Repair of DNA double-strand breaks (DSBs) in mammalian cells by nonhomologous end-joining (NHEJ) is initiated by the DNA-PK protein complex. Recent studies have shown inositol hexakisphosphate (InsP6) is a potent cofactor for DNA-PK activity in NHEJ. Specifically, InsP6 binds to the Ku component of DNA-PK, where it induces a conformational change and a corresponding increase in DNA end-joining activity. However, the effect of InsP6 on the dynamics of Ku, such as its mobility in the nucleus, is unknown. Importantly, these dynamics reflect the character of Ku's interactions with other molecules. To address this question, the diffusion of Ku was measured by fluorescence photobleaching experiments using cells expressing green fluorescent protein (GFP)-labeled Ku. InsP6 was depleted by treating cells with calmodulin inhibitors, which included the compounds W7 and chlorpromazine. These treatments caused a 50% reduction in the mobile fraction of Ku-GFP, and this could be reversed by replenishing cells with InsP6. By expressing deletion mutants of Ku-GFP, it was determined that its W7-sensitive region occurred at the N-terminus of the dimerization domain of Ku70. These results therefore show that InsP6 enhances Ku mobility through a discrete region of Ku70, and modulation of InsP6 levels in cells represents a potential avenue for regulating NHEJ by affecting the dynamics of Ku and hence its interaction with other nuclear proteins.

Polarization of membrane rafts and signaling proteins to form an immunological synapse is a hallmark of T cell stimulation. However, the kinetics of raft polarization and associated proteins in relation to the initial contact of the T cell with the APC are poorly defined. We addressed this question by measuring the distribution of membrane-targeted fluorescent protein markers during initial T cell interactions with B cell APCs. Experiments with unpulsed B cells lacking cognate Ag demonstrated an MHC class II-independent capping that was specific to membrane raft markers and required actin rearrangements and signals from Src kinases and PI3K. By live cell imaging experiments, we identified a similar specific polarization of membrane raft markers before TCR-dependent stop signals, and which occurred independently of cognate peptide-MHC class II. T cells conjugated to unpulsed B cells exhibited capping of CD4 and microclusters of the TCR ζ-chain, but only the CD4 enrichment was cholesterol dependent. Furthermore, raft association of CD4 was necessary for its efficient targeting to the Ag-independent caps. Interestingly, anergic Vβ8+ T cells isolated from staphylococcal enterotoxin B-injected mice did not exhibit Ag-independent capping of membrane rafts, showing that inhibition of these early, Ag-independent events is a property associated with tolerance. Altogether, these data show that membrane raft capping is one of the earliest events in T cell activation and represents one avenue for promoting and regulating downstream peptide-MHC-dependent signaling within the T cell.

All-trans-retinoic acid may be an important molecular signal in the postnatal control of eye size. The goal of this study was to identify retinoic acid-binding proteins secreted by the choroid and sclera during visually guided ocular growth. Following photoaffinity labeling with all-trans-[11,12-3 H]retinoic acid, the most abundant labeled protein detected in the conditioned medium of choroid or sclera had an apparent M r of 27,000 Da. Following purification and mass spectrometry, the M r 27,000 band was identified as apolipoprotein A-I. Affinity capture of the radioactive M r 27,000 band by anti-chick apolipoprotein A-I antibodies confirmed its identity as apolipoprotein A-I. Photoaffinity labeling and fluorescence quenching experiments demonstrated that binding of retinoic acid to apolipoprotein A-I is 1) concentration-dependent, 2) selective for all-trans-retinoic acid, and 3) requires the presence of apolipoprotein A-I-associated lipids for retinoid binding. Expression of apolipoprotein A-I mRNA and protein synthesis were markedly upregulated in choroids of chick eyes during the recovery from induced myopia, and apolipoprotein A-I mRNA was significantly increased in choroids following retinoic acid treatment. Together, these data suggest that apolipoprotein A-I may participate in a regulatory feedback mechanism with retinoic acid to control the action of retinoic acid on ocular targets during postnatal ocular growth.

V(D)J recombination of lymphocyte antigen receptor genes occurs via the formation of DNA double strand breaks (DSBs) through the activity of RAG1 and RAG2. The coexistence of RAG-independent DNA DSBs generated by genotoxic stressors potentially increases the risk of incorrect repair and chromosomal abnormalities. However, it is not known whether cellular responses to DSBs by genotoxic stressors affect the RAG complex. Using cellular imaging and subcellular fractionation approaches, we show that formation of DSBs by treating cells with DNA damaging agents causes export of nuclear RAG2. Within the cytoplasm, RAG2 exhibited substantial enrichment at the centrosome. Further, RAG2 export was sensitive to inhibition of ATM, and was reversed following DNA repair. The core region of RAG2 was sufficient for export, but not centrosome targeting, and RAG2 export was blocked by mutation of Thr490. In summary, DNA damage triggers relocalization of RAG2 from the nucleus to centrosomes, suggesting a novel mechanism for modulating cellular responses to DSBs in developing lymphocytes.

T cells become polarized during initial interactions with an APC to form an antigen-independent synapse (AIS) composed of membrane rafts, TCR, and TCR-proximal signaling molecules. AISs occur temporally before TCR triggering, but their role in downstream TCR signaling is not understood. Using both human and murine model systems, we studied the signals that activate AIS formation, and the effect of these signals on TCR-dependent responses. We show that CD28 produces AISs detectable by spinning disc confocal microscopy seconds following initial interactions between the T cell and APC. AIS formation by CD28 coincided with co-stimulatory signaling, evidenced by a cholesterol-sensitive activation of the MAP kinase ERK that potentiated Ca2+ signaling in response to CD3 crosslinking. CD45 also enriched in AISs, but to modulate Src kinase activity, since localization of CD45 at the cell interface reduced the activation of proximal Lck. In summary, we show that signaling by CD28 during first encounters between the T cell and APC both sensitizes TCR Ca2+ signaling by an Erk-dependent mechanism, and drives formation of an AIS that modulates the early signaling until TCR triggering occurs. Thus, early Ag-independent encounters are an important window for optimizing T cell responses to antigen by CD28.