analysis of 2C T cell extracts prepared after the mitochondrial drug treatments at concentrations around IC80s for the pMV- absorption (Figure 2A), the ratio of concentration of ATP to that of entire pool of adenine nucleotides decreased by a maximum of about 25%, which occurs when the cells were treated with Deguelin, and by an average of 15%. Changes in cellular energy balance were hardly detected when 2C T cells were treated with the drugs, except for Deguelin and Phenformin, at concentrations near the IC50s; even with Deguelin and Phenformin, decrease in the ratio of concentration of ATP to that of entire pool of adenine nucleotides was only about 4%. Given the marginal changes in the ATP ratio after the mitochondrial drug treatments, it seems unlikely that ATP depletion is a main reason for inhibition by the mitochondrial antagonists of the pMV-absorption. Dose-dependent activation of AMPKa1 after the mitochondrial drug treatments was expected. Nonetheless, it is of note that significant levels of AMPKa1 activation were observed after treatments of 2C T cells with the respective mitochondrial drugs at concentrations near the IC50s where changes in cellular energy balance were almost undetectable (Figure 2A and B). With regard to that discrepancy, one may postulate that the cellular energy balance affected by the mitochondrial drug treatments was quickly restored to a homeostatic level following the AMPKa1 activation. That is, activated AMPKa1 suppressed ATP-consuming meta- bolic processes to balance the rate of ATP production and consumption and thereby the ratio among adenine nucleotides was maintained at the constant level [32]. Alternatively, it is also conceivable that the subtle change in cellular energy level hardly detected by UV absorption HPLC detector was able to induce phosphorylation of AMPKa1. It is puzzling that mitochondrial antagonists with different modes of action have different effects on superoxide production (Figure 2C). It was, however, shown by studies using isolated mitochondria in vitro that the component of electron transport chain predominantly responsible for the production of SO varies depending on energy state of mitochondria. According to those studies, while complex III is the main complex for SO production in mitochondria of high respiratory activity (state III), complex I plays a major role in mitochondria of low respiratory activity (state IV). Results shown in Figure 2C could be understood in the context of those findings [10,33,34]. We examined the SO level to find whether changes in the SO level by the mitochondrial drug treatments could account for the ubiquitous inhibition of the pMV-absorption by those treatments. Given the differential effects, it seems difficult to explain the inhibition of the pMV-absorption with the perturbation of SO production by the drug treatments. Phosphorylation of Akt and PLC-c1 and entry of extracellular Ca2+, key signaling events for T cell activation, are preceded by activation of multiple upstream signaling molecules immediately following TCR triggering, which include PTK(s) and PI3K[35]. F- actin also plays a role in those signaling events. Dynamic F-actin rearrangement results in directional relocation of signaling molecules bringing them into close proximity and facilitating inter-molecular interactions among them [30]. In addition, costimulatory receptor/ ligand interactions (e.g., CD28/B7-1 and LFA-1/ICAM-1) also make strong contribution to the phosphorylation of Akt and PLC-c1 as well as entry of extracellular Ca2+ [26,27,28]. Thus, although results that the mitochondrial drug treatments inhibited the phosphorylation of Akt and PLC-c1 and Ca2+ entry suggested a mitochondria-mediated control mechanism for membrane-proximal TCR signaling events (Figure 3), it was difficult to know whether the inhibition resulted from down-regulation of a specific signaling event or suppression of overall signaling process. Mitochondria and TCR Signaling Here, results from the experiments for F-actin polymerization provide an important clue (Figure 4). Different from phosphory- lation of Akt and PLC-c1, F-actin polymerization in 2C T cells cultured with QL9-loaded LdB7-1ICAM-1 pMVs is solely dependent on 2C TCR/Ld/QL9 interaction. Thus, extents of the F-actin polymerization are not affected by mAb blocking of either CD28/B7-1 or LFA-1/ICAM-1 interaction [15]. F-actin polymerization proceeds via a complex signaling process including activation of kinases (i.e., PTKs and PI3K), adapter proteins (i.e., Vav) and a WASP family protein (i.e., WAVE), etc [36,37]. Together, results that the mitochondrial drug treatments had little effects on the F-actin polymerization (Figure 4) lead us to draw a conclusion that a branch of TCR signaling events progresses normally even after the mitochondrial drug treatments. In agreement with the results from the flow cytometric analysis, microscopic analysis of 2C T cells cultured with the QL9-loaded pMVs showed enhanced cortical F-actin staining regardless of the mitochondrial drug treatments (Figure 5). Nevertheless, clear differences in distribution of cortical F-actin and in cell morphology were observed between DMSO- and mitochondrial drug-treated 2C T cells, indicating a process for dynamic rearrangement of F-actin was impaired by the drug treatments. Proper rearrangement of F-actin is the key for cell movement and directional relocation of signaling proteins [30,38]. Dynamic rearrangement of F-actin proceeds via a series of signaling events involving kinases, phosphatases, adopter molecules and a host of actin binding proteins [39]. Taken together, we hypothesize that the mitochondrial drug treatments result in, by an unknown mechanism, inhibition of a specific signaling mechanism involved in dynamic rearrangement of F-actin but not in F-actin polymerization. We may understand the inhibition of the phosphorylation of Akt and PLC-c1 in the same context. Thus, even though upstream signaling cascades commonly involved in phosphorylation of Akt and PLC-c1 and F- actin polymerization (e.g., activation of PTKs and PI3K) operates normally after the mitochondrial drug treatments, the drug treatments attenuate F-actin rearrangement to compromise directional relocation of signaling molecules in close proximity to each other and thereby inhibit the activation of downstream signaling molecules such as Akt and PLC-c1. Importance of AMPK activation in inhibition by the mito- chondrial antagonists of the pMV-absorption is yet to be resolved. Even though the strong inhibition by AICAR (5-aminoimidazole- 4-carboxamide-1-b-riboside), a well-known AMPK activator [40], of the pMV-absorption by 2C T cells indicates the role of AMPKa1 in control of TCR signaling for LFA-1 activation (authors’ unpublished data), that result has to be interpreted with caution. Studies by others have shown that AICAR treatment could have effects on certain metabolic processes independent of AMPK activation [41]. Studies using 2C T cells genetically deficient in AMPKa1 expression may help to understand the importance of AMPKa1 in regulation of TCR signaling and LFA- 1 activation [42]. Signaling via TCR is crucial not only for activation and clonal expansion of T cells encountering foreign peptide/MHC com- plexes but also for survival of resting T cells. TCR signaling for the T cell survival is instigated as T cells encounter specific self peptide/MHC complexes that direct development (positive selection) of those T cell in thymus and are also presented by neighboring cells in secondary lymphoid organs (lymph nodes and spleen) [31]. The TCR/pMHC interactions are not strong enough to induce activation of downstream signaling events for cell cycle progression but sufficient to induce signaling events for maintain- ing integrity of cells. Given those, expected was the decrease in PLoS ONE | www.plosone.org 8 November 2009 | Volume 4 | Issue 11 | e7738