoprotein at the apparent molecular weight for chimeric
, verifying the activation-dependent requirement of phosphorylation of chimeric
and the
lack of intramolecular phosphate-binding sites in T
trunc (data not shown). We also determined that the tyrosine kinase inhibitor, herbimycin A (1 µM), inhibited
stimulation-dependent cellular phosphorylation (data not
shown).
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H/T (Fig. 3 B, top and bottom rows) and H/T
trunc
(middle row) cells, expressing ZAP-70 GFP and Lck F505
and including herbimycin A (1 µM, bottom row), were PV
stimulated and monitored using digital imaging confocal
microscopy. As expected, cells expressing full-length T
were successful in recruiting ZAP-70 to the plasma membrane with kinetics similar to those defined earlier (Fig. 1).
In contrast, H/T
trunc cells showed no evidence of ZAP-70 redistribution to the cell surface even after 15 min PV
stimulation (middle row), similarly to HeLa cells without a TCR chain (Fig. 1, first and third rows). While herbimycin
A pretreatment prevented ZAP-70 redistribution to the
plasma membrane, there was some redistribution of the
chimera under these conditions (Fig. 3, bottom row). This
drug is likely causing several effects in the cells, many of
which may be tyrosine kinase dependent. However, the
lack of redistribution of ZAP-70 to the plasma membrane in the presence of herbimycin A is consistent with its inhibition of ITAM phosphorylation. Thus, the cytosolic tail
of TCR containing ITAMs and tyrosine phosphorylation
after activation were critical for movement of ZAP-70 to
the membrane. TT
and T
appeared to perform equivalently for ZAP-70 recruitment, suggesting that TCR
dimerization has no effect on the binding efficiency for
ZAP-70 in this system (data not shown).
Intact Microtubules or Actin Cytoskeleton Are Not Required for ZAP-70 Translocation to the Cell Surface
Biochemical studies on ZAP-70 have not addressed how
this kinase accumulates at the plasma membrane upon cellular stimulation. In fact, the model suggesting that it
moves from the cytosol to the cell surface has only been an
assumption. Our results confirm that translocation indeed
occurs. To explore the mechanism of this translocation, we
tested whether microtubules or the actin cytoskeleton
were required. H/TT cells were treated with nocodazole, which leads to disassembly of the intracellular microtubule
lattice. Antitubulin antibody staining of cells showed complete disassembly of the microtubule array in cells treated
with nocodazole (33 mM, Fig. 4 b), but not in untreated
cells (Fig. 4 a). Time-lapse imaging of ZAP-70 GFP- and
Lck F505-transfected H/TT
cells indicated that ZAP-70
translocated to the plasma membrane in response to cellular stimulation in both nocodazole-treated and untreated cells with similar kinetics (Fig. 4 d, nocodazole-treated,
and c, untreated, both 15 min PV, and data not shown).
Other cells from the population, treated identically, were
stained with antitubulin mAb to confirm that nocodazole
had disrupted the microtubule lattice. Moreover, treatment of cells with up to 100 µM nocodazole still did not
impair ZAP-70 translocation to the cell surface. In addition, the breakdown of actin filaments by cytochalasin B
had no effect on ZAP-70 redistribution (data not shown).
These data indicate that the microtubule array and the actin cytoskeleton are not required for the intracellular
translocation of ZAP-70.
Redistribution of ZAP-70 from the Cytosol to the Cell Surface Correlates with Its Conversion to a Less Mobile State
The ability to photobleach GFP chimeras makes them attractive tools for studying molecular dynamics in real time
proteins (Cole et al., 1996). Using photobleaching techniques, we initially took a qualitative approach to determine whether there were any gross changes in ZAP-70 diffusibility after its redistribution within the cell. For this, we
repetitively photobleached a small area within the cell and
looked for fluorescence loss in the entire cellular compartment due to diffusional exchange of unbleached with
bleached molecules (Cole et al., 1996
; Ellenberg et al., 1997
). The length of time required for fluorescence loss
under these conditions depends on the diffusional mobility
of the fluorescent protein and the extent of continuity of
the cellular compartments. This approach, termed FLIP,
was used to compare the dynamics of cytosolic and nuclear
ZAP-70 in resting H/TT
cells expressing ZAP-70 GFP
(Fig. 5 A). Both pools of ZAP-70 were extremely mobile, with a rapid loss of fluorescence in cells repetitively bleached in the cytosol and in the nucleus. Fluorescence was significantly depleted after only two bleaches (<60 s), and a
complete loss was apparent after five bleaches (Fig. 5 A,
bottom right cell). In cells in which the bleached region encompassed only the cytosol, nuclear ZAP-70 GFP remained detectable for a longer time period (Fig. 5 A, top
right cell). These data indicate that cytosolic and nuclear
ZAP-70 are both highly mobile and that they are not freely interchangeable with each other. Also, the coexpression of Lck F505 had no effect on the mobility of
ZAP-70 during the time frame of the experiment (Fig. 5,
compare B to A).
|
The time-lapse imaging studies showed that ZAP-70 redistributed from the cytosol to a more peripheral location
in TT-expressing cells in response to stimulation (Fig. 1).
This phenotype was biochemically consistent with a specific molecular interaction between ZAP-70 and the
chimera (Figs. 2 and 3). We wanted to determine if this shift
of ZAP-70 to the periphery correlated with a change in its
diffusional properties. H/TT
cells expressing ZAP-70
GFP and Lck F505 were stimulated with PV for 12 min before the commencement of FLIP to induce maximal ZAP-70 redistribution to the cell surface. After repetitive photobleaching of a region of the cell, it was obvious that the
diffusional mobility of ZAP-70 had been altered (Fig. 5
C). Specifically, surface-localized ZAP-70 was less mobile
and no longer able to exchange with the intracytoplasmic pool. A strong fluorescent signal was still apparent at the
end of the bleaching sequence (~300 s). There are two
possible explanations for this. Either all of the cytosolic
ZAP-70 redistributes to the cell periphery, or some remains in the cytosol but converts to a less mobile phenotype, perhaps because of an activation-induced interaction
with cytoskeletal proteins. The first hypothesis is the more
likely since the conversion to the less mobile state only occurs after stimulation of TT
-expressing cells, which is consistent with a molecular interaction between these two
molecules (data not shown). Regardless, activation-dependent redistribution of ZAP-70 to the cell surface is accompanied by a decrease in its mobility.
Membrane-associated ZAP-70 Is More Mobile
than TCR
Biochemical studies have indicated that cell surface-located
ZAP-70 is physically bound to TCR, via the tandem SH2
domains of ZAP-70 and the phospho-ITAMs of TCR
(Figs. 2 and 3) (Isakov et al., 1995
; Bu et al., 1995
). One
prediction from these data would be that peripherally located ZAP-70 would acquire the same diffusion mobility
as TCR
. We therefore compared the diffusion mobility of
the translocated ZAP-70 to that of the
chimera, T
. A
T
-GFP fusion protein was constructed and expressed in HeLa cells and was expressed exclusively on the cell membrane, as expected for an integral membrane protein (Fig.
6 C). H/TT
cells expressing ZAP-70 GFP alone or together with Lck F505 and HeLa cells expressing T
-GFP
were then stimulated with PV for 12 min, and FLIP was
performed to compare the mobilities of the two proteins at
the cell surface (Fig. 6). As before, peripheral ZAP-70 was
relatively stable, showing a similar phenotype whether Lck
F505 was coexpressed or not. However, when repetitive
photobleaching inside the rectangular box was continued,
complete loss of ZAP-70 GFP fluorescence outside the
box occurred between 750 and 870 s (Fig. 6, A and B). In
contrast, fluorescence of T
-GFP remained prominently
visible on the cell membrane after repetitive photobleaching for 30 min (Fig. 6 C, and data not shown). The mobility
of T
-GFP was not affected by PV stimulation since it
photobleached at a comparable rate in untreated cells.
Furthermore, when a TT
-GFP chimera was used, a similar phenotype was noted, indicating that
dimerization
has no effect on its mobility in the plasma membrane (data
not shown). Thus, it was apparent that the peripherally located ZAP-70 was more mobile than the integral membrane
chimera.
|
FLIP is limited to determining qualitative assessments
since the rate at which a cell loses its fluorescence signal is
a function of both the diffusion out of the surrounding areas to the bleach zone as well as the size of the bleach zone
relative to the entire cell. To better understand the nature
of the interaction between ZAP-70 and TCR, we wanted
to be able to make quantitative measurements of protein
diffusion. For this, we used the FRAP technique, in which
fluorescence recovery into a bleached region of the cell after a single photobleach is monitored until recovery is
complete (Edidin, 1994
; Ellenberg et al., 1997
; Lippincott-Schwartz, 1998). Experimental data are then plotted versus time and are fit to an empirical formula (Fig. 7 c) to
determine the diffusion coefficient, D, for the protein. A
representative plot of fluorescence recovery versus time is
shown for both T
-GFP and membrane-associated ZAP-70 GFP (Fig. 7 a). These data indicate that T
moves
slowly within the membrane, since a plateau of fluorescence recovery is not approached until >440 s for T
compared with about 120 s for ZAP-70. Moreover, the D
value derived for T
indicated that it diffused slowly in
the membrane and was similar to those derived for other
plasma membrane proteins (Fig. 7 b; 0.011 ± 0.001 µm2/s)
(Pal et al., 1991
). However, the D value derived for peripherally located ZAP-70 was >20-fold higher (Fig. 7 b;
0.234 ± 0.036 µm2/s). These data thus provide strong evidence against the possibility that ZAP-70 irreversibly
binds to TCR subunits at the plasma membrane and instead suggest that it interacts dynamically with TCR
, continually exchanging on and off the plasma membrane. The
extremely rapid motion of cytosolic ZAP-70 made it difficult to determine an accurate D value with our experimental setup, but it is greater than 1 µm2/s.
The equation used for FRAP D value measurements assumes one-dimensional recovery since the membranes are
bleached all across their length and entire depth. To assess
the effect of geometry, the calculated D values were
checked against a numerical simulation that used the prebleach intensity of the entire cell as input to simulate diffusion recovery into the bleached strip (Ellenberg et al.,
1997; Sciaky et al., 1997
). For T
GFP, the experimental D value correlated well with that derived from the simulated calculation, which was 0.016 µm2/s (compared with
0.011 µm2/s). In contrast, the D value derived experimentally for ZAP-70 GFP (0.234 µm2/s) was much faster than
its simulated counterpart (0.090 µm2/s). Moreover, the fits
generated by the simulation for ZAP-70 GFP were poor
and variable. These observations, together with the fact
that
-associated ZAP-70 moves 20-fold faster than TCR
, supports the hypothesis that the movement of ZAP-70 at
the plasma membrane is more complex than simple diffusion and likely involves other dynamic parameters, such as
exchange of ZAP-70 with the cytosolic pool.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have used GFP technology to study the movement, kinetics, and associations of key proteins used in signal
transduction pathways coupled to the T cell antigen receptor. Biochemical data have suggested that ZAP-70 undergoes rapid intracellular translocation (Chan et al., 1991;
Wange et al., 1992
). Thus, we were curious to study its intracellular distribution and visualize its changes in response to cellular stimulation in live cells. Our earlier report provided preliminary data defining where ZAP-70
resides in resting cells and how it translocates to the cell
surface upon cellular stimulation (Sloan-Lancaster et al.,
1997
). However, the specific nature of the translocation,
and the subsequent binding to TCR subunits at the plasma
membrane, could not be investigated. Here we analyze
ZAP-70-TCR
interaction in real time and the intramolecular properties required for this association. The kinetics of ZAP-70 translocation to the plasma membrane and
its diffusional mobility in the different intracellular locations were measured.
The activation-induced movement of ZAP-70 to the cell
surface in HeLa cells expressing TT, but not in HeLa, was
a strong indication that we were monitoring the specific
molecular interaction of two proteins over real time. This
redistribution was much more dramatic than that in COS 7 cells, with an impressive clearing from the cytosol accompanying a uniform redistribution to the plasma membrane.
Biochemically, the interaction of ZAP-70 and the ITAMs
has been shown to require the tandem SH2 domains of ZAP-70 and a doubly phosphorylated ITAM (Wange et
al., 1993
; Iwashima et al., 1994
; Koyasu et al., 1994
). The
crystal structure of the SH2 domains of ZAP-70 bound to
a phospho-ITAM verified the molecular properties of this
association (Hatada et al., 1995
). Based on these data, we
designed mutants of ZAP-70 and truncated T
constructs to study the interaction of the two molecules in living cells. Our results validate the imaging approach taken, since
they correlated exactly with the biochemical analyses.
Moreover, they have allowed us to monitor for the first
time the dynamics of these two proteins together in living
cells. The tandem SH2 domains of ZAP-70 and an ITAM
with phosphorylated tyrosines are clearly critical for any
association to occur since there was no detectable redistribution at any time point for more than an hour after stimulation unless both of these criteria were met (Figs. 2 and 3
and data not shown).
How does ZAP-70 translocate to and remain at the
plasma membrane? Because it is a cytosolic protein without any identified retention signal, it is likely freely diffusible within the cell, a phenotype supported by our FLIP
data (Fig. 5). As the kinase moves randomly throughout
the cytoplasm, it will be in a state in which some molecules
are in close proximity to the cell surface at any time, and
should therefore bind any available unoccupied phospho-ITAMs. After TCR engagement, when the CD3 and
TCR ITAMs quickly become phosphorylated, the likelihood that ZAP-70 will bind to them and be retained at the
membrane increases tremendously. This should result in
an accumulation of ZAP-70 at the cell surface and a reciprocal decrease in its cytosolic concentration, as seen in our
time-lapse imaging studies (Fig. 1). Thus, the phosphorylation of the TCR ITAMs seems to be the only triggering
event for ZAP-70 redistribution. Our data using mutant
ZAP-70 GFPs indicate that the domains required for its
redistribution and membrane retention parallel those required to bind TT
(Fig. 2). Moreover, the enhancement of ZAP-70 redistribution accompanied by coexpression of
Lck F505 indicates that simply increasing the level of
ITAM phosphorylation results in more ZAP-70 at the
plasma membrane (Fig. 1). Finally, neither the organized
microtubular network nor the actin microfilaments are required for successful movement of ZAP-70 to the cell surface (Fig. 4).
The data reported by Huby et al. (1998) indicated that
nocodazole treatment of T cells can prevent ZAP-70 activation, independently of the location of ZAP-70 at the cell
surface. Moreover, in that study ZAP-70 appeared to be in
a membrane proximal region in resting T cells, before cellular activation. The differences between this study and
our data might reflect the presence of additional proteins in T cells that engage ZAP-70 and affect its dynamics and
activation. Our studies in HeLa cells represent an early effort in studying these molecules in real time. The dynamic
properties of ZAP-70 must now be analyzed with these
methods in T cells.
The kinetics by which ZAP-70 relocated to the HeLa
cell surface were much slower than would be predicted
from the biochemical analyses in T cells, which indicate
that ZAP-70 binds TCR within seconds of TCR cross-linking (Chan et al., 1991
; Wange et al., 1992
). Surprisingly, there was a significant delay between cellular activation and any detectable, redistributed ZAP-70 (Fig. 1). Perhaps the rate-limiting step is the tyrosine phosphorylation of proteins within the cells due to time required for
PV, when delivered in the media, to be incorporated into
the cells. Moreover, the live cell experiments were conducted at room temperature, and we anticipate that increasing the temperature to 37°C would also result in a
faster translocation initiation time. Of course, the system
employed here using HeLa cells and recombinant proteins is a simplification of the complexity of early T cell signaling events, in which multiple protein-protein interactions
participate to initiate signal transduction. In the T cell environment, such interactions might affect the mobility of
both ZAP-70 and TCR
. However, in the HeLa system,
once ZAP-70 began to translocate to the cell surface it
quickly reached a steady state, without evidence of any reaccumulation in the cytosol even as long as several hours
after PV addition (data not shown). Whether it moves back
after the pharmacological or physiological stimulus ceases or is degraded at the plasma membrane is not known.
The data derived using the photobleaching techniques
allowed us not only to qualitatively compare the movement of different pools of ZAP-70 with itself and with chimeric , but also to calculate diffusion constants for these
molecules. While a role for nuclear ZAP-70 has still not
been defined, it clearly is not rapidly interchangeable with
the cytosolic pool (Fig. 5 A). Both nuclear and cytosolic ZAP-70 are extremely mobile, so we could not determine
a lower limit for their diffusion constants. This suggests
that the protein is not associated with any anchoring molecules in these compartments. However, membrane-associated ZAP-70 in stimulated cells has dramatically different
characteristics in that it is much less mobile. Clearly, membrane-associated ZAP-70 moves slowly relative to the cytosolic pool of unstimulated cells and is likely part of a
large multiprotein lattice under the cell surface, containing
many of the downstream molecules involved in intracellular signaling.
The peripherally located ZAP-70 had a faster diffusion
rate than the chimeric molecule. This was confirmed
when the diffusion constants were determined, indicating
that ZAP-70 moved ~20-fold faster than
(Fig. 7). This
indicated that the binding between
and ZAP-70 is more
complex than an irreversible and stationary interaction.
The simulation data also confirmed that the movement of
ZAP-70 at or near the membrane is not explained by a single diffusion constant. Instead, it seems that the SH2-phosphotyrosine interaction is dynamic, with specific on- and
off-rates. Indeed, this dynamic relationship could explain
how an immune response is regulated at the cellular level.
Once initiated, T cell activation must eventually be turned
off as antigen is cleared from the system. ITAM phosphorylation is a key initiating event of intracellular T cell
activation, but dephosphorylation of these domains is critical for the disassembly of the activating lattice under the
membrane. In fact, a proposed role of ZAP-70 is that it
protects the phosphates of the TCR ITAMs by binding via
its SH2 domains, thus maintaining the receptor in an "on"
state (Iwashima et al., 1994
). Only if ZAP-70 has a dynamic relationship with TCR
will the phospho-ITAMs be
exposed to phosphatases, which will then have an opportunity to dephosphorylate the tyrosine residues. As a result,
the now dephosphorylated ITAMs will no longer be suitable targets for ZAP-70, which may eventually recycle to
the cytosol or be degraded over time. As fewer active
ZAP-70 molecules remain at the cell surface, all subsequent signaling events in the cell will also sequentially be
turned off, until the cell returns to its quiescent state.
The ability to study intracellular signal transduction in
real time now provides one with the tools to begin to answer many unaddressed questions. With the availability of
several GFP variants that excite and emit at different
wavelengths (Heim et al., 1994; Heim and Tsien, 1996
;
Ormo et al., 1996
), the movements of several proteins
have been successfully monitored simultaneously by time-lapse imaging (Rizzuto et al., 1995
; Ellenberg et al., 1998
).
Moreover, the relationship of protein location and second
messenger stimulation has also been studied (Miyawaki
et al., 1997
; Oancea et al., 1998
; Stauffer et al., 1998
). Fluorescence resonance energy transfer to assess protein-
protein interactions will provide detailed information regarding how intracellular networks are established and
maintained (Miyawaki et al., 1997
; Romoser et al., 1997
;
Tsien and Miyawaki, 1998
). As these techniques are refined
and applied, more studies on how intracellular complexes
form in many signaling systems should be performed.
![]() |
Footnotes |
---|
Received for publication 16 July 1998 and in revised form 11 September 1998.
J. Sloan-Lancaster is a fellow of the Damon Runyon-Walter Winchell
Cancer Research Fund. T. Yamazaki is a fellow of the Japan Society for
the Promotion of Science.
J. Sloan-Lancaster's current address is Division of Research Technologies
and Proteins, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285.
Address all correspondence to Lawrence E. Samelson, NICHD,
CBMB, Bldg. 18T, Rm 101, Bethesda, MD 20892. Tel.: (301) 496-6368. Fax: (301) 402-0078. E-mail: samelson{at}helix.nih.gov
![]() |
Abbreviations used in this paper |
---|
FLIP, fluorescence loss in photobleaching;
FRAP, fluorescence recovery after photobleaching;
GFP, green fluorescent protein;
ITAM, immunoreceptor tyrosine-based activation motif;
MHC, major histocompatibility complex;
PV, pervanadate;
ROI, region of interest;
TCR, T cell antigen receptor;
TT, Tac Tac zeta;
T
, Tac zeta zeta.
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