Nematode Infection Triggers the de Novo Formation of Unloading Phloem That Allows Macromolecular Trafficking of Green Fluorescent Protein into Syncytia

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

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Destabilizing Interactions Among [PSI +] and [PIN +] Yeast Prion Variants

Genetics ◽

10.1093/genetics/165.4.1675 ◽

2003 ◽

Vol 165 (4) ◽

pp. 1675-1685 ◽

Cited By ~ 2

Author(s):

Michael E Bradley ◽

Susan W Liebman

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

De Novo ◽

Human Diseases ◽

Yeast Prion ◽

Fluorescence Pattern ◽

Prion Strains ◽

Green Fluorescent

AbstractThe yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi–]or[pin–], respectively, or the multiple infectious amyloid-like forms called [PSI+]or[PIN+] prion variants (or prion strains). It was previously shown that [PSI+] and [PIN+] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI+] and [PIN+] disrupt each other's stable inheritance. Acquiring [PSI+] often impedes the inheritance of particular [PIN+] variants. Conversely, the presence of some [PIN+] variants impairs the inheritance of weak [PSI+] but not strong [PSI+] variants. These same [PIN+] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN+] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI+] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.

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Compartmentalization of VP16 in Cells Infected with Recombinant Herpes Simplex Virus Expressing VP16-Green Fluorescent Protein Fusion Proteins

Journal of Virology ◽

10.1128/jvi.78.15.8002-8014.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 8002-8014 ◽

Cited By ~ 56

Author(s):

Sylvie La Boissière ◽

Ander Izeta ◽

Sophie Malcomber ◽

Peter O'Hare

Keyword(s):

Herpes Simplex Virus ◽

Green Fluorescent Protein ◽

Herpes Simplex ◽

Fluorescent Protein ◽

De Novo ◽

Structural Protein ◽

Dynamic Features ◽

Protein Fusion ◽

Green Fluorescent ◽

Simplex Virus

ABSTRACT VP16 is an essential structural protein of herpes simplex virus. It plays important roles in immediate-early transcriptional regulation, in the modulation of the activities of other viral components, and in the pathway of assembly and egress of infectious virions. To gain further insight into the compartmentalization of this multifunctional protein we constructed and characterized recombinant viruses expressing VP16 linked to the green fluorescent protein (GFP). These viruses replicate with virtually normal kinetics and yields and incorporate the fusion protein into the virion, resulting in autofluorescent particles. De novo-synthesized VP16-GFP was first detected in a diffuse pattern within the nucleus. Nuclear VP16-GFP was progressively recruited to replication compartments, which coalesced into large globular domains. By 10 to 12 h after infection additional distinct foci containing VP16-GFP could be seen, almost exclusively located at the periphery of the replication compartments. At the same time pronounced accumulation was observed in the cytoplasm, first in a diffuse pattern and then accumulating in vesicle-like compartments which were concentrated in an asymmetric fashion reminiscent of the Golgi. Inhibition of DNA replication resulted in prolonged diffuse nuclear distribution with minimal cytoplasmic accumulation. Treatment with brefeldin disrupted the cytoplasm vesicular pattern, resulting in redistributed large foci. Time-lapse microscopy demonstrated various dynamic features of infection, including the active induction of very long cellular projections (up to 100 μM). Vesicular clusters containing VP16 were transported within projections to the termini, which developed bulbous ends and appeared to embed into the membranes of adjacent uninfected cells.

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Aequorea’s secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing

PLoS Biology ◽

10.1371/journal.pbio.3000936 ◽

2020 ◽

Vol 18 (11) ◽

pp. e3000936

Author(s):

Gerard G. Lambert ◽

Hadrien Depernet ◽

Guillaume Gotthard ◽

Darrin T. Schultz ◽

Isabelle Navizet ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Polypeptide Chain ◽

De Novo ◽

Fluorescent Proteins ◽

Transcriptome Assembly ◽

X Ray ◽

X Ray Crystallography ◽

Green Fluorescent ◽

New Generation

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

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Analytical ultracentrifugation as a tool in the studies of aggregation of the fluorescent marker, Enhanced Green Fluorescent Protein

Acta Biochimica Polonica ◽

10.18388/abp.2020_5081 ◽

2020 ◽

Author(s):

Aleksandra Dawidziak-Pakula ◽

Joanna Krasowska ◽

Beata Wielgus-Kutrowska

Keyword(s):

Green Fluorescent Protein ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Analytical Ultracentrifugation ◽

De Novo ◽

Continuous Distribution ◽

Fluorescent Marker ◽

Low Concentrations ◽

Green Fluorescent ◽

De Novo Folding

Enhanced green fluorescent protein (EGFP) is a fluorescent marker used in bio-imaging applications, including as an indicator of folding or aggregation of a fused partner. However, the limited maturation, low folding efficiency, and presence of non-fluorescent states of EGFP can influence the interpretation of experimental data. To measure aggregation associated with de novo folding of EGFP from a high GdnHCl concentration, the analytical ultracentrifugation method was used. Absorption detection at 280 nm allowed to monitor the presence of monomers and aggregated forms. Fluorescence detection enabled the observation of only properly folded molecules with a functional chromophore. The results showed intensive aggregation of EGFP in low concentrations of GdnHCl with a continuous distribution of aggregated forms. The properly folded monomers with mature chromophore were fluorescent, while the conglomerates of EGFP molecules were not. These facts are essential for a proper interpretation of data obtained with EGFP labelling.

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Intracellular Localization of the Peanut Clump Virus Replication Complex in Tobacco BY-2 Protoplasts Containing Green Fluorescent Protein-Labeled Endoplasmic Reticulum or Golgi Apparatus

Journal of Virology ◽

10.1128/jvi.76.2.865-874.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 865-874 ◽

Cited By ~ 31

Author(s):

Patrice Dunoyer ◽

Christophe Ritzenthaler ◽

Odile Hemmer ◽

Pierre Michler ◽

Christiane Fritsch

Keyword(s):

Endoplasmic Reticulum ◽

Green Fluorescent Protein ◽

Golgi Apparatus ◽

Laser Scanning ◽

Fluorescent Protein ◽

De Novo ◽

Intracellular Localization ◽

Viral Rna ◽

Replication Complex ◽

Green Fluorescent

ABSTRACT RNA-1 of Peanut clump virus (PCV) encodes the proteins P131 and P191, containing the signature motifs of replication proteins, and P15, which regulates viral RNA accumulation. In PCV-infected protoplasts both P131 and P191 were immunodetected in the perinuclear region. Laser scanning confocal microscopy (LSCM) showed that P131 and P191 colocalized with neosynthesized 5-bromouridine 5′-triphosphate-labeled RNA and double-stranded RNA, demonstrating that they belong to the replication complex. On the contrary, the P15 fused to the enhanced green fluorescent protein (EGFP) never colocalized with the two proteins. In endoplasmic reticulum (ER)-GFP transgenic BY-2 protoplasts, the distribution of the green fluorescent-labeled ER was strongly modified by PCV infection. LSCM showed that both P131 and P191 colocalized with ER green fluorescent bodies accumulating around the nucleus during infection. The replication process was not inhibited by cerulenin and brefeldin A, suggesting that PCV replication does not depend on de novo-synthesized membrane and does not require transport through the Golgi apparatus. Electron microscopy of ultrathin sections of infected protoplasts showed aggregates of broken ER but also visualized vesicles, some of which resembled modified peroxisomes. The results suggest that accumulation of PCV during infection is accompanied by specific association of PCV RNA-1-encoded proteins with membranes of the ER and other organelles. The concomitant extensive rearrangement of these membranous structures leads to the formation of intracellular compartments in which synthesis and accumulation of the viral RNA occur in defined areas.

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Incorporation of sensing modalities into de novo designed fluorescence-activating proteins

Nature Communications ◽

10.1038/s41467-020-18911-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jason C. Klima ◽

Lindsey A. Doyle ◽

Justin Daho Lee ◽

Michael Rappleye ◽

Lauren A. Gagnon ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Fluorescent Protein ◽

De Novo ◽

Relative Ease ◽

Gfp Reporter ◽

Wide Range ◽

Reporter Systems ◽

Green Fluorescent ◽

Direct Readout

AbstractThrough the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

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