Multi-domain GFP-like proteins from two species of marine hydrozoans.

Proteins homologous to Green Fluorescent Protein (GFP) are widely used as genetically encoded fluorescent labels. Many developments of this technology were spurred by discoveries of novel types of GFP-like proteins (FPs) in nature. Here we report two proteins displaying primary structures never before encountered in natural FPs: they consist of multiple GFP-like domains repeated within the same polypeptide chain. A two-domain green FP (abeGFP) and a four-domain orange-fluorescent FP (Ember) were isolated from the siphonophore Abylopsis eschscholtzii and an unidentified juvenile jellyfish (order Anthoathecata), respectively. Only the most evolutionary ancient domain of Ember is able to synthesize an orange-emitting chromophore (emission at 571 nm), while the other three are purely green (emission at 520 nm) and putatively serve to maintain the stability and solubility of the multidomain protein. When expressed individually, two of the green Ember domains form dimers and the third one exists as a monomer. The low propensity for oligomerization of these domains would simplify their adoption as in vivo labels. Our results reveal a previously unrecognized direction in which natural FPs have diversified, suggesting new avenues to look for FPs with novel and potentially useful features.

Multi-colored homologs of the green fluorescent protein from hydromedusa Obelia sp.

The presence of green fluorescent protein (GFP) within the bioluminescent system of Obelia (Cnidaria, Hydrozoa, Campanulariidae) was inferred shortly after the discovery of GFP in Aequorea. Despite the enormous success of Aequorea GFP as a genetically encoded fluorescent label, Obelia GFP thus far has been defeating attempts to clone it from the hydroid life cycle stage. Here, we report cloning of three GFP-like fluorescent proteins (FPs) from Obelia medusa, representing cyan, green, and yellow spectral types. Such color diversity has never been detected outside class Anthozoa, suggesting a more general function for multi-colored fluorescence in cnidarians than has been previously hypothesized. An unusual property of the new FPs is the formation of large soluble complexes of well-defined sizes and molecular weights, corresponding to up to 128 individual polypeptides. This aligns well with the earlier observation that luminescence in Obelia, unlike in Aequorea, is localized within subcellular granules, which prompts further inquiry into the self-assembly properties of the new FPs and their interactions with the photoprotein. The discovery of Obelia FPs fills the four-decade-old gap in the knowledge of cnidarian bioluminescence and provides experimental material to further investigate the details of its molecular mechanism.

Nuclear speckle-associated protein Pnn/DRS binds to the transcriptional corepressor CtBP and relieves CtBP-mediated repression of the E-cadherin gene.

Previously, we have shown that pinin/DRS (Pnn), a 140-kDa nuclear and cell adhesion-related phosphoprotein, is involved in the regulation of cell adhesion and modulation of the activity of multiple tumor suppressor genes. In the nucleus Pnn is concentrated in the "nuclear speckles," zones of accumulation of transcriptional and mRNA splicing factors, where Pnn is involved in mRNA processing. Alternatively, other roles of Pnn in gene regulation have not yet been established. By utilizing in vitro pull-down assays, in vivo interaction studies, and immunofluorescence in combination with overexpression and RNA interference experiments, we present evidence that Pnn interacts with the known transcriptional corepressor CtBP1. As a consequence of this interaction Pnn was capable of relieving the CtBP1-mediated repression of E-cadherin promoter activity. Our results suggest that the interaction of Pnn with the corepressor CtBP1 may modulate repression of transcription by CtBP1. This interaction may reflect the existence of coupling factors involved in CtBP-mediated transcriptional regulation and mRNA processing events.

Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis.

Mycoplasma alligatoris causes acute lethal cardiopulmonary disease of susceptible hosts. A survey of its genome implicated sialidase and hyaluronidase, synergistic regulators of hyaluronan receptor CD44-mediated signal transduction leading to apoptotic cell death, as virulence factors of M. alligatoris. In this study, after the existence of a CD44 homolog in alligators was established by immunolabeling primary pulmonary fibroblasts with monoclonal antibody IM7 against murine CD44, the sialidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was used to examine the effects of sialidase on fibroblast apoptosis following in vitro infection with M. alligatoris. While their CD44 expression remained constant, infected cells exhibited morphologic changes characteristic of apoptosis including decreased size, rounding, disordered alpha-tubulin, and nuclear disintegration compared to untreated controls. DANA was a potent, non-toxic inhibitor of the sialidase activity, equivalent to about 1mU of Clostridium perfringens Type VI sialidase, expressed by M. alligatoris in the inoculum. Although DANA did not measurably reduce the proportion of infected fibroblasts labeled by a specific ligand of activated caspases, co-incubation with DANA protected (P<0.01) fibroblasts in a concentration-dependent fashion from the M. alligatoris-induced trends toward increased apoptosis receptor CD95 expression, and increased 5-bromo-2'-deoxyuridine incorporation measured in a terminal dUTP nick end-labeling apoptosis assay. In contrast, incubation with 200-fold excess purified C. perfringens sialidase alone did not affect CD95 expression or chromatin integrity, or induce fibroblast apoptosis. From those observations we conclude that interaction of its sialidase with hyaluronidase or another virulence factor(s) is necessary to elicit the pro-apoptotic effects of M. alligatoris infection.

Reduction of Pnn by RNAi induces loss of cell-cell adhesion between human corneal epithelial cells.

PURPOSE: Pinin (Pnn/DRS/memA) plays an important role in regulating cell-cell adhesion of corneal epithelial cells. In the nucleus, Pnn interacts with both transcriptional repressor and pre-mRNA processing machinery. Here we investigated the consequences of "knocking down" Pnn expression with short hairpin RNAi (shRNAi) on the corneal epithelial cell phenotype. METHODS: Cultured human corneal epithelial (HCE-T) cells were cotransfected with a shRNAi-expressing construct containing an inverted repeat of a Pnn specific 21 nucleotide sequence (Pnn shRNAi) and a GFP vector as a marker of transfected cells. After 24-48 h, cells were fixed and immunostained with antibodies against Pnn, keratin, desmoplakin, desmoglein, E-cadherin, ZO-1, SR-proteins, and SRm300. To demonstrate specificity of the Pnn knock down, a rescue vector was designed by incorporating three conservative nucleotide substitutions within the Pnn-shRNAi targeting sequences of the full length Pnn-GFP construct, thus generating a Pnn construct to produce mRNA that Pnn shRNAi could not target (Pnn-CS3-GFP). RESULTS: HCE-T cells were cotransfected with Pnn shRNAi and GFP vectors and after 24 and 48 h exhibited significantly reduced immunostaining for Pnn. Western blot analyses of Pnn and E-cadherin protein expression in cells transfected with Pnn-shRNAi and GFP vectors revealed marked reduction in levels of both proteins compared to those observed in cells transfected with GFP alone. The cells receiving Pnn-shRNAi appeared to be less adherent to neighboring nontransfected cells, often exhibited altered cell shape, downregulated cell adhesion and cell junction molecules, and escaped from the epithelium. The Pnn shRNAi transfected cells exhibited fewer keratin filaments anchored to desmosomes and a concurrent increase in the perinuclear bundling of filaments. SR proteins and SRm300 showed an altered distribution in the Pnn knock down cells. Cotransfection of Pnn-CS3-GFP with Pnn shRNAi demonstrated that the conservatively mutated Pnn could maintain cell-cell adhesion. CONCLUSIONS: Our results indicate that knocking down Pnn expression leads to a loss of epithelial cell-cell adhesion, changes in cell shape, and movement of Pnn shRNAi transfected cells out of the epithelium. We suggest that Pnn plays an integral role in the establishment and maintenance of epithelial cell-cell adhesion via its activity within nuclear multi-protein complexes.

Very bright green fluorescent proteins from the Pontellid copepod Pontella mimocerami.

BACKGROUND: Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development. RESULTS: Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M(-1) cm(-)) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness. CONCLUSIONS: The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.

Diversity and evolution of coral fluorescent proteins.

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.