Dendritic cells (DCs) either boost the immune system (enhancing immunity) or dampen it (leading to tolerance). This dual effect explains their vital role in cancer development and progression. DCs have been tested as a predictor of outcomes for cancer progression. Eight studies evaluated tumour-infiltrating DCs (TIDCs) as a predictor for colorectal cancer (CRC) outcomes. The detection of TIDCs has not kept pace with the increased knowledge about the identification of DC subsets and their maturation status. For that reason, it is difficult to draw a conclusion about the performance of DCs as a predictor of outcome for CRC. In this review, we comprehensively examine the evidence for the in situ immune response due to DC infiltration, in predicting outcome in primary CRC and how such information may be incorporated into routine clinical assessment.

Cell Communication and Adhesion has been fortunate to enlist two pioneers of epidermal and cardiac cell junctions, Kathleen Green and Mario Delmar, as Guest Editors of a two part series on junctional targets of skin and heart disease. Part 2 of this series begins with an overview from Dipal Patel and Kathy Green comparing epidermal desmosomes to cardiac area composita junctions, and surveying the pathogenic mechanisms resulting from mutations in their components in heart disease. This is followed by a review from David Kelsell on the role of desmosomal mutation in inherited syndromes involving skin fragility. Agnieszka Kobeliak discusses how structural deficits in the epidermal barrier intersect with the NFkB signaling pathway to induce inflammatory diseases such as psoriasis and atopic dermatitis. Farah Sheikh reviews the specialized junctional components in cardiomyocytes of the cardiac conduction system and Robert Gourdie discusses how molecular complexes between sodium channels and gap junction proteins within the perijunctional microdomains within the intercalated disc facilitate conduction. Glenn Radice evaluates the role of N-cadherin in heart. Andre Kleber and Chris Chen explore new approaches to study junctional mechanotransduction in vitro with a focus on the effects of connexin ablation and the role of cadherins, respectively. To complement this series of reviews, we have interviewed Werner Franke, whose systematic documentation the tissue-specific complexity of desmosome composition and pioneering discovery of the cardiac area composita junction greatly facilitated elucidation of the role of desmosomal components in the pathophysiology of human heart disease.

Astroctyes express a set of three connexins (Cx26, Cx30, and Cx43) that are contained in astrocyte-to-astrocyte (A/A) gap junctions; oligodendrocytes express a different set of three connexins (Cx29, Cx32, and Cx47) that are contained in the oligodendrocyte side of necessarily heterotypic astrocyte-to-oligodendrocyte (A/O) gap junctions, and there is little ultrastructural evidence for gap junction formation between individual oligodendrocytes. In addition, primarily Cx29 and Cx32 are contained deeper in myelin sheaths, where they form autologous gap junctions at sites of uncompacted myelin. The presence of six connexins in macroglial cell populations has revealed unprecedented complexity of potential connexin coupling partners, and with restricted deployment of gap junctional intercellular communication (GJIC) within the "pan-glial" syncytium. New implications for the organization and regulation of spatial buffering mediated by glial GJIC are derived from recent observations of the existence of separate astrocyte anatomical domains, with only narrow regions of overlap between astrocyte processes at domain borders. Thus, widespread spatial buffering in the CNS may occur not successively through a multitude of processes arising from different astrocytes, but rather in a more orderly fashion from one astrocyte domain to another via intercellular coupling that occurs only at restricted regions of overlap between astrocyte domains, augmented by autocellular coupling that occurs within each domain.

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.

We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxy-terminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.

Connexin43 (Cx43), a component of gap junctions, has a relatively large carboxy-terminal region with multiple proteomic interactions. Proteomic interactions with its cytoplasmic loop, however, are poorly defined. The goal of this study is to examine proteomic interactions involving the cytoplasmic loop (CL) of Cx43. The authors utilized various techniques, including glutathione-S-transferase (GST) pull-down, immunoblot analysis, two-dimensional (2D) gel electrophoresis, and mass spectrometry, to elucidate binding partners for Cx43-CL. The authors identified novel interactions with Cx43-CL involving α- and β-tubulin, myelin basic protein, and Purα. Because tubulin interacts with the C-terminus of Cx43 (Cx43-CT), the authors further investigated the nature of the interaction between β-tubulin and Cx43-CL. β-Tubulin binds with the full length of Cx43-CL with approximately one-fifth the affinity of the interaction between Cx43-CT and β-tubulin. This study demonstrates novel proteomic interactions involving Cx43-CL that may lead to a more complete understanding of trafficking and gating of gap junction channels.

Pannexins are mammalian orthologs of innexins and have a predicted topological folding pattern similar to that of connexins, except they are glycosylated. Rat pannexin 1 is glycosylated at N254 and this residue is important for plasma membrane targeting. Here we demonstrate that cell surface expression levels of the rat pannexin 1 N254Q mutant are rescued by coexpression with the wild-type protein. In paired Xenopus oocytes, the functional effect of this rescue is inconsequential; however, cell surface deglycosylation by PNGase F significantly enhanced functional gap junction formation. In mammalian cells, wild-type oligomers traffic at a slower rate than Myc-or tetracysteine domain-tagged versions, a behavior opposite to that of tagged connexins. The temporal differences of Panx1 trafficking correlate with spatial differences of intracellular localizations induced by Golgi blockage by Brefeldin-A or glycosylation prevention by tunicamycin. Therefore, Panx1 has kinetics and dynamics that make it unique to serve distinct functions separate from connexin-based channels.

Connexin oligomerization and trafficking are regulated processes. To identify proteins that control connexin 43 (Cx43), a screen was designed using HeLa cells expressing a Cx43 construct with di-lysine endoplasmic reticulum (ER)-retention/retrieval motif, Cx43-HKKSL. At moderate levels of expression, Cx43-HKKSL is retained in the ER as monomers; however, Cx43-HKKSL stably overexpressed by HeLa cells localizes to the perinuclear region and oligomerizes. HeLa/Cx43-HKKSL overexpressors were transiently transfected with pooled clones from a human kidney cDNA library and used immunofluorescence microscopy to identify cDNAs that enabled overexpressed Cx43-HKKSL to convert from a perinuclear to ER localization pattern. Using this approach, a small molecular weight GTPase, rab20, was identified as a candidate protein with the ability to regulate Cx43 trafficking. Enhanced green fluorescent protein (EGFP)-tagged rab20 showed a predominantly perinuclear and ER localization pattern and caused wild-type Cx43 to be retained inside the cell. By contrast, mutant EGFP-rab20T19N, which lacks the ability to bind GTP, had no effect on Cx43. These results suggest Cx43 is transported through an intracellular compartment regulated by rab20 along the secretory pathway.

It is often difficult to determine molecular mechanisms leading to early embryonic lethality of genetically modified mice due to lack of cells for further analyses. The authors describe here establishment of mouse embryonic fibroblast (MEF) cell lines from gastrulation stage embryos. In this example, using a combination of in vivo and in vitro techniques, the authors successfully generated MEF cell lines that lack both fibronectin (FN) and focal adhesion kinase (FAK).