Cytoplasmic GFP Research Articles

Regulatory B10 cells activated in vivo transiently produce IL-10 before differentiating into antibody-secreting cells with a diverse BCR repertoire (62.4)

Abstract B cells with potent IL-10-dependent regulatory functions (B10 cells) expand in mice and humans during inflammation and autoimmunity. In this study, the fate of B10 cells following in vivo expansion was examined using two distinct il-10 reporter strains: Tiger mice that express cytoplasmic GFP and 10BiT mice that express cell surface Thy1.1. B10 cells from Tiger mice express cytoplasmic IL-10 and GFP with similar kinetics following stimulation in vitro and in vivo. By contrast, B10 cell Thy1.1 expression in 10BiT mice was delayed relative to IL-10 production and persisted even after B10 cells had lost the capacity to produce IL-10. Thy1.1 also served as a marker for B10 cells that expressed plasma cell-associated transcription factors and had the capacity to differentiate into CD138+B220LO antibody-secreting plasma cells in vivo and in vitro. Antibodies secreted by B10 cells included broadly reactive “natural antibodies” and autoantibodies, and were antigen-specific in immunized mice. At the molecular level, B10 cells predominantly expressed a germline-encoded IgM antibody repertoire with diverse VH and VL gene utilization. These results indicate that B10 cells can differentiate following IL-10 secretion to produce broadly-reactive germline-encoded antibodies that may function to rapidly clear antigens and further reduce inflammation and immunopathology.

Time-Resolved, Single-Cell Study of the Attack of the Antimicrobial Peptide Ll-37 on Live E. Coli Cells

Human LL-37 is an antimicrobial peptide whose amphiphilic helices selectively degrade bacterial membranes by a mechanism that is poorly understood. We are using single-cell, two-color fluorescence microscopy to directly observe the attack of rhodamine-LL-37 on live E. coli cells in real time. The cells express either periplasmic or cytoplasmic GFP. This enables quantitative correlation of the extent of LL-37 adsorption with leakage or lysing of GFP from the two different compartments, while simultaneously monitoring cell growth. At 15 uM, LL-37 lyses the periplasm to GFP and halts growth in 2-4 min, long before the cytoplasm lyses to GFP at 20-30 min. At 6 uM, rhodamine-LL-37 binding occurs in three distinct waves, with Wave 2 correlating in time with the halting of cell growth (t = 7-10 min). Wave 1 coats the cell periphery uniformly, but Wave 2 preferentially attacks the septal region and slowly spreads outward towards the poles. This suggests that the cell division machinery may be a target of LL-37-induced cell death. We will use FRET to discern the penetration depth of LL-37 during the different waves of attack and a variety of mutant strains to correlate the LL-37 attack with formation of the Z-ring and additional parts of the divisome. These methods will enable quantitative comparison of antimicrobial attack on real bacterial membranes with studies of lysing of synthetic lipid bilayers. They will be applicable to a wide variety of antimicrobial agents and bacterial species.

Global Transcriptional Repression in C. elegans Germline Precursors by Regulated Sequestration of TAF-4

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Arabidopsis COLD SHOCK DOMAIN PROTEIN2 is a RNA chaperone that is regulated by cold and developmental signals

Bacterial cold shock proteins (CSPs) are RNA chaperones that unwind RNA secondary structures. Arabidopsis COLD SHOCK DOMAIN PROTEIN2 (AtCSP2) contains a domain that is shared with bacterial CSPs. Here we showed that AtCSP2 binds to RNA and unwinds nucleic acid duplex. Heterologous expression of AtCSP2 complemented cold sensitivity of an Escherichia coli csp quadruple mutant, indicating that AtCSP2 function as a RNA chaperone in E. coli. AtCSP2 mRNA and protein levels increased during cold acclimation, but the protein accumulation was most prominent after 10 days of cold treatment. AtCSP2 promoter::GUS transgenic plants revealed that AtCSP2 is expressed only in root and shoot apical regions during vegetative growth but is expressed in reproductive organs such as pollens, ovules and embryos. These data indicated that AtCSP2 is involved in developmental processes as well as cold adaptation. Localization of AtCSP2::GFP in nucleolus and cytoplasm suggested different nuclear and cytosolic RNA targets.

A Nuclear Export Sequence Located on a β-Strand in Fibroblast Growth Factor-1

Receptor-bound and endocytosed fibroblast growth factor-1 (FGF-1) is able to cross the vesicle membrane and translocate to cytosol and nucleus. This suggests an intracellular role of FGF-1, which also signals by activating transmembrane FGF receptors. Phosphorylation of internalized FGF-1 by nuclear protein kinase C delta induces rapid export from the nuclei by a leptomycin B-sensitive pathway. In the present work, we have searched for and identified a Leu-rich nuclear export sequence (NES) at the C terminus of FGF-1 required for its nuclear export and able to confer nuclear export activity to a reporter protein in an in vivo system. Mutants where hydrophobic amino acids within the NES were exchanged for alanine exhibited reduced or abolished nuclear export. As demonstrated in co-immunoprecipitation experiments, a complex containing FGF-1, exportin-1, and its co-factor Ran-GTP, was formed in vitro. Formation of this complex in vivo was demonstrated by a peroxisomal targeting assay. Formation of the FGF-1-exportin-1-Ran-GTP complex in vitro as well as nuclear export of FGF-1 in vivo was dependent on phosphorylation of FGF-1, and it was abolished by leptomycin B. The FGF-1 NES was found to be situated along a beta-strand, which has not been reported before, since NESs usually are alpha-helical.

FUS1Regulates the Opening and Expansion of Fusion Pores between Mating Yeast

Mating yeast cells provide a genetically accessible system for the study of cell fusion. The dynamics of fusion pores between yeast cells were analyzed by following the exchange of fluorescent markers between fusion partners. Upon plasma membrane fusion, cytoplasmic GFP and DsRed diffuse between cells at rates proportional to the size of the fusion pore. GFP permeance measurements reveal that a typical fusion pore opens with a burst and then gradually expands. In some mating pairs, a sudden increase in GFP permeance was found, consistent with the opening of a second pore. In contrast, other fusion pores closed after permitting a limited amount of cytoplasmic exchange. Deletion of FUS1 from both mating partners caused a >10-fold reduction in the initial permeance and expansion rate of the fusion pore. Although fus1 mating pairs also have a defect in degrading the cell wall that separates mating partners before plasma membrane fusion, other cell fusion mutants with cell wall remodeling defects had more modest effects on fusion pore permeance. Karyogamy is delayed by >1 h in fus1 mating pairs, possibly as a consequence of retarded fusion pore expansion.

Down-regulation of cytokine secretion and repression of apoptosis hDaxx in macrophages

Objective: To investigate the regulation effects on LPS-mediated cytokine secretion and dexamethasone- induced apoptosis in macrophages by transient overexpression of hDaxx. Methods: An eukaryotic expression vector pEGFP/hDaxx, which could express a fusion protein GFP-Daxx, was transfected into macrophages. The expression and localization of GFP-hDaxx fusion protein was analyzed by fluorescent microscope and western blot. The effects of transient overexpression of GFP-hDaxx fusion protein on the lipopolysaccharide(LPS)-mediated secretion of TNF-α and IL-1β were determined by ELISA. Moreover, the dexamethasone-induced apoptosis was determined morphologically by Giemsa stain. Results: The results observed showed that GFP-hDaxx fusion protein overexpressed in macrophages and localized in nuclei but GFP in cytoplasm under fluorescent microscope. The overexpression of GFP-hDaxx fusion protein could be detected by Western blot with an antibody against C-terminal of hDaxx. In the group with overexpressed GFP-hDaxx fusion protein, the LPS-mediated cytokine secretion decreased remarkably at 1 h, 3 h, 6 h respectively after LPS stimulation, and the dexamethasone- induced apoptosis reduced notably at 6 h, 12 h and 24 h respectively after addition of dexamethasone. There were remarkable difference between pEGFP/hDaxx group and control group (P<0.01) at different time. Conclusion: Transient overexpression of hDaxx down-regulates LPS-mediated cytokine secretion in macrophages and inhibits dexamethasone-induced macrophages apoptosis.

812. Hydroporation as the Mechanism Accounting for Efficient Gene Transfer by Hydrodynamic Delivery

Top of pageAbstract We have reported that hydrodynamic delivery of plasmid DNA into a mouse results in a high level of transgene expression in many organs with the highest in the liver. Gene transfer efficiency by this procedure is dictated by a combined effect of large volume and high injection speed. The current study was designed to elucidate the underlying mechanism of the hydrodynamic delivery. Using a catheter inserted to the point of vena cava through a renal vein in retrograde, we observed a sharp increase in venous pressure and then a rapid drop to a level of 10-15 mmHg above the original baseline pressure, followed by a much slower decline. Electrocardiogram (ECG) of a mouse undergoing the hydrodynamic injection showed an irregular cardiac rhythm, which lasted about 60 sec. The heart rate decreased from approximately 510 beats/min to 280 beats/min 3 sec after the injection and returned to normal in 2 min. SEM and TEM examination of the liver sections revealed partial disruption of endothelial lining of liver capillaries, enlargement of liver fenestrae and more importantly membrane pores on hepatocytes. Using Evans blue and reporter gene containing plsmids as a probe we found that these membrane pores were transient and resealed within a few min. We also demonstrated that proteins (beta-galactosidase), fluorescent dye (Evans blue), reporter plasmids and 125I-labeled DNA can all be hydroynamically delivered into hepatocytes with very similar efficiency. Using cytoplasmic microinjection technique and GFP as a reporter, we demonstrated that a large volume of DNA solution entering the cytoplasm helps transgene expression. For example, GFP positive cells increased from approximately 26% of injected cells (n=339) at 5-10% cytoplasmic volume to 50% at 50-70% of cytoplasmic volume (n=303). We conclude from these results that hydrodynamic delivery is a physical process that does not involve membrane receptors for DNA transfer into cells. The mechanism underlying this efficient procedure can be described as follows. A rapid injection of a large bolus of DNA solution via the tail vein causes a transient cardiac congestion, resulting in an accumulation of the injected solution in the inferior vena cava. Subsequently, a high pressure develops in this venous region and causes a retrograde movement of the solution into the liver, an organ with enriched vascular structure and direct connection to the inferior vena cava. A large portion of DNA solution enters the liver via the hepatic vein under a hydrodynamic force, pushing the blood in hepatic circulation to move backward toward the portal blood vessels. As a consequence of this hydrodynamic influence (mechanic or/and pressure influence due to hydrodynamic injection) the pore sizes of the liver fenestrae are enlarged and the membrane pores on hepatocytes are generated. Consequently, DNA is driven into the hepatocytes and membrane of hepatocytes reseals with time, trapping the DNA molecules inside. In a short time, the cardiovascular system adapts to the volume load and normal circulatory homeostasis is restored.

Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.

The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: implications for spore dormancy.

Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased approximately 2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.

Basolateral localization of organic cation transporter 2 in intact renal proximal tubules.

The localization of organic cation transporter 2 (OCT2) within renal cells is the subject of considerable controversy, resulting in marked uncertainty as to its function. To resolve this issue, we made an OCT2/green fluorescent protein (GFP) fusion construct (rOCT2-GFP) and determined its localization within Xenopus laevis oocytes and renal cells using confocal microscopy. Oocytes expressing rOCT2-GFP exhibited plasma membrane fluorescence as well as greatly increased specific, potential-driven uptake of [(14)C]tetraethylammonium (TEA). Polarized monolayers of renal epithelial cell lines [LLC-PK(1) and Madin-Darby canine kidney (MDCK)] transiently transfected with pEGFP-C3, which codes for a cytoplasmic GFP, showed a diffuse, evenly distributed cytoplasmic signal with no plasma membrane fluorescence. In contrast, cells transiently transfected with pEGFP-C3/rOCT2 (the vector coding for rOCT2-GFP) showed predominantly plasma membrane fluorescence, which was most prominent in the lateral membrane. MDCK cells stably expressing rOCT2-GFP (MDCK/rOCT2-GFP) maintained in long-term culture showed a greatly increased basal and lateral membrane fluorescence. When grown on porous supports, MDCK/rOCT2-GFP monolayers showed specific, potential-driven TEA uptake from the basal side. Finally, expression and distribution of rOCT2-GFP were investigated in isolated killifish (Fundulus heteroclitus) renal proximal tubules. On expression of rOCT2-GFP, transfected tubules showed marked basal and lateral membrane fluorescence, with no detectable signal at the apical membrane. In contrast, tubules expressing a luminal sodium-dicarboxylate cotransporter (rbNaDC-1)-GFP construct showed apical membrane fluorescence, and tubules expressing cytoplasmic GFP had a diffuse cytoplasmic fluorescence. These results indicate that rOCT2 is basolateral in renal proximal tubule cells.

An Integral Membrane Green Fluorescent Protein Marker, Us9-GFP, Is Quantitatively Retained in Cells during Propidium Iodide-Based Cell Cycle Analysis by Flow Cytometry

Previously, we described GFP-spectrin, a membrane-localized derivative of the green fluorescent protein that can be employed as a marker during the simultaneous identification of transfected cells and cell cycle analysis by flow cytometry (Kalejtaet al., Cytometry29: 286–291, 1997). A membrane-anchored GFP fusion protein is necessary because the ethanol permeabilization step required to achieve efficient propidium iodide staining allows cytoplasmic GFP to leach out of the cell. However, viable cells expressing GFP-spectrin are not as bright as cells expressing cytoplasmic GFP and their fluorescence intensity is further diminished after ethanol treatment. Here, we demonstrate that the fluorescence intensity of cells expressing an integral membrane GFP fusion protein (Us9-GFP) is similar to that of cells expressing cytoplasmic GFP and is quantitatively maintained in cells after ethanol treatment. By allowing an accurate assessment of the expression level of GFP, Us9-GFP allows a more precise analysis of the effects of a cotransfected plasmid on the cell cycle and thus represents an improvement upon the original membrane-associated GFP fusion proteins employed in this assay.