Red Fluorescent Protein Signals Research Articles

Generation of Vgll4-DreER transgenic mouse for visualizing and manipulating VGLL4-expressing cells in vivo.

Vestigial like family member 4 (VGLL4), a member of the Hippo pathway, is a transcriptional cofactor involved in many biological processes, such as tumor progression, postnatal heart growth, and muscle regeneration. However, the VGLL4 expression pattern in vivo remains unclear. To detect and trace Vgll4-expressing cells and their progeny, we generated and characterized a new tamoxifen-inducible Dre knock-in mouse line, Vgll4-DreER. This mouse line expressed DreER (Dre recombinase fused to the estrogen receptor) under the control of the endogenous Vgll4 promoter. After crossing the Vgll4-DreER mouse line with the Dre-responsive reporter H11-rRFP, Dre-mediated recombination in the tissue was monitored on the basis of red fluorescent protein (RFP) signals, which indicated the distribution of VGLL4-positive cells in vivo. Our data revealed that VGLL4 is widely expressed in various cell types at embryonic and neonatal stages. After comparison with our previously reported Vgll4-GFP mouse, we found that the RFP signal profile was wider than the green fluorescent protein (GFP) pattern, indicating that Vgll4-DreER is more sensitive for labeling VGLL4-expressing cells. We next used a dual-recombination system to simultaneously label VGLL4- and keratin 5 (KRT5)-positive cell populations, and no crosstalk was observed in the Krt5-CreER;Vgll4-DreER;R26-rGlR mice. Taken together, the Vgll4-DreER mouse line is a valuable new tool for examining the precise VGLL4 expression profile and conditional manipulating of VGLL4-expressing cells and their progeny.

Extracellular DEK Activates NRF2 Antioxidant Targets and Drives Expansion of Functional and ROS-Low Human Hematopoietic Stem Cells

Umbilical cord blood (CB) is a promising source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation (HCT), a life-saving treatment, due to lower incidence of graft versus host disease and its availability. CB HCT is limited by low numbers of HSCs, thus there is a strong need to increase numbers of engraftable HSCs, for instance by expanding functionally competent HSCs ex vivo. We have shown that extracellular DEK protein, an epigenetic regulator that is secreted in response to stress and inflammation, is a novel hematopoietic regulatory chemokine. Treatment with extracellular DEK in growth stimulating media significantly enhances the expansion of engraftable HSCs in mouse bone marrow (BM) and human CB (Capitano, et al. JCI 2019; 129(6):2555-2570). To examine mechanisms driving enhanced expansion of functional HSCs after DEK treatment, we performed RNA-sequencing on CB HSCs, multipotent progenitor cells (MPPs), common myeloid progenitors (CMPs), and granulocyte macrophage progenitors (GMPs) after overnight DEK treatment in expansion media. All cell populations upregulated gene programs related to antioxidant responses upon DEK treatment. Indeed, 7/10 genes upregulated by DEK in all HSC/HPC populations (NMRAL2P, PIR, NQO1, SELENOW, FTL, RIT1, and QPCT) promote antioxidant activity. Overnight DEK treatment significantly reduced mean fluorescence of the activated reactive oxygen species (ROS) indicator CM-H2DCFDA in HSCs, MPPs, CMPs, and GMPs. Thus, DEK may induce antioxidant activity. We next found that all 10 genes commonly upregulated in HSCs/HPCs are either direct transcriptional targets of NRF2 (NMRAL2P, LUCAT1,PIR, NQO1, FTL, and QPCT) or are directly affected by changes in NRF2 activation (SELENOW, EBI3, TNFRSF9, and RIT1). Further, several genes highly upregulated by DEK treatment in HSCs (i.e., CCL2, KYNU, and CXCL8) are NRF2 targets. To examine if DEK activates NRF2 transcriptional activity, we utilized a transgene reporter assay where expression of red fluorescent protein (RFP) is driven by activation of promoter antioxidant response elements (ARE), to which active NRF2 binds. DEK treatment of hematopoietic derived cell lines (mouse MLL-AF9 AML and human HL-60 APL) expressing the transgene significantly increased RFP signal, indicating that DEK could activate NRF2. We further examined whether DEK treatment affects mitochondrial metabolism, a source of ROS and tightly regulated process in hematopoiesis. Seahorse XFe analysis, which measures metabolic flux, showed DEK treatment of MLL-AF9 cells significantly reduced mitochondrial oxygen consumption rate, suggesting a reduced reliance on oxidative phosphorylation as an energy source in DEK treated cells. These data suggest that DEK treatment during expansion of primitive hematopoietic cells drives activation of NRF2 transcription, upregulation of antioxidant response genes, reduction of intracellular ROS levels, and may reduce the mitochondrial metabolic rate. These effects in a sense "mimic” the in vivo niche of HSCs, where they reside in low oxygen tensions, have low intracellular ROS, and exhibit low levels of oxidative phosphorylation. To test this hypothesis, we examined if DEK treatment has similar effects to maintaining HSCs in physiologic oxygen tensions (3%) compared to HSCs exposed to extraphysiologic ambient air (21%), which drives oxidative stress (Mantel, et al. Cell 2015; 161(7):1553-65). Isolating and maintaining mouse BM in physiologic oxygen tensions or treating mice in vivo with recombinant DEK preserved HSC numbers compared to exposing the BM cells to ambient air or treating with vehicle. However, combining in vivo DEK treatment with physiologic isolation of HSCs/HPCs had no additive or synergistic effect, suggesting that the overall effects of DEK treatment and maintaining low oxygen tensions may be similar. Thus, extracellular DEK treatment leads to expansion of functionally competent HSCs and reduction of oxidative stress. These data have important implications for the regulation of HSCs/HPCs in stress conditions, where DEK is most likely to be secreted. Further, DEK may present a novel way to mimic physiologic oxygen conditions for enhancement of functional HSC expansion, and the elucidated mechanisms may be exploited for more efficient targeting of molecular programs critical for functional HSC competency, potentially providing new ways to enhance HCT.

Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage.

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.

Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L.

Efficient transformation system for genetic improvement is essential in Crocus sativus, as it lacks sexual reproduction. This is the first report wherein an efficient protocol is developed for the transformation of Crocus sativus L. by Agrobacterium rhizogenes strain ARqua1 with a transformation efficiency of 78.51%. The ARqua1 strain harboring both Ri plasmid and binary vector plasmid pSITE-4NB, and marker genes for red fluorescent protein (RFP) and a β-glucuronidase (GUS) reporter gene were used for selection. Transformation was confirmed by RFP signal, GUS reporter assay and polymerase chain reaction (PCR) analysis of the test samples after 21days post inoculation. These results confirm the establishment of protocol for hairy root transformation in C. sativus that can be further used for gene transfer or gene editing in Crocus for its genetic improvement.

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice.

The transmission of Buruli ulcer (BU), caused by Mycobacterium ulcerans (MU), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that Acanthamoeba species may play a role in BU transmission. We cocultured MU N2 and MU 1615 which expresses red fluorescent protein (RFP) and Acanthamoeba polyphaga (AP), and confirmed infected AP by Ziehl-Neelsen (ZN) staining. We tested for viability of MU inside AP and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following; MU N2 only (2.25 x 106 colony forming units [CFU] / ml), MU N2:AP coculture (2.96 x 104 CFU: 1.6 x 106 cells/ml), AP only (1.6 x 106 cells/ml), PYG medium and sterile distilled water. Both MU N2 only and MU N2:AP elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of MU N2 only and MU N2:AP, and compare their virulence, the standard mouse footpad inoculation method was used. MU N2:AP elicited reddening in footpads by D 3 compared to D 14 with MU N2 only of the same dose of MU N2 (2.96 x 104 CFU). ZN-stained MU were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable MU N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice MU transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of MU with AP enhances pathogenesis.

Transgene chgH-rfp expression at developmental stages and reproductive status in marine medaka (Oryzias dancena)

The transgenic approach using estrogen-responsive regulator in fish has been given much attention as a potential means to detect and/or address estrogen-related aquatic pollutions. In order to address the development stage- and reproduction status-dependent expression patterns of the chgH-rfp transgene (red fluorescent protein transgene driven by choriogenin H promoter) in marine medaka Oryzias dancena, naturally occurring red fluorescent protein (RFP) signals under non-exposed conditions as well as the transgenically induced RFP signals under estrogen-exposed conditions were assayed. Female transgenics begun to show naturally occurring RFP signals from the age of 7 weeks post hatching (WPH) without experimental estrogen exposure. Afterward, these RFP signals in female transgenics became robust with the progress of ovarian maturation. On the other hand, male transgenics did not show any naturally occurring RFP signal under non-exposed conditions irrespective of developmental stages and maturation statue. Upon exposures using estradiol-17β (E2) and 17α-ethinylestradiol (EE2), RFP signals were significantly induced specifically in the livers of transgenic males. Male chgH-rfp transgenics were able to keep the “off” state of RFP expression during their entire life cycle unless exposed to exogenous estrogens. Owing to their tight regulation capability of estrogen-responsive transgene, transgenesis of chgH-rfp in male marine medaka could offer a useful model system for future ecotoxicogenomic studies regarding estrogenicity-related issues in aquatic and marine environments.

Safe engineering of CAR T cells for adoptive cell therapy of cancer using long-term episomal genetransfer.

Chimeric antigen receptor (CAR) T‐cell therapy is a new successful treatment for refractory B‐cell leukemia. Successful therapeutic outcome depends on long‐term expression of CAR transgene in T cells, which is achieved by delivering transgene using integrating gamma retrovirus (RV) or lentivirus (LV). However, uncontrolled RV/LV integration in host cell genomes has the potential risk of causing insertional mutagenesis. Herein, we describe a novel episomal long‐term cell engineering method using non‐integrating lentiviral (NILV) vector containing a scaffold/matrix attachment region (S/MAR) element, for either expression of transgenes or silencing of target genes. The insertional events of this vector into the genome of host cells are below detection level. CD19 CAR T cells engineered with a NILV‐S/MAR vector have similar levels of CAR expression as T cells engineered with an integrating LV vector, even after numerous rounds of cell division. NILV‐S/MAR‐engineered CD19 CAR T cells exhibited similar cytotoxic capacity upon CD19+ target cell recognition as LV‐engineered T cells and are as effective in controlling tumor growth in vivo. We propose that NILV‐S/MAR vectors are superior to current options as they enable long‐term transgene expression without the risk of insertional mutagenesis and genotoxicity.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice

Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue.

Therapeutic effect of Artemia enriched with Escherichia coli expressing double-stranded RNA in the black tiger shrimp Penaeus monodon

We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E. coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E. coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P. monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11–17weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P. monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.

Phenotypic Characterization of Transgenic Japanese Medaka (Oryzias latipes) That Express a Red Fluorescent Protein in Hepatocytes

Transgenic organisms that express fluorescent proteins are used frequently for in vivo visualization of proteins and cells. The phenotype of a transgenic medaka (Oryzias latipes) strain that expresses a red fluorescent protein (RFP) in hepatocytes was characterized using light and fluorescence microscopy, immunohistochemistry, and transmission electron microscopy (TEM). Expression of RFP was first detected by confocal fluorescence microscopy in the location of the liver bud of live medaka embryos at 60 hr postfertilization (developmental stage 27). Subsequently, RFP signal was observed exclusively in hepatocytes throughout life using fluorescence microscopy in live fish and immunohistochemistry in formalin-fixed, paraffin-embedded liver sections. As the fish aged, prominent intracytoplasmic eosinophilic inclusions immunoreactive for RFP were observed by light microscopy and were correlated with membrane-bound electron dense inclusions on TEM. These results define the onset and location of RFP expression in the Tg(zf.L-fabp:DsRed) medaka and characterize a histologic phenotype that results from RFP expression in hepatocytes.

Molecular Characterization of Cytoskeletal Beta-Actin and its Promoter in the Javanese Ricefish Oryzias javanicus

We characterized the cytoskeletal beta-actin (<TEX>${\beta}$</TEX>-ACT) gene (actb) and its 5'-upstream regulatory region in the Javanese ricefish Oryzias javanicus. The gene and protein structures were deduced from amino acid sequences of the actb gene and conserved in the teleost lineage. The O. javanicus actb gene has common transcription factor binding motifs in its regulatory region found in teleostean orthologues. Following quantitative reverse transcription-PCR, actb gene transcripts were detected in all tissues examined; however, the basal expression levels were different. During early development, O. javanicus actb mRNA levels showed a gradual increase and peaked between late somitogenesis and the heartbeat stage. Microinjection of O. javanicus embryos with the actb gene promoter-driven red fluorescent protein (RFP) gene reporter vector showed a ubiquitous distribution of RFP signals, although most exhibited a mosaic pattern of transgene expression. A small number of microinjected embryos displayed a wide distribution of RFP signals over their entire body, which resembled the expression pattern of endogenous actb. Data from this study provide a basis to develop a transgenic system with ubiquitous expression of foreign genes in O. javanicus.

Genomic Organization, Intronic Duplications, and Promoter Characteristics of the Fast Skeletal Myosin Light Chain-2 Gene (mlc2f) from Javanese Ricefish Oryzias javanicus

The present study characterized the fast skeletal myosin light chain-2 gene (mlc2f) in the euryhaline Javanese ricefish (Oryzias javanicus: Beloniformes). Coding nucleotide and deduced amino acid sequences of Javanese ricefish mlc2f were well conserved in the vertebrate lineage. Javanese ricefish mlc2f showed a typical seven-exon structure, and its promoter exhibited transcription factor binding motifs common to most muscle-specific genes. However, Javanese ricefish mlc2f also displayed tandem duplications of intronic sequences in both intron 1 and intron 3. Based on quantitative reverse transcription-polymerase chain reaction, the mlc2f transcripts were highly predominant in skeletal muscles of adults and were differentially modulated during embryonic development. Microinjection of the mlc2f promoter-driven red fluorescent protein (RFP) reporter construct successfully exhibited heterologous expression of the fluorescent reporter, primarily in muscular areas of hatchlings, although the distribution pattern of RFP signals was not uniform due to the mosaic nature of the introduced transgene. Data from this study indicate that the Javanese ricefish mlc2f gene has undergone "intra-intronic" duplication events in a species-specific manner and that the mlc2f regulator may also be useful in heterologous expression assays of the skeletal muscles of this species.

Characterization of estrogen-responsive transgenic marine medaka Oryzias dancena germlines harboring red fluorescent protein gene under the control by endogenous choriogenin H promoter

Transgenic marine medaka (Oryzias dancena) germlines were generated by the microinjection of the red fluorescent protein (RFP) reporter gene (rfp) driven by the endogenous choriogenin H gene (chgH) promoter. The selected transgenic lines contained multiple copies of the transgene (3-42 copies per cell) in their genomes. Although all the founders were mosaic, the transgene was stably transmitted from the F1 generation to all subsequent generations following a Mendelian pattern. Different transgenic lines showed different responsiveness to estradiol-17β (E2) exposure at the mRNA and protein levels, and the expression efficiency was dependent upon the transgene copy number. The induction of RFP was significantly affected by the developmental stage of transgenic larvae: later-stage larvae (older than 7 days post-hatching) showed higher sensitivity to E2 exposure than earlier-stage larvae. The response of transgenic expression to E2 was fairly dependent upon the E2 dose (200-3,200 ng/L) and exposure period (1-7 days), according to both a microscopic examination of RFP intensity and a qRT-PCR assay. The transgenic marine medaka showed similar transgenic responses to E2 under freshwater, brackish, and seawater conditions. In addition to E2, the transgenic RFP signal was also successfully induced during 1-week exposure to various other natural (1 μg/L estrone and 10 μg/L estriol) and synthetic (xeno)estrogens (0.1 μg/L 17α-ethynylestradiol, 1 μg/L diethylstilbestrol, and 10 mg/L bisphenol A). The efficiency of transgene expression varied greatly among the chemicals tested. The results of this study suggest that the chgH-rfp transgenic marine medaka species will be useful in the in vivo detection of waterborne estrogens under a wide range of salinity conditions.

Targeting Tumor Stroma Using Engineered Mesenchymal Stem Cells Reduces the Growth of Pancreatic Carcinoma

To analyze the efficacy of engineered mesenchymal stem cell based therapy directed towards pancreatic tumor stroma. Mesenchymal stem cells (MSC) are actively recruited to tumor stroma where they enhance tumor growth and metastases. Upregulation of chemotactic cytokine (CCL5) by MSCs within the tumor stroma has been shown to play a central role in this process. Murine MSCs were engineered to express reporter genes or therapeutic genes under control of the CCL5 promoter and adoptively transferred into mice with growing pancreatic tumors. The effect on tumor growth and metastases was then evaluated. MSCs isolated from bone marrow of C57/Bl6 p53 mice were stably transfected with red fluorescent protein (RFP), enhanced green fluorescent protein (eGFP), or herpes simplex virus (HSV) thymidine kinase (Tk) gene driven by the RANTES promoter. MSCs were intravenously applied once per week over 3 weeks to mice carrying an orthotopic, syngeneic pancreatic Panc02 tumor. eGFP and RFP signals driven by the CCL5 promoter were detected by fluorescence in treated pancreatic tumor samples. The HSV-Tk therapy group treated intraperitoneal with the prodrug ganciclovir 5 to 7 days after stem cell application lead to a 50% reduction of primary pancreatic tumor growth (P < 0.0003, student t test) and reduced liver metastases (0% vs. 60%). The active homing of MSCs into primary pancreatic tumor stroma and activation of the CCL5 promoter was verified using eGFP- and RFP-reporter genes. In the presence of ganciclovir, HSV-Tk transfected MSCs led to a significant reduction of primary pancreatic tumor growth and incidence of metastases.

Innate Immunity to Intraphagosomal Pathogens Is Mediated by Interferon Regulatory Factor 8 (IRF-8) That Stimulates the Expression of Macrophage-specific Nramp1 through Antagonizing Repression by c-Myc

Macrophages are a central arm of innate immune defense against intracellular pathogens. They internalize microbes into phagosomes where the invaders are being killed by oxygen and nitrogen reactive species. Despite this battery of antimicrobial molecules, some are able to thrive within the phagosome thus termed intraphagosomal pathogens among which are Salmonella, Leishmania, and Mycobacteria. In mice, a single dominant gene termed Nramp1/Slc11a1 controls innate resistance to such pathogens. This gene is expressed exclusively in myeloid cells. Previously, we have shown that the restricted expression of Nramp1 is regulated by a myeloid cell-specific transcription factor termed IRF-8/ICSBP. It is demonstrated here that the induction of Nramp1 expression in activated macrophages is accompanied by a promoter shift from a repression state elicited by c-Myc to an activation state elicited by the induction of IRF-8 in activated macrophages. This transition from repression to activation is facilitated by a competitive protein-protein interaction with the transcription factor Miz-1. To show that IRF-8 is directly involved in the elimination of intraphagosomal pathogens through the regulation of Nramp1 gene expression, we bred wild type as well as IRF-8 and Nramp1 null mouse strains and examined macrophages derived from bone marrow and peritoneum. Our results clearly show that the absence of IRF-8 and Nramp1 leads to the same phenotype; defective killing of intraphagosomal Salmonella enterica serovar typhimurium and Mycobacterium bovis. Thus, interplay between repression and activation state of the Nramp1 promoter mediated by IRF-8 provides the molecular basis by which macrophages resist intraphagosomal pathogens at early stage after infection.

Advanced Generation Adenoviral Virotherapy Agents Embody Enhanced Potency Based upon CAR-Independent Tropism

In this current issue of Clinical Cancer Research , Reddy et al. ([1][1]) constructed an adenoviral virotherapy agent that uses the human E2F-1 gene promoter to regulate the adenoviral E1A gene. The E2F transcription factor plays an important role in cell proliferation and is repressed by the

Protein Aggregates are Transported to Vacuoles by Macroautophagic Mechanism in Nutrient-Starved Plant Cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated with nutrient limit medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit authophagic processes, inhibited the transport of RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP–NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to otherorganelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation occurs in plant cells.