Understanding the Structure and Function of the Immunological Synapse

The immunological synapse has been an area of very active scientific interest over the last decade. Surprisingly, much about the synapse remains unknown or is controversial.  Here we review some of these current issues in the field:  how the synapse is defined, its potential role in T-cell function, and our current understanding about how the synapse is formed.T cells are activated when they recognize peptide-MHC complexes on the surface of antigen presenting cells (APC) (Babbitt et al. 1985). But the exact process regarding how antigenic pMHC complexes are recognized and transduced into signals is still incompletely understood. Naïve T cells enter secondary lymphoid organs such as the lymph node and scan dendritic cells for the presence of rare specific pMHC complexes (Miller et al. 2004). After recognizing less than 10 specific pMHC complexes, naïve T cells maintain long contacts (6–18 h) with dendritic cells before being committed to enter cell cycle and differentiate into effector T cells (Iezzi et al. 1998; Irvine et al. 2002; Mempel et al. 2004).The immunological synapse (IS) refers to the organization of membrane proteins that occurs at the interface between the T cell and the APC during these long contacts and also during the effector phase (Grakoui et al. 1999; Monks et al. 1998). Interest in studying the IS stems from ideas that the supramolecular structures that form at the IS underlies the high sensitivity of T cell recognition and that understanding these structures will lead to better insights into how antigen recognition leads to the decision of a T cell to proliferate, differentiate, and function.Springer first put forward the concept that receptors would segregate laterally during cell interactions (Springer 1990). Subsequently, Kupfer was the first to show that proteins in the contact area between a T cell and APC segregate laterally (Monks et al. 1998). Specifically, he noted that the integrin, LFA-1, became concentrated in an outer ring, known as the peripheral supramolecular activation complex (pSMAC) and the TCR became concentrated in the center, in a zone known as the central supramolecular activation complex (cSMAC) (Monks et al. 1998)(Fig. 1). We showed that CD2 could segregate from LFA-1 and concentrate in the center of a hybrid cell-planar bilayer junction and suggested that these patterns and those described by Monks et al. (1998) provided evidence for the previously hypothesized immunological synapse (Dustin et al. 1998; Norcross 1984). The function of this receptor segregation is still not completely understood but it was initially hypothesized that formation of this pattern might be related to T-cell activation and constitute a “molecular machine” that would be formed in response to the presence of antigenic ligand and that this “molecular machine” might function to sustain signaling for long periods of time and direct subsequent T-cell differentiation (Grakoui et al. 1999).Open in a separate windowFigure 1.Structure of the immunological synapse. The basic structure of the “organized” immunological synapse with SMACs is shown (left). In the center is the central supramolecular activation complex or cSMAC, which contains receptors like the TCR, CD28, CD4, CD8, and CD2. Newer studies suggest that the cSMAC may be divided into an outer area containing CD28 and an inner area containing the TCR (not shown). The ring that surrounds the cSMAC is called the peripheral supramolecular activation complex or pSMAC. This domain is mainly populated by the integrin molecule LFA-1. Outside of the pSMAC is another domain known as the distal supramolecular activation complex. Originally the dSMAC was thought not be important and contain all of the molecules that are not specifically recruited to the cSMAC or pSMAC but it is increasingly becoming appreciated that the dSMAC is an area of active membrane movement. This suggests that the pSMAC and dSMAC may be analogous to the actin structures known as the lamellae and lamellipodia, respectively (right).     

Downregulation of ADIPOQ and PPARγ2 Gene Expression in Subcutaneous Adipose Tissue of Obese Adolescents With Hepatic Steatosis

Hepatic steatosis is associated with hypoadiponectinemia. The mechanism(s) resulting in lower serum adiponectin levels in obese adolescents with fatty liver is unknown. In two groups of equally obese adolescents, but discordant for hepatic fat content, we measured adiponectin, leptin, peroxisome proliferator–activated receptor γ 2 (PPARγ2) and tumor necrosis factor‐α (TNFα) gene expression in the abdominal subcutaneous adipose tissue (SAT). Twenty six adolescents with similar degrees of obesity underwent a subcutaneous periumbilical adipose tissue biopsy, in addition to metabolic (oral glucose tolerance test, and hyperinsulinemic—euglycemic clamp), and imaging studies (magnetic resonance imaging (MRI), DEXA). Using quantitative real‐time‐PCR; adiponectin, PPARγ2, TNFα, and leptin mRNA were measured. Based on a hepatic fat content (hepatic fat fraction, HFF) >5.5%, measured by fast MRI, the subjects were divided into low and high HFF group. In addition to the hypoadiponectinemia in the high HFF group, we found that the expression of adiponectin as well as PPARγ2 in the SAT was significantly decreased in this group. No differences were noted for TNFα and leptin plasma or mRNA levels between the groups. An inverse relationship was observed between adiponectin or PPARγ2 expression and hepatic fat content, whereas, adiponectin expression was positively related to PPARγ2 expression. Independent of overall obesity, a reduced expression of adiponectin and PPARγ2 in the abdominal SAT is associated with high liver fat content, as well as with insulin resistance in obese adolescents.     

Ecomorph or Endangered Coral? DNA and Microstructure Reveal Hawaiian Species Complexes: Montipora dilatata/flabellata/turgescens & M. patula/verrilli

M. dilatata, M. flabellata, and M. patula and 80 other scleractinian corals were petitioned to be listed under the US Endangered Species Act (ESA), which would have major conservation implications. One of the difficulties with this evaluation is that reproductive boundaries between morphologically defined coral species are often permeable, and morphology can be wildly variable. We examined genetic and morphological variation in Hawaiian Montipora with a suite of molecular markers (mitochondrial: COI, CR, Cyt-B, 16S, ATP6; nuclear: ATPsβ, ITS) and microscopic skeletal measurements. Mitochondrial markers and the ITS region revealed four distinct clades: I) M. patula/M. verrilli, II) M. cf. incrassata, III) M. capitata, IV) M. dilatata/M. flabellata/M. cf. turgescens. These clades are likely to occur outside of Hawai'i according to mitochondrial control region haplotypes from previous studies. The ATPsβ intron data showed a pattern often interpreted as resulting from hybridization and introgression; however, incomplete lineage sorting may be more likely since the multicopy nuclear ITS region was consistent with the mitochondrial data. Furthermore, principal components analysis (PCA) of skeletal microstructure was concordant with the mitochondrial clades, while nominal taxa overlapped. The size and shape of verrucae or papillae contributed most to identifying groups, while colony-level morphology was highly variable. It is not yet clear if these species complexes represent population-level variation or incipient speciation (CA<1MYA), two alternatives that have very different conservation implications. This study highlights the difficulty in understanding the scale of genetic and morphological variation that corresponds to species as opposed to population-level variation, information that is essential for conservation and for understanding coral biodiversity.     

The E3 ubiquitin ligase NEDD4 is an LC3-interactive protein and regulates autophagy

The MAP1LC3/LC3 family plays an essential role in autophagosomal biogenesis and transport. In this report, we show that the HECT family E3 ubiquitin ligase NEDD4 interacts with LC3 and is involved in autophagosomal biogenesis. NEDD4 binds to LC3 through a conserved WXXL LC3-binding motif in a region between the C2 and the WW2 domains. Knockdown of NEDD4 impaired starvation- or rapamycin-induced activation of autophagy and autophagosomal biogenesis and caused aggregates of the LC3 puncta colocalized with endoplasmic reticulum membrane markers. Electron microscopy observed gigantic deformed mitochondria in NEDD4 knockdown cells, suggesting that NEDD4 might function in mitophagy. Furthermore, SQSTM1 is ubiquitinated by NEDD4 while LC3 functions as an activator of NEDD4 ligase activity. Taken together, our studies define an important role of NEDD4 in regulation of autophagy.     

Crystal structure of a novel non-Pfam protein PF2046 solved using low resolution B-factor sharpening and multi-crystal averaging methods

Sometimes crystals cannot diffract X-rays beyond 3.0 ? resolution due to the intrinsic flexibility associated with the protein. Low resolution diffraction data not only pose a challenge to structure determination, but also hamper interpretation of mechanistic details. Crystals of a 25.6 kDa non-Pfam, hypothetical protein, PF2046, diffracted X-rays to 3.38 ? resolution. A combination of Se-Met derived heavy atom positions with multiple cycles of B-factor sharpening, multi-crystal averaging, restrained refinement followed by manual inspection of electron density and model building resulted in a final model with a R value of 23.5 (R_free=24.7). The asymmetric unit was large and consisted of six molecules arranged as a homodimer of trimers. Analysis of the structure revealed the presence of a RNA binding domain suggesting a role for PF2046 in the processing of nucleic acids.     

Visualisation of stromules in transgenic wheat expressing a plastid-targeted yellow fluorescent protein

Stromules are stroma-filled tubules that extend from the plastids in all multicellular plants examined to date. To facilitate the visualisation of stromules on different plastid types in various tissues of bread wheat (Triticum aestivum L.), a chimeric gene construct encoding enhanced yellow fluorescent protein (EYFP) targeted to plastids with the transit peptide of wheat granule-bound starch synthase I was introduced by Agrobacterium-mediated transformation. The gene construct was under the control of the rice Actin1 promoter, and EYFP fluorescence was detected in plastids in all cell types throughout the transgenic plants. Stromules were observed on all plastid types, although the stromule length and abundance varied markedly in different tissues. The longest stromules (up to 40 μm) were observed in epidermal cells of leaves, whereas only short beak-like stromules were observed on chloroplasts in mesophyll cells. Epidermal cells in leaves and roots contained the highest proportion of plastids with stromules, and stromules were also abundant on amyloplasts in the endosperm tissue of developing seeds. The general features of stromule morphology and distribution were similar to those shown previously for tobacco (Nicotiana tabacum L.) and arabidopsis (Arabidopsis thaliana (L.) Heynh.).     

A hormone-dependent module regulating energy balance

Under fasting conditions, metazoans maintain energy balance by shifting from glucose to fat burning. In the fasted state, SIRT1 promotes catabolic gene expression by deacetylating the forkhead factor FOXO in response to stress and nutrient deprivation. The mechanisms by which hormonal signals regulate FOXO deacetylation remain unclear, however. We identified a hormone-dependent module, consisting of the Ser/Thr kinase SIK3 and the class IIa deacetylase HDAC4, which regulates FOXO activity in Drosophila. During feeding, HDAC4 is phosphorylated and sequestered in the cytoplasm by SIK3, whose activity is upregulated in response to insulin. SIK3 is inactivated during fasting, leading to the dephosphorylation and nuclear translocation of HDAC4 and to FOXO deacetylation. SIK3 mutant flies are starvation sensitive, reflecting FOXO-dependent increases in lipolysis that deplete triglyceride stores; reducing HDAC4 expression restored lipid accumulation. Our results reveal a hormone-regulated pathway that functions in parallel with the nutrient-sensing SIRT1 pathway to maintain energy balance.