The use of siRNA silencing techniques both in Jurkat and primary T cells and the isolation of a Jurkat cell variant lacking MAL expression allowed us to address the role and mechanism of action of MAL in T cells. Conjugates with APC were efficiently formed using MAL-depleted T cells, but TCR, Lck, ZAP-70, and PKC-θ did not redistribute to the IS. The requirement for MAL for IS formation was confirmed in T cell–APC conjugates formed in the presence of the SEB superantigen or a specific HA peptide. In addition to deficient IS formation, MAL-depleted cells were unable to activate signaling pathways leading to up-regulation of IL-2 promoter transcription in response to SEE and showed reduced levels of Lck at the cell periphery. All these defects were corrected by exogenous expression of MAL. Therefore, our results establish the requirement for MAL in the processes of IS formation and T cell activation.
Given the crucial role of MAL as an element of the specialized machinery for apical transport in epithelial cells (12
), an obvious aspect of interest concerning MAL function in T cells was its involvement in specialized membrane trafficking to the cell surface (15
). During our search for abnormally distributed plasma membrane proteins in MAL-deficient cells, we found that although Lck was distributed at both the cell periphery and in endosomes in normal Jurkat cells, Lck was practically absent from the plasma membrane and instead was concentrated in a compact intracellular structure in Jurkat cells lacking MAL expression. It is of particular note that Lck distributed normally when MAL levels were reconstituted by exogenous expression. Consistent with these findings, knockdown of MAL levels in primary T cells reduced the presence of Lck at the plasma membrane with concomitant intracellular accumulation compared with the exclusive peripheral expression of Lck in control cells. Therefore, MAL appears to be crucial for normal targeting of Lck to the T cell surface.
Lck is known to rely on membrane-trafficking mechanisms for targeting to the cell surface (6
). Expression of Lck at the plasma membrane is essential for T cell function because Lck mutants that are unable to reach the plasma membrane are defective in T cell signaling (7
). The presence of a pool of Lck in endosome structures and their colocalization with MAL in normal Jurkat cells are consistent with a possible role for MAL in Lck exocytosis. This view was supported by our observations of time-lapse videomicroscopy in living cells, in which Lck and MAL traveled in the same vesicles destined for the cell surface. The vesicles were large and formed discontinuously. It is worth noting that the formation of the vesicles containing Lck was impaired in the MAL-deficient Jurkat variant, and only a few much smaller vesicles were sporadically formed. The absence of MAL did not provoke a general block on the generation of transport vesicles because vesicles containing Fyn-GFP or p75-GFP formed efficiently in JTIM cells. Our results indicate that MAL mediates a specialized route of transport in T cells that is used for targeting Lck to the plasma membrane.
Membrane microdomains were originally postulated as being specialized platforms for apical transport (8
). Although controversial (25
), it is sometimes assumed that the membrane fraction resistant to detergent solubilization is enriched in membrane microdomains (26
). Approximately 30–50% of Lck is isolated in detergent-insoluble membrane fractions (28
). Consistent with the observed colocalization of MAL and Lck in endosome structures in Jurkat cells, both proteins cofractionate in a detergent-insoluble subendosomal membrane fraction (11
). It is of particular note that the inability of Lck to reach the plasma membrane in MAL-deficient Jurkat cells is associated with its exclusion from detergent-insoluble membrane fractions. Similar to the strict link between plasma membrane targeting and detergent insolubility for Lck found in WT, MAL-deficient, and MAL-reconstituted Jurkat cells, polarized apical targeting of HA in Mardin-Darby canine kidney cells is associated with HA insolubility in a MAL expression–dependent manner (12
). This latter observation, together with the association found between MAL and HA, was interpreted as meaning that one of MAL's functions is to recruit HA to membrane platforms for apical exocytosis (12
). Consistent with this view, the transport defect observed for Lck in the absence of MAL expression may be explained by a membrane alteration that prevents incorporation of Lck into specific membrane platforms acting in Lck exocytosis. Because MAL associates with Lck and exogenous expression of MAL fully restored not only the blocking of the partitioning into detergent-insoluble membranes but also the transport of Lck, it might be that a primary function of MAL in T cells is to recruit Lck to specialized membranes involved in the formation of transport carriers and the subsequent targeting of Lck to the plasma membrane.
In addition to the redistribution of receptors and signaling machinery, IS formation implies recruitment of actin cytoskeleton elements, which form a specialized network of actin filaments while retaining their apparently normal subcortical organization in the rest of the cell membrane, and repositioning of MTOC, which controls the position of the secretory and endocytic compartments toward the IS (30
). Repositioning of these compartments could have consequences on the activation process that could bring Fyn, Pyk2, and other signaling molecules closer to IS (31
). It has also been reported that a pool of Lck and LAT that is associated with recycling endosomes translocates to mature synapses (32
). Therefore, the absence of MTOC reorientation in MAL-deficient cells probably affects polarized targeting from the secretory/endocytic compartments to IS (32
Both actin and talin redistributed to the IS regardless of whether MAL was expressed, implying that MAL is not required for this process. Remarkably, unlike the TCR, which required MAL for IS targeting, ICAM-3 was normally recruited in the absence of MAL. This indicates that ICAM-3 targeting to IS takes place by MAL-independent mechanisms that are most probably linked to the actin cytoskeleton rearrangement. In this regard, association of ICAM-3 with ERM proteins has been previously documented (35
). In contrast to the remodeling of the actin cytoskeleton, the MTOC did not reorient to the IS in the absence of MAL. MTOC reorientation is impaired in Lck- or ZAP-70–deficient Jurkat cells (36
) and requires phosphorylation in tyrosine of immunoreceptor tyrosine-based activation motifs that are present at the cytoplasmic tail of the CD3 subunits (37
). To analyze whether the absence of Lck at the plasma membrane can by itself fully account for the failure to trigger the signals required for proper IS formation, we forced the expression of Lck at the plasma membrane by expression of CD4/Lck and LAT/Lck transmembrane chimeras in JTIM cells. It is of particular note that the chimeras were able to restore the targeting of TCR, ZAP-70, and PKC-θ to IS, indicating that these transmembrane forms of Lck, regardless of their level of partitioning into detergent-insoluble membranes, were equally able to initiate TCR-induced signaling even in the absence of MAL expression. Despite this, however, both chimeras did not correct MTOC reorientation or TCR-induced downstream signaling events such as ERK activation and CD69 up-regulation. These findings indicate that the deficient targeting of Lck to the plasma membrane is not the sole cause of the effects observed in MAL-deficient cells and implies that, in addition to transporting Lck, MAL mediates other functions in the cell, probably related to the transport of other specific molecules and the repositioning of the MTOC.
Jurkat cells lacking MAL expression were unable to activate transcription of the IL-2 promoter in response to stimulation with SEE. Consistent with this observation, the signaling pathways leading to activation of transcription factors AP-1, NF-κB, and NFAT, or to increased tyrosine phosphorylation of specific substrates, were defective. Reconstitution of MAL levels by exogenous expression made these pathways fully operative and rescued normal response of the IL-2 promoter to T cell stimulation. Restoration of these pathways is probably mediated by correction of the abnormal targeting of Lck, and probably of other signaling molecules, that is observed in the absence of MAL expression.
A constitutive MAL KO mouse line was generated to investigate the role of MAL on myelin formation (38
). The mice were viable, had a normal life span, and appeared grossly normal. However, a closer inspection revealed defects in axon–glia interactions. Importantly, although MAL is expressed in oligodendrocytes and Schwann cells, alterations were observed only in the central nervous system, whereas the peripheral nervous system did not seem to be affected. The possible existence of a compensatory mechanism of another as yet unknown MAL family protein was claimed that might replace the functional role of endogenous MAL at the peripheral nervous system. Unlike humans, thymus, primary T cells, and T cell lines of mouse origin do not express the MAL
gene. The most plausible explanation is that another member of the MAL protein family or of the MARVEL domain–containing protein superfamily does the job in mouse T cells. This reminds us of the caution that must be exercised in making definitive assignments of physiological function across species. Although animal models are undoubtedly quite useful for biomedical research, it is clear that studies in human cells need to be performed to learn about human cell functioning.
Progress in understanding MAL function as a component of the machinery for specialized routes of protein transport was first made possible in epithelial cells by taking advantage of the seminal discoveries made in epithelial cells concerning protein sorting (39
). In this study, using gene silencing in Jurkat T cells and primary human T lymphocytes, and a Jurkat cell clone with deficient MAL expression, we have found an essential role for MAL in exocytic transport of Lck to the plasma membrane. In addition, our results highlight the similarity of the Lck transport process of T cells to that of the specialized MAL-mediated apical route of protein exocytosis of polarized epithelial cells. Normal functioning of this novel transport pathway is essential for T cell activation and IS formation.