PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Molecular basis for CD40 signaling mediated by TRAF3

Tumor necrosis factor receptors (TNFR) are single transmembrane-spanning glycoproteins that bind cytokines and trigger multiple signal transduction pathways. Many of these TNFRs rely on interactions with TRAF proteins that bind to the intracellular domain of the receptors. CD40 is a member of the TNFR family that binds to several different TRAF proteins. We have determined the crystal structure of a 20-residue fragment from the cytoplasmic domain of CD40 in complex with the TRAF domain of TRAF3. The CD40 fragment binds as a hairpin loop across the surface of the TRAF domain. Residues shown by mutagenesis and deletion analysis to be critical for TRAF3 binding are involved either in direct contact with TRAF3 or in intramolecular interactions that stabilize the hairpin. Comparison of the interactions of CD40 with TRAF3 vs. TRAF2 suggests that CD40 may assume different conformations when bound to different TRAF family members. This molecular adaptation may influence binding affinity and specific cellular triggers.

Roles for genomic imprinting and the zygotic genome in placental development

The placenta contains several types of feto-maternal interfaces where zygote-derived cells interact with maternal cells or maternal blood for the promotion of fetal growth and viability. The genetic factors regulating the interactions between different cell types within feto-maternal interfaces and the relative contributions of the maternal and zygotic genomes are poorly understood. Genomic imprinting, the epigenetic process responsible for parental origin-dependent functional differences between homologous chromosomes, has been proposed to contribute to these events. Previous studies showed that mouse conceptuses with an absence of imprinted differences between the two copies of chromosome 12 (upon paternal inheritance of both copies) die late in gestation and have a variety of defects, including placentomegaly. Here we examined the role of chromosome 12 imprinting in these placentae in more detail. We show that the spatial interactions between different cell types within feto-maternal interfaces are defective and identify abnormal behaviors in both zygote-derived and maternal cells that are attributed to the genome of the zygote but not the mother. These include compromised invasion of the maternal decidualized endometrium and the central maternal artery situated within it by zygote-derived trophoblast, abnormalities in the wall of the central maternal artery, and defects within the zygote-derived cellular layer of the labyrinth, which is in direct contact with maternal blood. These findings demonstrate multiple roles for chromosome 12 imprinting in the placenta that have not previously been associated with imprinting effects. They provide insights into the function of imprinting in placental development and have evolutionary and clinical implications.

Intracellular pH in Arbuscular Mycorrhizal Fungi1 : A Symbiotic Physiological Marker

A method was developed to perform real-time analysis of cytosolic pH of arbuscular mycorrhizal fungi in culture using dye and ratiometric measurements (490/450 nm excitations). The study was mainly performed using photometric analysis, although some data were confirmed using image analysis. The use of nigericin allowed an in vivo calibration. Experimental parameters such as loading time and concentration of the dye were determined so that pH measurements could be made for a steady-state period on viable cells. A characteristic pH profile was observed along hyphae. For Gigaspora margarita, the pH of the tip (0–2 μm) was typically 6.7, increased sharply to 7.0 behind this region (9.5 μm), and decreased over the next 250 μm to a constant value of 6.6. A similar pattern was obtained for Glomus intraradices. The pH profile of G. margarita germ tubes was higher when cultured in the presence of carrot (Daucus carota) hairy roots (nonmycorrhizal). Similarly, extraradical hyphae of G. intraradices had a higher apical pH than the germ tubes. The use of a paper layer to prevent the mycorrhizal roots from being in direct contact with the medium selected hyphae with an even higher cytosolic pH. Results suggest that this method could be useful as a bioassay for studying signal perception and/or H+ cotransport of nutrients by arbuscular mycorrhizal hyphae.

TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors

To achieve an efficient intracellular drug and DNA delivery, attempts were made to target microparticulate drug carriers into cytoplasm bypassing the endocytotic pathway. TAT peptides derived from the HIV-1 TAT protein facilitate intracellular delivery of proteins and small colloidal particles. We demonstrated that relatively large drug carriers, such as 200-nm liposomes, can also be delivered into cells by TAT peptide attached to the liposome surface. Liposomes were fluorescently labeled with membranotropic rhodamine-phosphatidylethanolamine or by entrapping FITC-dextran. Incubation of fluorescent TAT liposomes with mouse Lewis lung carcinoma cells, human breast tumor BT20 cells, and rat cardiac myocyte H9C2 results in intracellular localization of certain liposomes. Steric hindrances for TAT peptide⋅cell interaction (attachment of TAT directly to the liposome surface without spacer or the presence of a high MW polyethylene glycol on the liposome surface) abolish liposome internalization, evidencing the importance of direct contact of TAT peptide with the cell surface. Low temperature or metabolic inhibitors, sodium azide or iodoacetamide, have little influence on the translocation of TAT liposomes into cells, confirming the energy-independent character of this process. The approach may have important implications for drug delivery directly into cell cytoplasm.

Antisense ribosomes: rRNA as a vehicle for antisense RNAs.

Although rRNA has a conserved core structure, its size varies by more than 2000 bases between eubacteria and vertebrates, mostly due to the size variation of discrete variable regions. Previous studies have shown that insertion of foreign sequences into some of these variable regions has little effect on rRNA function. These properties make rRNA a potentially very advantageous vehicle to carry other RNA moieties with biological activity, such as "antisense RNAs." We have explored this possibility by inserting antisense RNAs targeted against one essential and two nonessential genes into a site within a variable region in the Tetrahymena thermophila large subunit rRNA gene. Expression of each of the three genes tested can be drastically reduced or eliminated in transformed T. thermophila lines containing these altered rRNAs. In addition, we found that only antisense rRNAs containing RNA sequences complementary to the 5' untranslated region of the targeted mRNA were effective. Lines containing antisense rRNAs targeted against either of the nonessential genes grow well, indicating that the altered rRNAs fulfill their functions within the ribosome. Since functional rRNA is extremely abundant and stable and comes into direct contact with translated mRNAs, it may prove to be an unparalleled vehicle for enhancing the activity of functional RNAs that act on mRNAs.

Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events and antigenic spread in systemic lupus erythematosus.

The restriction of phosphatidylserine (PtdSer) to the inner surface of the plasma membrane bilayer is lost early during apoptosis. Since PtdSer is a potent surface procoagulant, and since there is an increased incidence of coagulation events in patients with systemic lupus erythematosus (SLE) who have anti-phospholipid antibodies, we addressed whether apoptotic cells are procoagulant and whether anti-phospholipid antibodies influence this. Apoptotic HeLa cells, human endothelial cells, and a murine pre-B-cell line were markedly procoagulant in a modified Russell viper venom assay. This procoagulant effect was entirely abolished by addition of the PtdSer-binding protein, annexin V, confirming that it was PtdSer-dependent. The procoagulant effect was also abolished by addition of IgG purified from the plasma of three patients with anti-phospholipid antibody syndrome, but not IgG from normal controls. Confocal microscopy of apoptotic cells stained with fluorescein-isothiocyanate-conjugated-annexin V demonstrated (Ca2+)-dependent binding to the surface of membrane blebs o apoptotic cells, but not to intracellular membranes. Recent data indicate that the surface blebs of apoptotic cells constitute an important immunogenic particle in SLE. We propose that the PtdSer exposed on the outside of these blebs can induce the production of anti-phospholipid antibodies, which might also enhance the immunogenicity of the bleb contents. When apoptosis occurs in a microenvironment in direct contact with circulating plasma, the unique procoagulant consequences of the apoptotic surface may additionally be expressed. This might explain the increased incidence of pathological intravascular coagulation events that occur in some lupus patients who have anti-phospholipid antibodies.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Interactions between the retinoid X receptor and a conserved region of the TATA-binding protein mediate hormone-dependent transactivation.

The retinoid X receptor (RXR) participates in a wide array of hormonal signaling pathways, either as a homodimer or as a heterodimer, with other members of the steroid and thyroid hormone receptor superfamily. In this report the ligand-dependent transactivation function of RXR has been characterized, and the ability of RXR to interact with components of the basal transcription machinery has been examined. In vivo and in vitro experiments indicate the RXR ligand-binding domain makes a direct, specific, and ligand-dependent contact with a highly conserved region of the TATA-binding protein. The ability of mutations that reduce ligand-dependent transcription by RXR to disrupt the RXR-TATA-binding protein interaction in vivo and in vitro suggests that RXR makes direct contact with the basal transcription machinery to achieve activation.

Segregation of Two Spectrin Isoforms: Polarized Membrane-binding Sites Direct Polarized Membrane Skeleton Assembly

Spectrin isoforms are often segregated within specialized plasma membrane subdomains where they are thought to contribute to the development of cell surface polarity. It was previously shown that ankyrin and β spectrin are recruited to sites of cell–cell contact in Drosophila S2 cells expressing the homophilic adhesion molecule neuroglian. Here, we show that neuroglian has no apparent effect on a second spectrin isoform (αβH), which is constitutively associated with the plasma membrane in S2 cells. Another membrane marker, the Na,K-ATPase, codistributes with ankyrin and αβ spectrin at sites of neuroglian-mediated contact. The distributions of these markers in epithelial cells in vivo are consistent with the order of events observed in S2 cells. Neuroglian, ankyrin, αβ spectrin, and the Na,K-ATPase colocalize at the lateral domain of salivary gland cells. In contrast, αβH spectrin is sorted to the apical domain of salivary gland and somatic follicle cells. Thus, the two spectrin isoforms respond independently to positional cues at the cell surface: in one case an apically sorted receptor and in the other case a locally activated cell–cell adhesion molecule. The results support a model in which the membrane skeleton behaves as a transducer of positional information within cells.

Two contact regions between Stat1 and CBP/p300 in interferon γ signaling

Interferon γ (IFN-γ) induces rapid tyrosine phosphorylation of the latent cytoplasmic transcription factor, Stat1, which then forms homodimers, translocates to the nucleus and participates in IFN-γ-induced transcription. However, little is known of the interactions between Stat1 and the general transcription machinery during transcriptional activation. We show here that Stat1 can directly interact with the CREB-binding protein (CBP)/p300 family of transcriptional coactivators. Specifically, two interaction regions were identified: the amino-terminal region of Stat1 interacts with the CREB-binding domain of CBP/p300 and the carboxyl-terminal region of Stat1 interacts with the domain of CBP/p300 that binds adenovirus E1A protein. Transfection experiments suggest a role for these interactions in IFN-γ-induced transcription. Because CBP/p300-binding is required for the adenovirus E1A protein to regulate transcription of many genes during viral replication and cellular transformation, it is possible that the anti-viral effect of IFN-γ is based at least in part on direct competition by nuclear Stat1 with E1A for CBP/p300 binding.