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–TCRζ 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 TTζ, 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 Tζζ 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. and 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. ). 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 TCRζ 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. ). 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 TTζ (Fig. ). 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. ). Finally, neither the organized microtubular network nor the actin microfilaments are required for successful movement of ZAP-70 to the cell surface (Fig. ).
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 TCRζ 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. ). 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 TCRζ. 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 ζ, 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. 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 ζ molecule. This was confirmed when the diffusion constants were determined, indicating that ZAP-70 moved ~20-fold faster than ζ (Fig. ). This indicated that the binding between ζ 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 TCRζ 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.