Patterning of Caenorhabditis elegans posterior structures by the Abdominal-B homolog, egl-5.
(9/8843)
The Caenorhabditis elegans body axis, like that of other animals, is patterned by the action of Hox genes. In order to examine the function of one C. elegans Hox gene in depth, we determined the postembryonic expression pattern of egl-5, the C. elegans member of the Abdominal-B Hox gene paralog group, by means of whole-mount staining with a polyclonal antibody. A major site of egl-5 expression and function is in the epithelium joining the posterior digestive tract with the external epidermis. Patterning this region and its derived structures is a conserved function of Abd-B paralog group genes in other animals. Cells that initiate egl-5 expression during embryogenesis are clustered around the presumptive anus. Expression is initiated postembryonically in four additional mesodermal and ectodermal cell lineages or tissues. Once initiated in a lineage, egl-5 expression continues throughout development, suggesting that the action of egl-5 can be regarded as defining a positional cell identity. A variety of cross-regulatory interactions between egl-5 and the next more anterior Hox gene, mab-5, help define the expression domains of their respective gene products. In its expression in a localized body region, function as a marker of positional cell identity, and interactions with another Hox gene, egl-5 resembles Hox genes of other animals. This suggests that C. elegans, in spite of its small cell number and reproducible cell lineages, may not differ greatly from other animals in the way it employs Hox genes for regional specification during development. (+info)
Tagging the human immunodeficiency virus gag protein with green fluorescent protein. Minimal evidence for colocalisation with actin.
(10/8843)
The assembly and budding of human immunodeficiency virus type 1, encoded solely in the Gag protein precursor Pr55Gag, occur at the plasma membrane of infected cells. However, little is known about the routing of the Gag molecule from its site of synthesis in the cytoplasm to the site of budding, with past studies suggesting that the cytoskeleton, particularly actin, may be involved in the translocation. We have constructed a T7 promoter-driven gag gene fusion with green fluorescent protein (GFP) that expresses Gag-GFP in both cells and supernatant. The distribution of Gag-GFP was the same as Gag only, suggesting that cellular routing was not affected by fusion to GFP, and using colabelling techniques, Gag-GFP was shown to have no particular colocalisation with actin. After detergent extraction of expressing cells, Gag and Gag-GFP remained cell associated, whereas GFP only was wholly released. These data suggest that Gag may associate with other cytoskeletal components or, perhaps more likely, that a partial assembly to a large-molecular-weight intermediate occurs before localisation at the plasma membrane. (+info)
Native display of complete foreign protein domains on the surface of hepatitis B virus capsids.
(11/8843)
The nucleocapsid of hepatitis B virus (HBV), or HBcAg, is a highly symmetric structure formed by multiple dimers of a single core protein that contains potent T helper epitopes in its 183-aa sequence. Both factors make HBcAg an unusually strong immunogen and an attractive candidate as a carrier for foreign epitopes. The immunodominant c/e1 epitope on the capsid has been suggested as a superior location to convey high immunogenicity to a heterologous sequence. Because of its central position, however, any c/e1 insert disrupts the core protein's primary sequence; hence, only peptides, or rather small protein fragments seemed to be compatible with particle formation. According to recent structural data, the epitope is located at the tips of prominent surface spikes formed by the very stable dimer interfaces. We therefore reasoned that much larger inserts might be tolerated, provided the individual parts of a corresponding fusion protein could fold independently. Using the green fluorescent protein (GFP) as a model insert, we show that the chimeric protein efficiently forms fluorescent particles; hence, all of its structurally important parts must be properly folded. We also demonstrate that the GFP domains are surface-exposed and that the chimeric particles elicit a potent humoral response against native GFP. Hence, proteins of at least up to 238 aa can be natively displayed on the surface of HBV core particles. Such chimeras may not only be useful as vaccines but may also open the way for high resolution structural analyses of nonassembling proteins by electron microscopy. (+info)
Dynamic and quantitative Ca2+ measurements using improved cameleons.
(12/8843)
Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling. (+info)
Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin-green fluorescent protein.
(13/8843)
Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN-green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites. (+info)
CLIP-170 highlights growing microtubule ends in vivo.
(14/8843)
A chimera with the green fluorescent protein (GFP) has been constructed to visualize the dynamic properties of the endosome-microtubule linker protein CLIP170 (GFP-CLIP170). GFP-CLIP170 binds in stretches along a subset of microtubule ends. These fluorescent stretches appear to move with the growing tips of microtubules at 0.15-0.4 microm/s, comparable to microtubule elongation in vivo. Analysis of speckles along dynamic GFP-CLIP170 stretches suggests that CLIP170 treadmills on growing microtubule ends, rather than being continuously transported toward these ends. Drugs affecting microtubule dynamics rapidly inhibit movement of GFP-CLIP170 dashes. We propose that GFP-CLIP170 highlights growing microtubule ends by specifically recognizing the structure of a segment of newly polymerized tubulin. (+info)
GABA- and glutamate-activated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice.
(15/8843)
Mice were generated expressing green fluorescent protein (GFP) under the control of the gonadotropin-releasing hormone (GnRH) promoter. Green fluorescence was observed in, and restricted to, GnRH-immunopositive neuronal somata in the olfactory bulb, ganglion terminale, septal nuclei, diagonal band of Broca (DBB), preoptic area (POA), and caudal hypothalamus, as well as GnRH neuronal dendrites and axons, including axon terminals in the median eminence and organum vasculosum of the lamina terminalis (OVLT). Whole-cell recordings from GFP-expressing GnRH neurons in the OVLT-POA-DBB region revealed a firing pattern among GFP-expressing GnRH neurons distinct from that of nonfluorescent neurons. Nucleated patches of GFP-expressing GnRH neurons exhibited pronounced responses to fast application of GABA and smaller responses to L-glutamate and AMPA. One-fifth of the nucleated patches responded to NMDA. The GABA-A, AMPA, and NMDA receptor channels on GnRH neurons mediating these responses may play a role in the modulation of GnRH secretory oscillations. (+info)
Selective activation of heterologously expressed G protein-gated K+ channels by M2 muscarinic receptors in rat sympathetic neurones.
(16/8843)
1. G protein-regulated inward rectifier K+ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1- and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes. 2. Co-transfected cells had larger Ba2+-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mM [K+]o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone. 3. Carbachol (CCh, 1-30 microM) increased the inwardly rectifying current in 70 % of GIRK1+ GIRK2-transfected cells by 261 +/- 53 % (n = 6, CCh 30 microM) at -120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents. 4. The effect of CCh was antagonized by 6 nM tripitramine but not by 100 nM pirenzepine, consistent with activation of endogenous M2 muscarinic acetylcholine receptors. 5. In contrast, inhibition of the voltage-activated Ca2+ current by CCh was antagonized by 100 nM pirenzepine but not by 6 nM tripitramine, indicating that it was mediated by M4 muscarinic acetylcholine receptors. 6. We conclude that endogenous M2 and M4 muscarinic receptors selectively couple to GIRK currents and Ca2+ currents respectively, with negligible cross-talk. (+info)