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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 1, 2006 as doi:10.1096/fj.05-4760fje. |
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Developmental Neurobiology Group, Department of Biology, University of Konstanz, Konstanz, Germany;
* Biotechnology Institute Thurgau (BITg) at the University of Konstanz, Tägerwilen, Switzerland
1Correspondence: Universitätstrasse 10, Konstanz D-78457, Germany. E-mail: matthiaslanghorst{at}email.de
SPECIFIC AIMS
The lipid raft proteins reggie-1 and -2 (flotillin-2 and -1, respectively) exhibit a strikingly polarized localization in resting lymphocytes, where they accumulate in one aspect of the cell constituting a "preformed cap." The aim of this study was to further investigate this remarkable localization of the reggies/flotillins in lymphocytes and to test how the preassembled reggie/flotillin platforms facilitate T cell signaling.
PRINCIPAL FINDINGS
1. Reggie-1/flotillin-2 is immobilized in preformed caps but highly mobile at the rest of the plasma membrane
Using fluorescence recovery after photobleaching (FRAP) we investigated first the lateral mobility of reggie-1/flotillin-2 in the plasma membrane of PC12 cells, which do not possess preformed caps. A mobile fraction of MF = 0.8 ± 0.1 (±SDM, n=19) suggested largely unrestricted lateral diffusion. Similarly, reggie-1 outside of the preformed caps showed unrestricted lateral diffusion in Jurkat T lymphocytes (MF=0.72±0.12; ±SDM, n=15). By contrast, reggie-1 within the preassembled caps was almost completely immobile as indicated by a mobile fraction of only MF = 0.12 ± 0.09 (±SDM, n=12). Pharmacological disruption of the actin cytoskeleton led to a loss of preformed reggie caps and to a redistrubution of reggie-1 evenly along the plasma membrane.
Cross-linking of carbohydrate-bearing surface molecules by the mitogenic lectin concanavalin A led to the accumulation of the adaptor protein LAT and the src family kinase lck in the region of the reggie cap of Jurkat T cells. Reggie-1, however, remained immobilized in the cap during ConA stimulation as shown by a mobile fraction of MF = 0.09 ± 0.09 (n=15; ±SDM). Reggie-1 clearly colocalized with LAT within the immunological synapses of superantigen-bearing Raji B and Jurkat T lymphocytes, thus confirming the physiological relevance of the reggie platforms.
2. The C terminus of reggie-1 is essential for its incorporation into the preformed cap design of a trans-negative reggie-1 deletion mutant
Deletion mutants lacking the C-terminal regions of reggie-1 correctly localized to the plasma membrane, but were not incorporated into the preformed cap demarcated by endogenous reggies. The C terminus was previously shown to be necessary and sufficient for the oligomerization of reggie-1. Based on these insights we designed a reggie-1 deletion mutant (R1EA) encompassing only the C-terminal region implicated in oligomerization, which should interfere with oligomer and cap formation.
3. Inhibition of preformed cap assembly impairs raft polarization and macrodomain formation after stimulation and hampers stimulation-induced T cell spreading
Expression of the deletion mutant R1EA in Jurkat T cells inhibited the assembly of preformed reggie-caps (Fig. 1
A). More important, expression of R1EA significantly reduced the efficiency of raft polarization and macrodomain assembly in the cap region after GM1 cross-linking using cholera toxin (CTX) (Fig. 1B
). CTX staining was strongly patched but these patches did not coalesce to form a macrodomain in the cap. Similarly capping efficiency after cross-linking with the lectin ConA was significantly reduced in R1EA-expressing cells. ConA-staining also appeared patchy, and in contrast to control cells, the patches again did not concentrate in a macrodomain in the cap (Fig. 1C
). Thus, the trans-negative reggie-1 mutant R1EA inhibited the assembly of the preformed cap and, more important, macrodomain assembly after T cell activation.
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Next, we investigated the effects of the trans-negative reggie-1 mutant on stimulation-induced morphological changes and cytoskeletal rearrangements. T cells contacting a planar stimulatory surface spread out in order to maximize contact with the stimulus. Mock-transfected Jurkat T cells placed on ConA-coated coverslips spread out completely within 10 min and finally exhibited a regular footprint as observed by interference reflection microscopy (IRM) with a rim of mobile filopodia surrounding the cell (Fig. 1D, F
). In cells transfected with R1EA the spreading was slower and ended before a regular footprint was established (Fig. 1E
). R1EA-expressing cells exhibited an irregular footprint, often with long, randomly organized filopodia at the periphery (Fig. 1F
). The footprint of R1EA-expressing cells was significantly smaller compared with control cells. Thus, reggie-1 apparently is important for the regulation of stimulation-induced cytoskeletal remodeling.
4. Reggie-1 regulates Vav localization
Expression of the trans-negative reggie-1 mutant R1EA did not affect the activation of the MAP kinases ERK1/2 or Ca2+ signaling after ConA stimulation (Fig. 2
A, B), suggesting that reggie function is important for specialized pathways most probably involved in the regulation of cytoskeletal dynamics, as both macrodomain assembly and spreading are dependent on extensive actin remodeling. The guanine nucleotide exchange factor Vav, a key regulator of cytoskeletal dynamics in T cells, is associated with reggie-1 as shown by coimmunoprecipiation and colocalization of the endogenous proteins (Fig. 2C, D
). In mock-transfected cells, Vav clustered at the plasma membrane contacting the stimulatory coverslip during spreading. Expression of R1EA led to an aberrant localization of Vav in spreading Jurkat T cells, explaining the severe spreading defects observed in these cells (Fig. 2F)
.
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CONCLUSIONS AND SIGNIFICANCE
T cell activation is controlled by the regulated assembly of the T cell receptor (TCR) signaling machinery. The formation of a stable, raft-like macrodomain surrounding engaged TCRs is thought to contribute to this complex process, so that aggregation or exclusion of molecules regulates signal strength and duration. This macrodomain assembly integrates signals from the TCR and from coreceptors and is crucially dependent on extensive remodeling of the cytoskeleton.
This is the first report showing a functional contribution of the preformed reggie/flotillin caps to T cell signaling, emphasizing the importance of the preexisting polarity in resting T cells. The preformed caps are exceptionally stable structures before as well as during cell stimulation. Reggie-1 stabilization in preformed caps of lymphocytes is dependent on direct or indirect anchorage to the actin cytoskeleton. Macrodomain assembly after activation takes place in the region of the reggie cap and interference with reggie cap assembly also inhibits raft polarization and formation of a stable macrodomain. The severe spreading defects in cells expressing the trans-negative reggie-1 mutant further support a functional role of the preformed reggie caps in T cell signaling. Both results suggest that inhibition of reggie function is associated with defects in stimulation-induced actin remodeling.
Our model is supported by the fact that reggie-1 associates with Vav and that a trans-negative reggie-1 mutant impairs the correct localization of Vav. Dominant-negative Vav mutants were recently shown to inhibit raft polarization and macrodomain assembly, similar to the trans-negative reggie-1 mutant described in the present study. We have previously shown that LAT and fyn are associated with the reggies. Both proteins are important regulators of Vav function and cytoskeletal dynamics in T cells. Therefore, we propose the following model: clusters of reggie proteins act as preexisting, polarized scaffolds, anchored to the cytoskeleton. Upon stimulation, signaling complexes already associated with the reggie scaffold containing Vav, LAT, lck, and fyn initiate raft polarization toward the reggie cap and the necessary remodeling of the cytoskeleton. During macrodomain formation, more and more signaling complexes assemble on the reggie scaffolds, further enhancing raft clustering. Thus, in a feed-forward mechanism macrodomain assembly is driven by proteins associated with the polarized reggie scaffold. Without the preformed reggie cap, spatial information important for macrodomain assembly in the cap is apparently missing. Although raft clustering still occurs, these small clusters do not coalesce to form a macrodomain in the cap. Therefore, the preformed reggie scaffold appears to be an important factor for cytoskeletal remodeling during T cell activation.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4760fje;
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