scholarly journals Expression of a Malassezia codon optimized mCherry fluorescent protein in a bicistronic vector

2020 ◽  
Author(s):  
Joleen P.Z. Goh ◽  
Giuseppe Ianiri ◽  
Joseph Heitman ◽  
Thomas L. Dawson
Keyword(s):  
Cellular Localization ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Targeted Mutagenesis ◽  
Peptide Sequence ◽  
Selection Marker ◽  
Specific Cell ◽  
Haploid Strain ◽  
2A Peptide ◽  
Bicistronic Vector

AbstractThe use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labelling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a model Malassezia species, the haploid strain M. furfur CBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies, Malassezia cellular localization, and co-expression of genes required for targeted mutagenesis.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

scholarly journals CRISPR/Cas9-mediated targeted mutagenesis in Japanese cedar (Cryptomeria japonica D. Don)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yoshihiko Nanasato ◽  
Masafumi Mikami ◽  
Norihiro Futamura ◽  
Masaki Endo ◽  
Mitsuru Nishiguchi ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cryptomeria Japonica ◽  
Fluorescent Protein ◽  
Japanese Cedar ◽  
Targeted Mutagenesis ◽  
Embryogenic Tissue ◽  
Breeding Period ◽  
Single Mutation ◽  
Knock Out ◽  
Guide Rna

AbstractCryptomeria japonica (Japanese cedar or sugi) is one of the most important coniferous tree species in Japan and breeding programs for this species have been launched since 1950s. Genome editing technology can be used to shorten the breeding period. In this study, we performed targeted mutagenesis using the CRISPR/Cas9 system in C. japonica. First, the CRISPR/Cas9 system was tested using green fluorescent protein (GFP)-expressing transgenic embryogenic tissue lines. Knock-out efficiency of GFP ranged from 3.1 to 41.4% depending on U6 promoters and target sequences. The GFP knock-out region was mottled in many lines, indicating genome editing in individual cells. However, in 101 of 102 mutated individuals (> 99%) from 6 GFP knock-out lines, embryos had a single mutation pattern. Next, we knocked out the endogenous C. japonica magnesium chelatase subunit I (CjChlI) gene using two guide RNA targets. Green, pale green, and albino phenotypes were obtained in the gene-edited cell lines. Sequence analysis revealed random deletions, insertions, and replacements in the target region. Thus, targeted mutagenesis using the CRISPR/Cas9 system can be used to modify the C. japonica genome.

scholarly journals LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

2000 ◽  
Vol 345 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Sean P. COLLINS ◽  
Junewai L. REOMA ◽  
David M. GAMM ◽  
Michael D. UHLER
Keyword(s):  
Protein Kinase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Multiple Cancer ◽  
Dependent Protein Kinase ◽  
Rnase Protection ◽  
Camp Dependent Protein Kinase ◽  
Increased Risk ◽  
Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

scholarly journals Focal Distribution of Baculovirus IE1 Triggered by Its Binding to the hr DNA Elements

2005 ◽  
Vol 79 (1) ◽  
pp. 39-46 ◽  
Author(s):  
Toshihiro Nagamine ◽  
Yu Kawasaki ◽  
Tetsutaro Iizuka ◽  
Shogo Matsumoto
Keyword(s):  
Fluorescent Protein ◽  
Direct Repeat ◽  
Single Copy ◽  
Targeted Mutagenesis ◽  
Homologous Region ◽  
Focus Formation ◽  
Primary Determinant ◽  
Dna Elements ◽  
Species Specific ◽  
Green Fluorescent

ABSTRACT In BmN cells infected with the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), IE1, a principal transcriptional activator, localizes to sites of viral DNA replication. IE1 initially displays focal distribution in BmNPV-infected cells prior to DNA synthesis, whereas the protein expressed by transfection with the ie1 gene is distributed throughout the nucleoplasm instead of localized to discrete subnuclear structures. To identify the inducer of focus formation for IE1, we conducted transfection experiments with an IE1-GFP construct and found that cotransfection with genomic DNA fragments bearing the homologous region (hr) sequences caused the formation of IE1-green fluorescent protein (GFP) foci. The transfection of insect cells with a single plasmid containing exclusively the hr3 sequence and the IE1-GFP gene was sufficient to form IE1-GFP foci. These results suggest that hr elements are a primary determinant of the focal distribution of IE1. An analysis of a series of hr3 deletion mutants showed that a single copy of the direct repeat could induce the formation of IE1 foci. Targeted mutagenesis within the hr-binding domain of IE1-GFP caused impairment of the hr-dependent IE1 localization, suggesting that binding of IE1 to the hr elements is essential for the onset of IE1 focus formation. The observation of BmNPV IE1 foci in non-BmNPV-susceptible cells suggests that no species-specific factors are required for hr-dependent IE1 focus formation.

scholarly journals Proprotein Convertase 5/6 Is Critical for Embryo Implantation in Women: Regulating Receptivity by Cleaving EBP50, Modulating Ezrin Binding, and Membrane-Cytoskeletal Interactions

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 5041-5052 ◽  
Author(s):  
Sophea Heng ◽  
Ana Cervero ◽  
Carlos Simon ◽  
Andrew N. Stephens ◽  
Ying Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Structural Changes ◽  
Current Knowledge ◽  
Embryo Implantation ◽  
Critical Role ◽  
Human Endometrium ◽  
Scaffolding Protein ◽  
Proprotein Convertase ◽  
Proteomic Approach

Establishment of endometrial receptivity is vital for successful embryo implantation; its failure causes infertility. Epithelial receptivity acquisition involves dramatic structural changes in the plasma membrane and cytoskeleton. Proprotein convertase 5/6 (PC6), a serine protease of the proprotein convertase (PC) family, is up-regulated in the human endometrium specifically at the time of epithelial receptivity and stromal cell decidualization. PC6 is the only PC member tightly regulated in this manner. The current study addressed the importance and mechanisms of PC6 action in regulating receptivity in women. PC6 was dysregulated in the endometrial epithelium during the window of implantation in infertile women of three demographically different cohorts. Its critical role in receptivity was evidenced by a significant reduction in mouse blastocyst attachment of endometrial epithelial cells after PC6 knockdown by small interfering RNA. Using a proteomic approach, we discovered that PC6 cleaved the key scaffolding protein, ezrin-radixin-moesin binding phosphoprotein 50 (EBP50), thereby profoundly affecting its interaction with binding protein ezrin (a key protein bridging actin filaments and plasma membrane), EBP50/ezrin cellular localization, and cytoskeleton-membrane connections. We further validated this novel PC6 regulation of receptivity in human endometrium in vivo in fertile vs. infertile patients. These results strongly indicate that PC6 plays a key role in regulating fundamental cellular remodeling processes, such as plasma membrane transformation and membrane-cytoskeletal interface reorganization. PC6 cleavage of a crucial scaffolding protein EBP50, thereby profoundly regulating membrane-cytoskeletal reorganization, greatly extends the current knowledge of PC biology and provides substantial new mechanistic insight into the fields of reproduction, basic cellular biology, and PC biochemistry.

Complex regulation and nuclear localization of JRK protein

2004 ◽  
Vol 32 (6) ◽  
pp. 920-923 ◽  
Author(s):  
R. Waldron ◽  
T. Moore
Keyword(s):  
Cellular Localization ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Distinct Pattern ◽  
Nuclear Bodies ◽  
Null Mouse ◽  
Fluorescence Confocal Microscopy ◽  
Pml Nuclear Bodies ◽  
Generalized Epilepsy ◽  
Green Fluorescent

The mouse jerky gene and its human orthologue, JRK/JH8, encode a putative DNA-binding protein with homology to the CENP-B (centromere-binding protein B). Disruption of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy. In addition (and similar to a cenp-b null mouse) jerky null mice exhibit postnatal weight loss and reduced fertility. Using fluorescence confocal microscopy, the cellular localization of a JRK–GFP fusion (where GFP stands for green fluorescent protein) was investigated in HeLa cells. JRK–GFP has a dynamic expression pattern in the interphase nucleus, localizing in a small number of punctate nuclear foci and in the nucleolus. The JRK–GFP foci number changes during the cell cycle, but a distinct pattern of three JRK–GFP foci is observed at G2. The endogenous protein behaves in a similar manner to the GFP-fusion protein. JRK–GFP was found to co-localize with CREST antigens (which recognize the centromere-binding proteins, CENP-A, -B and -C) through S and G2 phases of interphase and co-localized completely with a subset of PML nuclear bodies at G2. We speculate that JRK protein associates with a specific chromosomal centromeric locus in G2, where it associates fully with PML bodies. Research is underway to identify this locus.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

scholarly journals Ultrasound and Transcriptomics Identify a Differential Impact of Cisplatin and Histone Deacetylation on Tumor Structure and Microenvironment in a Patient-Derived In Vivo Model of Gastric Cancer

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1485
Author(s):  
Aina Venkatasamy ◽  
Eric Guerin ◽  
Anais Blanchet ◽  
Christophe Orvain ◽  
Véronique Devignot ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Microenvironment ◽  
Real Time ◽  
Histone Deacetylase Inhibitors ◽  
Structural Changes ◽  
Histone Deacetylation ◽  
In Vivo Model ◽  
Specific Cell ◽  
The Impact

The reasons behind the poor efficacy of transition metal-based chemotherapies (e.g., cisplatin) or targeted therapies (e.g., histone deacetylase inhibitors, HDACi) on gastric cancer (GC) remain elusive and recent studies suggested that the tumor microenvironment could contribute to the resistance. Hence, our objective was to gain information on the impact of cisplatin and the pan-HDACi SAHA (suberanilohydroxamic acid) on the tumor substructure and microenvironment of GC, by establishing patient-derived xenografts of GC and a combination of ultrasound, immunohistochemistry, and transcriptomics to analyze. The tumors responded partially to SAHA and cisplatin. An ultrasound gave more accurate tumor measures than a caliper. Importantly, an ultrasound allowed a noninvasive real-time access to the tumor substructure, showing differences between cisplatin and SAHA. These differences were confirmed by immunohistochemistry and transcriptomic analyses of the tumor microenvironment, identifying specific cell type signatures and transcription factor activation. For instance, cisplatin induced an “epithelial cell like” signature while SAHA favored a “mesenchymal cell like” one. Altogether, an ultrasound allowed a precise follow-up of the tumor progression while enabling a noninvasive real-time access to the tumor substructure. Combined with transcriptomics, our results underline the different intra-tumoral structural changes caused by both drugs that impact differently on the tumor microenvironment.

Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions

2003 ◽  
Vol 284 (6) ◽  
pp. C1633-C1644 ◽  
Author(s):  
Mohammed A. Khadeer ◽  
Zhihui Tang ◽  
Harriet S. Tenenhouse ◽  
Maribeth V. Eiden ◽  
Heini Murer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Bone Resorption ◽  
Protein Interactions ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Basolateral Membrane ◽  
Atp Synthesis ◽  
Cellular Distribution ◽  
Green Fluorescent ◽  
Carboxy Terminal

We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Picotransporters in the osteoclast plasma membrane provide Pifor ATP synthesis and that the osteoclast may utilize part of the Pireleased from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Picotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Picotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Picotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Picotransporters in the osteoclast.

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

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