Green Fluorescent Protein Chimeras Research Articles

Protein kinase A-regulated membrane trafficking of a green fluorescent protein-aquaporin 5 chimera in MDCK cells

The green fluorescent protein (GFP) of the jellyfish, Aeqorea victoria, was used as an autofluorescent tag to track the trafficking of aquaporin 5 (AQP5), an exocrine gland-type water channel. Two groups of chimeric proteins were constructed; one in which GFP was fused to the amino-terminus of AQP5 (GFP–AQP5) and the other, in which it was fused to the carboxyl terminus of it (AQP5–GFP). In each group, 2 chimeras were produced, a wild-type AQP5 with its normal sequence and a mutant AQP5 having a mutated amino acid at 259, i.e., GFP–AQP5–T259A and AQP5–GFP–T259A. They were used to transfect Madin–Darby canine kidney (MDCK) cells. The GFP–AQP5 chimera was localized in the intracellular vesicles, which trafficked to the plasma membrane in response to N 6, 2′- O-dibutyryladenosine 3′, 5′-cyclic monophosphate (dbcAMP). Membrane trafficking was inhibited by N-[2-( p-bromocinnamylamino)ethyl]-5-isoquimolinesulfonamide (H-89) but not by palmitoyl- dl-carnitine chloride (PCC). In contrast, the AQP5–GFP chimera expressed in MDCK cells was localized constitutively on the plasma membrane. The cellular localization of the latter chimera was not affected by stimulation with dbcAMP in the presence or absence of H-89 or PCC. Replacement of Thr-259 with Ala-259 did not affect the dbcAMP-induced translocation of the chimeric protein, suggesting that phosphorylation of Thr-259 was not necessary for AQP5 trafficking under the present experimental conditions. Thus, the GFP–AQP5 chimera will be a useful tool to study AQP5 trafficking in vitro, whereas the constitutive membrane localization of the AQP5–GFP chimera suggests the importance of the carboxyl terminus of the AQP5 protein for its sorting, whether it is translocated to intracellular vesicles or to the plasma membrane.

Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.

Docosahexaenoic acid selectively inhibits plasma membrane targeting of lipidated proteins

Membrane localization of lipidated cytosolic signaling proteins is mediated by interactions between specific lipid anchors and membranes, but little is known about the regulatory role of membrane composition in lipidated protein membrane targeting. Here, using green fluorescent protein (GFP) chimeras and quantitative fluorescence microscopy in living mouse colonocytes, we show that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid (PUFA) with membrane lipid-modifying properties, selectively inhibits plasma membrane (PM) targeting and increases the endomembrane localization of lipidated proteins that are cytoplasmic cargo in the exocytic pathway, without affecting the exocytic pathway itself. DHA selectivity seems to be dictated by the protein trafficking route, independent of the functional state of proteins and the location and composition of membrane anchors. DHA enrichment in cell membranes was required to elicit the inhibitory effect. These data reveal that membrane lipid composition influences cell signaling by modulating intracellular trafficking and localization of membrane proteins, providing a potential molecular mechanism for the documented health benefits of DHA.

Localization of a flavonoid biosynthetic polyphenol oxidase in vacuoles

Aureusidin synthase, a polyphenol oxidase (PPO), specifically catalyzes the oxidative formation of aurones from chalcones, which are plant flavonoids, and is responsible for the yellow coloration of snapdragon (Antirrhinum majus) flowers. All known PPOs have been found to be localized in plastids, whereas flavonoid biosynthesis is thought to take place in the cytoplasm [or on the cytoplasmic surface of the endoplasmic reticulum (ER)]. However, the primary structural characteristics of aureusidin synthase and some of its molecular properties argue against localization of the enzyme in plastids and the cytoplasm. In this study, the subcellular localization of the enzyme in petal cells of the yellow snapdragon was investigated. Sucrose-density gradient and differential centrifugation analyses suggested that the enzyme (the 39-kDa mature form) is not located in plastids or on the ER. Transient assays using a green fluorescent protein (GFP) chimera fused with the putative propeptide of the PPO precursor suggested that the enzyme was localized within the vacuole lumen. We also found that the necessary information for vacuolar targeting of the PPO was encoded within the 53-residue N-terminal sequence (NTPP), but not in the C-terminal sequence of the precursor. NTPP-mediated ER-to-Golgi trafficking to vacuoles was confirmed by means of the co-expression of an NTPP-GFP chimera with a dominant negative mutant of the Arabidopsis GTPase Sar1 or with a monomeric red fluorescent protein (mRFP)-fused Golgi marker (an H+-translocating inorganic pyrophosphatase of Arabidopsis). We identified a sequence-specific vacuolar sorting determinant in the NTPP of the precursor. We have demonstrated the biosynthesis of a flavonoid skeleton in vacuoles. The findings of this metabolic compartmentation may provide a strategy for overcoming the biochemical instability of the precursor chalcones in the cytoplasm, thus leading to the efficient accumulation of aurones in the flower.

Large-scale translocation reversal within the thylakoid Tat system in vivo

In vitro import assays have shown that the thylakoid twin-arginine translocase (Tat) system transports folded proteins in a unidirectional manner. Here, we expressed a natural substrate, pre-23K, and a 23K presequence–green fluorescent protein (GFP) chimera in vivo in tobacco protoplasts. Both are imported into chloroplasts, targeted to the thylakoids, and processed to the mature size by the lumen-facing processing peptidase. However, the vast majority of mature GFP and about half of the 23K are then returned to the stroma. Mutations in the twin-arginine motif block thylakoid targeting and maturation, confirming an involvement of the Tat apparatus. Mutation of the processing site yields membrane-associated intermediate-size protein in vivo, indicating a delayed reversal of translocation to the stroma and suggesting a longer lived interaction with the Tat machinery. We conclude that, in vivo, the Tat system can reject substrates at a late stage in translocation and on a very large scale, indicating the influence of factors that are absent in reconstitution assays.

The Steady‐State Distribution of Glycosyltransferases Between the Golgi Apparatus and the Endoplasmic Reticulum is Approximately 90:10

Several lines of evidence support a novel model for Golgi protein residency in which these proteins cycle between the Golgi apparatus and the endoplasmic reticulum (ER). However, to preserve the functional distinction between the two organelles, this pool of ER-resident Golgi enzymes must be small. We quantified the distribution for two Golgi glycosyltransferases in HeLa cells to test this prediction. We reasoned that best-practice, quantitative solutions would come from treating images as data arrays rather than pictures. Using deconvolution and computer calculated organellar boundaries, the Golgi fraction for both endogenous beta1,4-galactosyltransferase and UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase 2 fused with green fluorescent protein (GFP) was 91% by fluorescence microscopy. Immunogold labeling followed by electron microscopy and model analysis yielded a similar value. Values reflect steady-state conditions, as inclusion of a protein synthesis inhibitor had no effect. These data strongly suggest that the fluorescence of a GFP chimera with an organellar protein can be a valid indicator of protein distribution and more generally that fluorescent microscopy can provide a valid, rapid approach for protein quantification. In conclusion, we find the ER pool of cycling Golgi glycosyltransferases is small and approximately 1/100 the concentration found in the Golgi apparatus.

Processing of ovine [formula omitted] chimeras containing Asn 138 and Cys 151 polymorphisms

Polymorphisms in the prion protein, PrP C, affect the susceptibility of sheep to scrapie. Three rare polymorphisms, M137T, S138N, and R151C, have been found in Icelandic sheep. Observations suggest that R151C may be associated with lower scrapie susceptibility, whereas S138N is neutral. The effects of the S138N and R151C polymorphisms on the cellular processing of PrP C were examined in a model system consisting of the expression of ovine PrP C-EGFP (green fluorescent protein) chimeras in the mouse neuroblastoma cell line N2a. Chimeras with the haplotypes A 136R 154Q 171 (ARQ), AN 138RQ, and AC 151RQ were compared. The chimeras did not differ regarding their translocation into the secretory system, glycosylation, and transport to the cell surface. However, the AC 151RQ chimera differed from the other chimeras regarding disulfide bonding characteristics; furthermore, a slight difference was detected between AC 151RQ and the other chimeras by limited proteolysis. The processing of the ARQ and AN 138RQ chimeras was identical in the experiments performed consistent with observations that it is neutral.

Cross-species Sequence Analysis Reveals Multiple Charged Residue-rich Domains That Regulate Nuclear/Cytoplasmic Partitioning and Membrane Localization of A Kinase Anchoring Protein 12 (SSeCKS/Gravin)

A kinase anchoring proteins (AKAPs) assemble and compartmentalize multiprotein signaling complexes at discrete subcellular locales and thus confer specificity to transduction cascades using ubiquitous signaling enzymes, such as protein kinase A. Intrinsic targeting domains in each AKAP determine the subcellular localization of these complexes and, along with protein-protein interaction domains, form the core of AKAP function. As a foundational step toward elucidating the relationship between location and function, we have used cross-species sequence analysis and deletion mapping to facilitate the identification of the targeting determinants of AKAP12 (also known as SSeCKS or Gravin). Three charged residue-rich regions were identified that regulate two aspects of AKAP12 localization, nuclear/cytoplasmic partitioning and perinuclear/cell periphery targeting. Using deletion mapping and green fluorescent protein chimeras, we uncovered a heretofore unrecognized nuclear localization potential. Five nuclear localization signals, including a novel class of this type of signal termed X2-NLS, are found in the central region of AKAP12 and are important for nuclear targeting. However, this nuclear localization is suppressed by the negatively charged C terminus that mediates nuclear exclusion. In this condition, the distribution of AKAP12 is regulated by an N-terminal targeting domain that simultaneously directs perinuclear and peripheral AKAP12 localization. Three basic residue-rich regions in the N-terminal targeting region have similarity to the MARCKS proteins and were found to control AKAP12 localization to ganglioside-rich regions at the cell periphery. Our data suggest that AKAP12 localization is regulated by a hierarchy of targeting domains and that the localization of AKAP12-assembled signaling complexes may be dynamically regulated.

Mitochondrial Dok-4 Recruits Src Kinase and Regulates NF-κB Activation in Endothelial Cells

The downstream of kinase (Dok) family of adapter proteins consists of at least five members structurally characterized by an NH2-terminal tandem of conserved pleckstrin homology and phosphotyrosine binding domains linked to a unique COOH-terminal region. To determine the role of the novel adapter protein Dok-4 in endothelial cells, we first investigated the cell localization of Dok-4. Most surprisingly, immunofluorescence microscopy, cell fractionation studies, and studies with enhanced green fluorescent protein chimeras showed that wild type Dok-4 (Dok-4-WT) specifically localized in mitochondria. An NH2-terminal deletion mutant of Dok-4 (Dok-4-(deltaN11-29)), which lacks the mitochondrial targeting sequence, could not accumulate in mitochondria. Co-immunoprecipitation revealed an interaction of c-Src with Dok-4-WT in endothelial cells. Most interestingly, overexpression of Dok-4-WT, but not Dok-4-(deltaN1-99), increased mitochondrial c-Src expression, whereas knock-down of endogenous Dok-4 with a small interfering RNA vector greatly inhibited mitochondrial localization of c-Src, suggesting a unique function for Dok-4 as an anchoring protein for c-Src in mitochondria. Dok-4-WT significantly decreased 39-kDa subunit complex I expression. PP2, a specific Src kinase inhibitor, prevented the Dok-4-mediated complex I decrease, suggesting the involvement of Src kinase in regulation of complex I expression. Dok-4-WT enhanced tumor necrosis factor-alpha (TNF-alpha)-mediated reactive oxygen species (ROS) production, supporting the functional relevance of a Dok-4-Src-complex I/ROS signaling pathway in mitochondria. Finally, Dok-4 enhanced TNF-alpha-mediated NF-kappaB activation, whereas this was inhibited by transfection with Dok-4 small interfering RNA. In addition, Dok-4-induced NF-kappaB activation was also inhibited by transfection of a dominant negative form of c-Src. These data suggest a role for mitochondrial Dok-4 as an anchoring molecule for the tyrosine kinase c-Src, and in turn as a regulator of TNF-alpha-mediated ROS production and NF-kappaB activation.

Trafficking of the major virulence factor to the surface of transfected P falciparum–infected erythrocytes

AbstractAfter invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P falciparum–infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P falciparum–infected RBC membrane.

Giardia lamblia: stable expression of green fluorescent protein mediated by giardiavirus

Giardia lamblia, an early diverging eukaryote that infects several species including humans and a major agent of water-borne diarrhea throughout the world, can be infected with a double-stranded RNA virus, giardiavirus (GLV). A chimeric GLV cDNA and green fluorescent protein (GFP) according to the cis-acting signals of the GLV genome required for expression of foreign gene was constructed and its in vitro transcript was electroporated into GLV-infected G. lamblia trophozoites, GFP was expressed transiently. pGDH5/NEO/GLV was constructed by combining the neomycin resistance cassette in which the neomycin phosphotransferase gene was flanked by Giardia glutamate dehydrogenase (GDH) uncoding regions and the transcription cassette in which the chimera of GLV cDNA and GFP was located downstream from GDH gene promoter on a single plasmid. This plasmid was electroporated into G. lamblia and the transfectants persistently expressed GFP under G418 selection. This stable transfection system should provide a valuable tool for genetic study of G. lamblia.

Membrane type 1-matrix metalloproteinase promotes human prostate cancer invasion and metastasis

Development of metastases requires cancer cells to breach underlying basement membrane, migrate through interstitial stroma and gain access to blood or lymphatic vessels. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been linked with these processes. Expression of MT1-MMP in human prostate cancer correlates with the stage of this disseminated disease. The mechanism underlying this observation, however, still remains to be understood. To study the role of MT1-MMP in prostate cancer dissemination, endogenous and recombinant MT1-MMP expressed in human prostate cancer cell lines (DU-145 and LNCaP) were examined. Using FITC-labeled Matrigel, a soluble basement membrane extract coated coverslips, LNCaP cells stably expressing a chimera of MT1-MMP and Green Fluorescent Protein (MT1-GFP) degraded Matrigel and readily migrated over degraded substrates. The degradation of Matrigel by LNCaP cells expressing MT1-GFP was sensitive to MMP inhibitors, CT-1746 and TIMP-2, but not TIMP-1. Cell migration was dramatically enhanced by expression of MT1-MMP. By employing surgical orthotopic implantation of LNCaP cells stably expressing MT1-GFP into the prostate gland of immunodeficient mice, we demonstrated that MT1-MMP promotes lymph node and lung metastasis of prostate cancer cells. Together, these results emphasize the pivotal role of MT1-MMP in prostate cancer dissemination and confirm that MT1-MMP is a suitable target to prevent cancer metastasis.

Mechanisms of Dense Core Vesicle Recapture following “Kiss and Run” (“Cavicapture”) Exocytosis in Insulin-secreting Cells

The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic beta-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane.

Regulation of nucleocytoplasmic localization of the atDjC6 chaperone protein

The sequence of the atDjC6 chaperone protein includes three potential nuclear localization signal (NLS) sequences (A-C) and three potential nuclear export signal (NES) sequences (X-Z). The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green-fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. The localization of the atDjC6::GFP chimera was coincident with that of the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation sensitive. Site-directed mutagenesis of the NES sequences increased the amount of each chimera that was nuclearly localized, indicating a decrease in nuclear export. When any pair of NLS sequences were appended to GFP, the chimera were entirely nuclearly localized. Quantitative two-hybrid analysis was used to verify that the decoding of NLS sequence information involves interaction with the NLS-receptor protein importin-alpha. Each of the NLS sequences is flanked by a site of potential Ser phosphorylation, and recombinant atDjC6 could be phosphorylated in vitro. Mutagenesis of Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-alpha. Our results are consistent with a regulated nucleocytoplasmic localization of the atDjC6 chaperone protein.

LdARF1 in trafficking and structural maintenance of the trans-Golgi cisternal network in the protozoan pathogen Leishmania donovani.

Adenosine diphosphate ribosylation factors (ARFs) are small guanosine-5'-triphosphatases that are essential in vesicular trafficking and in the maintenance of the Golgi network. In this report, we identified a homolog of the mammalian ARF1 in the human pathogenic protozoan parasite, Leishmania donovani (Ld). Ld ARF1 is a 549 bp gene encoding a 183-amino acid deduced protein of approximately 20 kDa. We demonstrated by Southern blot analysis that there are at least two copies of ARF1 in the Ld genome. Moreover, Northern blot analysis revealed that Ld ARF1 is expressed on a 1.35 kb transcript in both the insect vector (promastigotes) and mammalian host (amastigotes) forms of this parasite. Fluorescent microscopy studies using Ld promastigotes episomally transfected with an ARF1::GFP (green fluorescent protein) chimeric construct showed that such chimeras appeared to localize to the Golgi region of these organisms. This observation was verified by immunoelectron microscopy using an anti-GFP antibody. Such studies also revealed that Ld ARF1::GFP chimeras localized to trans-Golgi vesicles, the flagellar pocket/reservoir and other vesicles located between the trans-Golgi network and flagellar pocket in these apically polarized cells. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching experiments revealed both the dynamic binding and releasing activity of Ld ARF1 from the Golgi network in these parasites. Further, episomal expression of a constitutively active ("on") ARF1 (Q71L mutation) resulted in the aberrant swelling and distended-structure of the trans-Golgi cisternae in these cells. These results show that Ld ARF1 is transiently associated with the Golgi network and plays a role in the structural maintenance of this organelle in these important human pathogens.

Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.

Mutation of Tyrosine in the Conserved NPXXY Sequence Leads to Constitutive Phosphorylation and Internalization, but Not Signaling, of the Human B2 Bradykinin Receptor

Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/beta-adrenergic-like receptors. Exchange of Tyr(305) in the corresponding NPLVY sequence of the bradykinin B(2) receptor (B(2)R) for Ala resulted in a mutant, termed Y305A, that internalized [(3)H]bradykinin (BK) almost as rapidly as the wild-type (wt) B(2)R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B(2)R. Confocal fluorescence microscopy revealed that, in contrast to the B(2)R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B(2)R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G(q/11) without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B(2)R. We conclude, therefore, that the Y305A mutation of B(2)R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G(q/11), thereby limiting the extent of ligand-independent receptor internalization.

Mitochondrial pH Monitored by a New Engineered Green Fluorescent Protein Mutant

We here describe a new molecularly engineered green fluorescent protein chimera that shows a high sensitivity to pH in the alkaline range. This probe was named mtAlpHi, for mitochondrial alkaline pH indicator, and possesses several key properties that render it optimal for studying the dynamics of mitochondrial matrix pH, e.g. it has an apparent pK(a) (pK(a)') around 8.5, it shows reversible and large changes in fluorescence in response to changes in pH (both in vitro and in intact cells), and it is selectively targeted to the mitochondrial matrix. Using mtAlpHi we could monitor pH changes that occur in the mitochondrial matrix in a variety of situations, e.g. treatment with uncouplers or Ca(2+) ionophores, addition of drugs that interfere with ATP synthesis or electron flow in the respiratory chain, weak bases or acids, and receptor activation. We observed heterogeneous pH increases in the mitochondrial matrix during Ca(2+) accumulation by this organelle. Finally, we demonstrate that Ca(2+) mobilization from internal stores induced by ionomycin and A23187 cause a dramatic acidification of the mitochondrial matrix.

Retrograde transport of the glucocorticoid receptor in neurites requires dynamic assembly of complexes with the protein chaperone hsp90 and is linked to the CHIP component of the machinery for proteasomal degradation

Here, we have used a chimera of green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to study retrograde movement of a model soluble (i.e., non-vesicle-associated) protein in axons and dendrites of cultured NT2-N neurons. It is known that in non-neuronal cells, the GFP–GR moves from cytoplasm to the nucleus in a steroid-dependent manner by a rapid, hsp90-dependent mechanism. When rapid movement is inhibited by geldanamycin (GA), a specific inhibitor of the protein chaperone hsp90, the GFP–GR translocates slowly to the nucleus by diffusion. Here we show that GFP–GR expressed in hormone-free neurons is localized in both cytoplasm and neurites, and upon treatment with dexamethasone (DEX), it moves to the nucleus. In neurites, movement by diffusion is not possible, and we show that movement of the GFP–GR from neurites is blocked by geldanamycin, suggesting that the hsp90-dependent movement machinery is required for retrograde movement. In cells treated with both dexamethasone and geldanamycin, the GFP–GR becomes concentrated in fluorescent globules located periodically along the neurites. Carboxyl terminus of Hsc70-interacting protein (CHIP), the E3 ubiquitin ligase for the GR, also concentrates in the same loci in a steroid-dependent and geldanamycin-dependent manner. If geldanamycin is removed, the GFP–GR exits the globules and continues its retrograde movement. However, in the continued presence of geldanamycin, the GFP–GR in the globules undergoes proteasomal degradation, suggesting that the globules function as degradasomes. This is the first evidence for a linkage between receptor trafficking along neurites and receptor degradation by the proteasome.

Pox, dyes, and videotape: making movies of GFP-labeled vaccinia virus.

The large size of poxvirus virions (approx 250 x 350 nm) makes them ideal candidates for microscopic studies. Recombinant vaccinia viruses that express a viral envelope-specific, green fluorescent protein (GFP) chimera produce enveloped virions that fluoresce green. This fluorescent labeling allows the live, real-time study of viral egress using a variety of microscopic techniques. The methods presented here describe how to image the movement of intracellular enveloped virions that are labeled with green fluorescent protein using time-lapse laser scanning confocal microscopy. Details are also provided for analyzing the images obtained and converting them into QuickTime movies suitable for presentation.