Yellow Fluorescent Protein Fluorescence Research Articles

Hypoglossal Motoneuron (XII MN) Survival Over the Lifespan in Pompe Disease

Glycogen Storage Disease II, more commonly known as Pompe disease, is a lysosomal storage disease characterized by a partial or complete loss of acid α‐glucosidase (GAA) activity. Lingual impairment and sleep disordered breathing are common symptoms of Pompe disease with a potential role of XII MN dysfunction in both cases. Through innervation of the tongue muscles, XII MNs contribute to swallowing, speech, and maintenance of upper airway patency. Studies of Pompe (Gaa‐/‐) mice have found molecular and histological markers of neurodegeneration, and a recent case study of a patient with late‐onset Pompe disease revealed atrophied and possible degenerating XII MNs. However, whether the absence of GAA activity in Pompe disease directly leads to death of XII MNs is not definitively established. Because the preponderance of evidence indicates progressive neurodegeneration in Pompe disease, here we tested the hypothesis that when XII MNs do not express GAA activity, there will be a progressive loss of these cells over the lifespan. A novel transgenic mouse model (ChAT‐cre+/+●Gaa+/‐●eYFP+/+●Flox‐Gaa+/+) was generated in which GAA activity is absent in ChAT positive neurons, which also carry a yellow fluorescent protein (YFP) marker. Thus MNs in this mouse model do not express GAA, but skeletal myofibers have normal GAA expression. Brainstem tissue was harvested in young (1‐3 months of age) and advanced age mice (21‐24 months). Tissues were cryosectioned at 20 μm increments across the entire XII motor nucleus. Sections were slide mounted and visualized under fluorescence microscopy to detect YFP. The XII motor nucleus was identified based on anatomical landmarks and MNs were identified based on location, size, morphology, and YFP fluorescence. Preliminary MN counts across the rostral‐caudal extent of the XII nucleus (n=3 per group) indicate an approximately 7% decline in detectable YFP‐positive XII MNs over the age span (young: 658±19; old 617±28). Further, the histological appearance of XII MNs showed striking differences between the two age groups. The MNs from older mice showed the prototypical hallmarks of neuronal lysosomal storage disease including a markedly enlarged soma and extensive vacuolization. These preliminary findings are consistent with the hypothesis that absence of GAA in XII MNs leads to a histopathological appearance and neurodegeneration. Ongoing studies are evaluating XII MNs in age matched wild type mice.

OR28-03 Drug Repurposing Identifies Inhibitors of the Proteostasis Network to Augment Radioiodine Uptake in Combinatorial Approaches Targeting Thyroid Cancer

New combinatorial drug strategies are urgently needed to improve radioiodine (RAI) uptake and efficiently ablate thyroid cancer cells, thereby reducing the risk of recurrent disease. Drug repurposing offers the promise of identifying already approved compounds capable of inducing sodium iodide symporter (NIS) function to enhance iodide uptake. However, a lack of thyroid cell-based assays amenable to high-throughput screening has limited progress. We utilised the mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide and screened the Prestwick Chemical Library (1200 drugs; 95% approved) for quenching of YFP fluorescence. This allowed us to identify putative candidate drugs which increased iodide uptake >2 SD above mean. Categorisation of these revealed a high proportion of drugs that modulate the proteostasis network (19/48; ~40%), including key processes in protein homeostasis such as endoplasmic reticulum-associated protein degradation (ERAD) and autophagy. Secondary screening validated the activity of proteostasis modulators in enhancing iodide uptake after ranking 73 leading compounds based on their pharmacologic (AUC, EMAX and EC50) and specificity of response (NIS+ve vs NIS-ve YFP-thyroid cells) at ten different drug doses (0.1 to 50 μM). Of importance, several repurposed drugs (e.g. ebastine, Prestwick N, Prestwick C and clotrimazole) in combination with the HDAC inhibitor vorinostat induced a robust enhancement in RAI uptake in thyroid cancer cells (TPC-1 and 8505C NIS+ve cells, up to 11-fold vs DMSO, P<0.001), which was significantly greater than using vorinostat alone (up to 3-fold, P<0.01). For clotrimazole, we designed 7 new chemical derivatives, 3 of which showed enhanced aqueous solubility and retained the ability to significantly enhance RAI uptake. TaqMan RT-PCR revealed that, in contrast to vorinostat, our repurposed drugs failed to alter NIS mRNA expression, highlighting post-transcriptional mechanisms. Critically, 11 repurposed drugs induced significant gains in RAI uptake in human primary thyroid cells (up to 4.1-fold; P<0.05), the most physiological setting for NIS function. In conclusion, we performed high-throughput screening and identified proteostasis modulators, as well as other repurposed drugs, that markedly enhance radioiodine uptake. Further clinical investigation of these drugs might offer new combinatorial approaches, especially with existing therapies, to improve the treatment of thyroid cancer.

Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory.

Feeding by chewing insects induces chemical defenses in plants that are regulated by the jasmonic acid (JA) pathway. Jasmonates are usually quantified by liquid chromatography–mass spectrometry (LC-MS) analysis of precursors and products in the biosynthetic pathway or inferred from the extraction and expression of genes known to respond to elevated levels of JA. Both approaches are costly and time consuming. To address these limitations, we developed a rapid reporter for the synthesis of JA based on the OPR3promoter:YFP-PTS1. Yellow fluorescent protein (YFP) fluorescence was increased by mechanical wounding and methyl jasmonate (MeJA) treatment and by caterpillar feeding. To develop an optimal sampling time for a quantitative bioassay, OPR3promoter:YFP-PTS1 plants were sampled at 1, 2, 3, and 24 h after treatment with 115 µM MeJA. The first increase in YFP fluorescence was detected at 2 h and remained elevated 3 and 24 h later; as a result, 3 h was chosen as the sampling time for a quantitative bioassay of jasmonate response to insect attack. Feeding by Pieris rapae caterpillars induced a 1.8-fold increase in YFP fluorescence, consistent with the known induction of JA production by this insect. We also assessed the utility of this reporter in studies of plant responses to caterpillar feeding vibrations, which are known to potentiate the JA-dependent production of chemical defenses. Pretreatment with feeding vibrations increased expression of the OPR3promoter:YFP-PTS1 in response to 14 µM MeJA. Feeding vibrations did not potentiate responses at higher MeJA concentrations, suggesting that potentiating effects of prior treatments can only be detected when plants are below a response threshold to the elicitor. The expression of OPR3 does not indicate levels of specific downstream jasmonates and quantification of specific jasmonates still requires detailed analysis by LC-MS. However, OPR3 expression does provide a rapid and inexpensive way to screen large numbers of plants for the involvement of jasmonate signaling in their response to a wide variety of treatments, and to study the induction and expression of AtOPR3.

Elucidating Carotenoid Biosynthetic Enzyme Localization and Interactions Using Fluorescent Microscopy.

Carotenoids are essential for survival of all plants, where these colorful pigments and derivatives are biosynthesized, as well as for humans and other species that obtain plant-derived carotenoids in their diets and rely upon them for vitamin biosynthesis or antioxidant actions. The plant carotenoid biosynthetic pathway consists of nuclear encoded enzymes that are imported into chloroplasts and other plastids. The pathway structural genes are known and have been targeted for metabolic engineering to improve carotenoid profiles or content. However, results are not always as expected because there remain fundamental gaps in understanding how the pathway is physically organized. Many of the enzymes have been found in high molecular weight complexes which are poorly described. Elucidation of enzyme localization as well as enzyme interactions in vivo are needed for advancing the carotenoid field and facilitating our understanding of the three-dimensional organization of this important pathway. Fluorescent protein fusions with carotenoid enzymes can provide in vivo information when these fusions are introduced and transiently expressed in plant cells. Current advances in fluorescent microscopy, especially confocal microscopy, provide the resolution needed to localize fluorescently tagged carotenoid enzymes within suborganellar locations of plastids. Interactions between carotenoid biosynthetic enzymes can be determined using bimolecular fluorescence complementation (BiFC), a method whereby genes of interest are fused with sequences encoding nonfluorescent N- and C-terminal halves of YFP (yellow fluorescent protein), and then introduced into plant protoplasts to allow expression and visualization by fluorescence microscopy. The YFP fluorescence is restored only if the N and C-terminal regions are brought together by interacting fusion partners. Here we describe the methodology, with extensive tips and notes, for determining in vivo carotenoid enzyme localization and enzyme interactions by transient expression of enzyme-fluorescent protein fusions.

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Visualization of PAX7 protein dynamics in muscle satellite cells in a YFP knock-in-mouse line

BackgroundSatellite cells are residential muscle stem cells that express a paired box protein, PAX7.ResultsHere, we report a knock-in mouse line expressing a PAX7-enhanced yellow fluorescent protein (YFP) fusion protein that enables visualization of PAX7 protein dynamics in living satellite cells through YFP fluorescence. The YFP fluorescence signals in Pax7-YFP knock-in mice clearly recapitulated the endogenous expression of PAX7 protein in satellite cells. YFP+ satellite cells were efficiently isolated from muscle tissues by fluorescence-activated cell sorting. Homozygous Pax7-YFP knock-in mice (Pax7YFP/YFP) were viable, grew and regenerated muscle normally, and Pax7YFP/YFP mouse-derived satellite cells proliferated, differentiated, and self-renewed as efficiently as those from wild-type (Pax7+/+) mice.ConclusionsTaken together, our Pax7-YFP mouse line is a useful tool to aid the development of stem-cell-based therapies for muscle diseases.

Inhibition of Human Papillomavirus Type 16 Infection Using an RNA Aptamer.

Human papillomavirus type 16 (HPV16) DNA has been found in ∼50% of cervical tumors worldwide. HPV infection starts with the binding of the virus capsid to heparan sulfate (HS) receptors exposed on the surface of epithelial basal layer keratinocytes. Previously, our group isolated a high-affinity RNA aptamer (Sc5c3) specific for HPV16 L1 virus-like particles (VLPs). In this study, we report the inhibition of HPV16 infection by Sc5c3 in a pseudovirus (PsVs) model. 293TT cells were infected by HPV16 PsVs containing the yellow fluorescent protein (YFP) as reporter gene. Incubation of HPV16 PsVs with Sc5c3 before infection resulted in a dose-dependent decrease in YFP fluorescence, suggesting infection inhibition. Aptamer degradation by RNase A restored PsVs infectivity, supporting the previous observation that Sc5c3 aptamer can inhibit infection. VLP mutants with removed HS binding sites were used in binding assays to elucidate the Sc5c3 blocking mechanism; however, no binding difference was observed between wild-type and mutant VLPs, suggesting that pseudoinfection inhibition relies on mechanisms additional to electrostatic HS binding site interaction. A DNA/RNA Sc5c3 version also inhibited HPV PsVs infection, suggesting that a modified, nuclease-resistant Sc5c3 may be used to inhibit HPV16 infection in vivo.

Defective interaction between p27 and cyclin A-CDK complex in certain human cancer cell lines revealed by split YFP assay in living cells.

Cyclin-cyclin dependent kinase (CDK) complex is negatively regulated by interaction with CDK inhibitors (CKIs). p27 protein is a major CKI in mammals and its down-regulation correlates with malignant transformation. However, some cancer cells express p27 at normal level, suggesting not only quantitative but qualitative control of p27, although little is known about such control. We analyzed the interaction between p27 and cyclin A (CycA)-CDK complex in living human cell lines, using a split yellow fluorescent protein (YFP) system in which the YFP fluorescence solely depends on p27-CycA binding. Introduction of this system into various cancer cell lines revealed that certain cell lines show no detectable YFP fluorescence. Furthermore, these cell lines exhibited reduced p27-CycA interaction as evaluated by immunoprecipitation, while they showed normal co-localization of both proteins. These results suggest that some cancer cells are defective for efficient interaction between p27 and CycA-CDK complex due to qualitative alteration(s).

Construction, characterization and application of a genome-wide promoter library in Saccharomyces cerevisiae

Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein (YFP) reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions. Open image in new window

Neural stem cells from mouse strain Thy1 YFP-16 are a valuable tool to monitor and evaluate neuronal differentiation and morphology

To analyse events following transplantation of stem cells in the brain robust tools for tracing stem cells are required. Here we took advantage of the mouse strain B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy1 YFP-16), where yellow fluorescent protein (YFP) is under control of the promoter of Thy1 gene. This allows visualising whole neurons, i.e. their cell body, axons and dendrites. In this work fluorescent cells were followed during embryonic development, in vitro differentiation, and after transplantation in the healthy and stroke-affected mouse brain. During embryonic development Thy1-YFP positive cells were first observed on E12.5 and subsequently located in the prosencephalon, rhombencephalon, spinal cord and peripheral nerves. Quantitative analysis by RT-PCR and immunocytochemistry revealed that Thy1-YFP positive cells during embryo development and in vitro differentiation were expressing nestin and SOX2 then MAP2, β3-tubulin and NeuN. Thy1-YFP positive cells isolated from E14.5 represented 21.88±053% (SD) of the cultivated neurons and this remained constant along in vitro differentiation. On the other hand, proportion of Thy1-YFP positive cells reached 50% of neurons in perinatal and one month old mouse brain. Neural stem cells isolated from Thy1 YFP-16 mouse strain transplanted near hippocampus of the healthy and stroke-affected brain were distinguishable by YFP fluorescence. They differentiated into mature neurons and were detectable even 14 weeks after transplantation, the end point of our experiment. In conclusion, stem cells originating from Thy1 YFP-16 mice represent an outstanding tool to monitor neurogenesis enabling morphological analyses of new neurons and their projections, in particular after transplantation in the brain.

Microwave fields have little effect on α‐synuclein aggregation in a Caenorhabditis elegans model of Parkinson's disease

Potential health effects of radiofrequency (RF) radiation from mobile phones arouse widespread public concern. RF fields from handheld devices near the brain might trigger or aggravate brain tumors or neurodegenerative diseases such as Parkinson's disease (PD). Aggregation of neural α-synuclein (S) is central to PD pathophysiology, and invertebrate models expressing human S have helped elucidate factors affecting the aggregation process. We have recently developed a transgenic strain of Caenorhabditis elegans carrying two S constructs: SC tagged with cyan (C) blue fluorescent protein (CFP), and SV with the Venus (V) variant of yellow fluorescent protein (YFP). During S aggregation in these SC+SV worms, CFP, and YFP tags are brought close enough to allow Foerster Resonance Energy Transfer (FRET). As a positive control, S aggregation was promoted at low Hg(2+) concentrations, whereas higher concentrations activated stress-response genes. Using two different exposure systems described previously, we tested whether RF fields (1.0 GHz CW, 0.002-0.02 W kg(-1); 1.8 GHz CW or GSM, 1.8 W kg(-1)) could influence S aggregation in SC+SV worms. YFP fluorescence in similar SV-only worms provided internal controls, which should show opposite changes due to FRET quenching during S aggregation. No statistically significant changes were observed over several independent runs at 2.5, 24, or 96 h. Although our worm model is sensitive to chemical promoters of aggregation, no similar effects were attributable to RF exposures.

Dual-Reporter β-Cell-Specific Male Transgenic Rats for the Analysis of β-Cell Functional Mass and Enrichment by Flow Cytometry.

Mouse β-cell-specific reporter lines have played a key role in diabetes research. Although the rat provides several advantages, its use has lagged behind the mouse due to the relative paucity of genetic models. In this report we describe the generation and characterization of transgenic rats expressing a Renilla luciferase (RLuc)-enhanced yellow fluorescent protein (YFP) fusion under control of a 9-kb genomic fragment from the rat ins2 gene (RIP7-RLuc-YFP). Analysis of RLuc luminescence and YFP fluorescence revealed that reporter expression is restricted to β-cells in the adult rat. Physiological characteristics including body weight, fat and lean mass, fasting and fed glucose levels, glucose and insulin tolerance, and β-cell mass were similar between two RIP7-RLuc-YFP lines and wild-type littermates. Glucose-induced insulin secretion in isolated islets was indistinguishable from controls in one of the lines, whereas surprisingly, insulin secretion was defective in the second line. Consequently, subsequent studies were limited to the former line. We asked whether transgene activity was responsive to glucose as shown previously for the ins2 gene. Exposing islets ex vivo to high glucose (16.7 mM) or in vivo infusion of glucose for 24 hours increased luciferase activity in islets, whereas the fraction of YFP-positive β-cells after glucose infusion was unchanged. Finally, we showed that fluorescence-activated cell sorting of YFP-positive islet cells can be used to enrich for β-cells. Overall, this transgenic line will enable for the first time the application of both fluorescence and bioluminescence/luminescence-based approaches for the study of rat β-cells.

Nonspecific labeling limits the utility of Cre-Lox bred CST-YFP mice for studies of corticospinal tract regeneration.

Studies of axon regeneration in the spinal cord often assess regeneration of the corticospinal tract (CST). Emx1-Cre x Thy1-STOP-YFP mice have been reported to have yellow fluorescent protein (YFP) selectively expressed in forebrain neurons leading to genetic labeling of CST axons in the spinal cord, and it was suggested that these CST-YFP mice would be useful for studies of CST regeneration. Because regeneration past a lesion may involve only a few axons, the presence of labeled non-CST axons compromises interpretation. We show here that in CST-YFP mice, some YFP-labeled axons are not from the CST. Specifically, YFP-labeled axons are present in regions beyond those with anterogradely labeled CST axons, most YFP-labeled axons beyond established CST locations do not undergo Wallerian degeneration following a large lesion of the sensorimotor cortex, some rubrospinal and reticulospinal neurons are labeled with YFP, and some YFP-labeled cells in the spinal gray matter have YFP-labeled projections into the spinal cord white matter. We further demonstrate that the density of YFP-labeled axon arbors hinders tracing of single axons to their point of origin in the main descending tracts. In light of recent advances in 3D imaging for visualizing axons in unsectioned blocks of spinal cord, we also assessed CST-YFP mice for 3D imaging and found that YFP fluorescence in CST-YFP mice is faint for clearing-based 3D imaging in comparison with fluorescence in Thy1-YFP-H mice and fluorescence of mini-ruby biotinylated dextran amine (BDA). Overall, the nonspecific and faint YFP labeling in CST-YFP mice limits their utility for assessments of CST axon regeneration.

Besides neuro-imaging, the Thy1-YFP mouse could serve for visualizing experimental tumours, inflammation and wound-healing.

The B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mouse strain, widely used to study neuronal development and regeneration, expresses the yellow fluorescent protein (YFP) in the peripheral nerves and the central nervous system under the control of regulatory sequences of the Thy1 gene. The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction. We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system. We demonstrated that the stroma of subcutaneous tumours induced by the injection of 4T1 or MC26 carcinoma cells into BALB/c(Thy1-YFP) mice, carrying the same construct, indeed expressed the YFP transgene. In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein. Local inflammation induced by subcutaneous injection of complete Freund's adjuvant, as well as the experimental wound-healing milieu, also triggered YFP fluorescence in both the BALB/c(Thy1-YFP) and B6.Cg-Tg(Thy1-YFP)16Jrs/J mice, pointing to eventual overlapping pathways of wound-healing, inflammation and tumour growth.

The localization of auxin transporters PIN3 and LAX3 during lateral root development in Arabidopsis thaliana

Fluorophore tagged proteins are used in Arabidopsis thaliana to understand their functional role in plant development. This requires the analysis of their spatial localization in planta. However, the localization analysis is often perturbed by a significant overlap of the fluorophores used to label proteins of interest and the optical filtering methods available on the confocal microscope. This problem can be addressed by the use of spectral imaging with linear unmixing the image data. We applied this method to help us identify double transgenic A. thaliana lines which expressed two fluorescently tagged auxin transporter proteins: the auxin efflux protein PIN-FORMED-3 (PIN3), tagged with green fluorescent protein (GFP), and the auxin influx protein LIKE-AUX1-3 (LAX3), tagged with yellow fluorescent protein (YFP). This method allows the reliable separation of overlapping GFP and YFP fluorescence signals and subsequent localization analysis highlighting the potential benefit of this methodology in studies of lateral root development.

Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination inPyricularia oryzae

To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.

Pollen tube growth regulation by free anions depends on the interaction between the anion channel SLAH3 and calcium-dependent protein kinases CPK2 and CPK20.

Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca(2+)-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca(2+)-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants.Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume.We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape.SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.

A sensitive tool to measure CFTR channel activity

cystic fibrosis transmembrane conductance regulator(CFTR, ABCC7) is a member of the human ABC protein fam-ily. The characteristic feature of ABC proteins is the similararchitecture of the ATP binding sites and the common mecha-nism of ATP binding and hydrolysis. In contrast to most ofthe ABC proteins, CFTR is not an active transporter, rather itis a phosphorylation and ATP hydrolysis gated chloride ionchannel and channel regulator (1,2). Mutations in both copiesof the CFTR gene may lead to a heritable genetic disease, i.e.cystic fibrosis (CF) with variable severity depending on thesite of the mutation. Certain mutations alter biogenesis or cel-lular processing of the CFTR protein, while others affect itschannel activity. Chemical chaperons, also called correctors(e.g., VX-809) can partially rescue the misprocessing, mostlikely by improving the folding of the protein at the endoplas-mic reticulum (2). Activators of CFTR may function throughelevating cytosolic cAMP (promoting CFTR phosphoryla-tion), inhibiting phosphatase activity (thus blocking CFTR de-phosphorylation), and/or interacting directly with the channelprotein (1). Since CFTR is expressed in the epithelial cells ofseveral organs, CF is a multi-organ disease that affects thesinopulmonary and male urogenital systems, alters pancreaticand biliary secretion, therefore the current average lifespan ofCF patients is approximately 40 years of age (1,2). In mostcases, the primary cause of early onset of death is the progres-sion of lung disease (2,3). The mechanisms by which CFTRmutations cause lung disease in CF patients are not fullyunderstood. It may include altered ion and water transportacross the airway epithelium and aberrant inflammatory andimmune responses to pathogens within the airways (3,4).Several techniques have been developed to study CFTRfunction or to search for CFTR activators or modulatorsincluding halide sensitive fluorescent dyes, electrophysiologi-cal approaches such as patch clamp, short-circuit measure-ments in Ussing chambers and influx or efflux measurementsapplying radioactive ions. In addition to the above techniquesa halide sensitive mutant form of yellow fluorescent protein(YFP) has been also utilized to probe CFTR function meas-uring the iodide-mediated quenching rate of YFP, since theopen state of CFTR is also permeable to iodide ions (5). YFPfluorescence intensity is both dependent on the intracellulariodide concentration and YFP expression level. Therefore, flu-orescence intensities measured in the presence of iodideshould be normalized to YFP fluorescence intensities meas-ured in the absence of iodide to decrease experimental vari-ability related to the differences in sensor expression levelamong individual cells.However, normalization to the un-quenched YFP fluores-cence intensity is problematic especially in flow cytometricassays. The co-expression of a halide insensitive fluorescentprotein can overcome the above problem as was demonstratedby Vijftigschild et al. published in the current issue of Cytome-try Part A (page 576). When the two fluorescent proteins wereexpressed as a fusion protein formation of aggregates wasobserved. To avoid this problem the two proteins were con-nected by an auto-cleavable polypeptide chain (6) resulting inphysically not connected proteins. Theoretically, the expres-sion level of the two proteins may change upon time due to