scholarly journals In vivo visualization of uterine mast cells by two-photon microscopy

Reproduction ◽  
2014 ◽  
Vol 147 (6) ◽  
pp. 781-788 ◽  
Author(s):  
Franziska Schmerse ◽  
Katja Woidacki ◽  
Monika Riek-Burchardt ◽  
Peter Reichardt ◽  
Axel Roers ◽  
...  
Keyword(s):  
Mast Cells ◽  
Intravital Microscopy ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Specific Cell ◽  
Enhanced Yellow Fluorescent Protein ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Custom Made

Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for the study ofin vivobehavior of these cells. We have recently reported that uterine mast cells (uMCs) are important for implantation and placentation. However, theirin vivolocalization in uterus before and during pregnancy is unknown. Herein, we report the direct observation of uMCsin vivousing double-transgenic C57BL/6JMcpt5-Cre ROSA26-EYFPmice with high expression of enhanced yellow fluorescent protein in MC protease 5 (Cma1(Mcpt5))-expressing cells by intravital two-photon microscopy. We were able to monitor MCs livein uteroduring the murine estrous cycle and at different days of pregnancy. We demonstrated that uMCs accumulated during the receptive phase of the female (estrus) and persisted in large numbers at early pregnancy stages and around mid-gestation and declined in number in non-pregnant animals at diestrus. This intravital microscopy technique, including a custom-made microscope stage and the adaption of the surgical procedure, allowed the access of the uterus and implantations for imaging. The introduced application of intravital microscopy to C57BL/6J-Mcpt5-Cre ROSA26-EYFPmice offers a novel and powerfulin vivoapproach to further address the evident relevance of uMCs to reproductive processes with obvious clinical implications.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles

2002 ◽  
Vol 157 (4) ◽  
pp. 615-629 ◽  
Author(s):  
Anthony K.L. Leung ◽  
Angus I. Lamond
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nucleolar Localization ◽  
Target Sequence ◽  
Enhanced Yellow Fluorescent Protein ◽  
Splicing Speckles

The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization.

Thick ascending limb-specific expression of Cre recombinase

2003 ◽  
Vol 285 (1) ◽  
pp. F33-F39 ◽  
Author(s):  
Peter K. Stricklett ◽  
Deborah Taylor ◽  
Raoul D. Nelson ◽  
Donald E. Kohan
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Yellow Fluorescent Protein ◽  
Cre Recombinase ◽  
Specific Gene ◽  
Thick Ascending Limb ◽  
Specific Expression ◽  
Enhanced Yellow Fluorescent Protein

Evaluation of thick ascending limb (TAL) function has been hindered by the limited ability to selectively examine the function of this nephron segment in vivo. To address this, a Cre/loxP strategy was employed whereby the Tamm-Horsfall (THP) promoter was used to drive Cre recombinase expression in transgenic mice. The THP gene was cloned from a mouse genomic library, and 3.7 kb of the mouse THP 5′-flanking region containing the first noncoding exon of the THP gene were inserted upstream of an epitope-tagged Cre recombinase (THP-CreTag). THP-CreTag transgenic mice were bred with ROSA26-enhanced yellow fluorescent protein (eYFP) mice (contain a loxP-flanked “STOP” sequence 5′ to eYFP), and doubly heterozygous offspring were analyzed. THP and eYFP were expressed in an identical pattern with predominant localization to the renal outer medulla without expression in nonrenal tissues. eYFP did not colocalize with thiazide-sensitive cotransporter (distal tubule) or neuronal nitric oxide synthase (macula densa) expression. THP mRNA expression was detected only in kidney, whereas CreTag mRNA was also present in testes. These data indicate that THP-CreTag transgenic mice can be used for TAL-specific gene recombination in the kidney.

scholarly journals Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits

2004 ◽  
Vol 15 (3) ◽  
pp. 990-1002 ◽  
Author(s):  
Nicola Susann Werner ◽  
Reinhard Windoffer ◽  
Pavel Strnad ◽  
Christine Grund ◽  
Rudolf Eberhard Leube ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Fluorescent Protein ◽  
Dynamic Equilibrium ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Epidermolysis Bullosa Simplex ◽  
Enhanced Yellow Fluorescent Protein ◽  
Dominant Negative Mutation

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.

scholarly journals Constitutive Somatostatin Receptor Subtype 2 Activity Attenuates GH Synthesis

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2399-2409 ◽  
Author(s):  
Anat Ben-Shlomo ◽  
Oxana Pichurin ◽  
Ramtin Khalafi ◽  
Cuiqi Zhou ◽  
Vera Chesnokova ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Fluorescent Protein ◽  
Somatostatin Receptor ◽  
Yellow Fluorescent Protein ◽  
Histone Deacetylation ◽  
Receptor Subtype ◽  
Mrna Levels ◽  
Enhanced Yellow Fluorescent Protein ◽  
Stable Transfectants

Abstract Somatostatin signals predominantly through somatostatin receptor (SSTR) subtype 2 to attenuate GH release. However, the independent role of the receptor in regulating GH synthesis is unclear. Because we had previously demonstrated constitutive SSTR2 activity in mouse corticotrophs, we now analyzed GH regulation in rat pituitary somatotroph (GC) tumor cells, which express SSTR2 exclusively and are devoid of endogenous somatostatin ligand. We demonstrate that moderately stable SSTR2 overexpression (GpSSTR2WT cells) was associated with decreased GH promoter activity, GH mRNA, and hormone levels compared with those of control transfectants (GpCon cells). In contrast, levels of GH mRNA and peptide and GH promoter activity were unchanged in GpSSTR2DRY stable transfectants moderately expressing DRY motif mutated SSTR2 (R140A). GpSSTR2DRY did not exhibit an enhanced octreotide response as did GpSSTR2WT cells; however, both SSTR2WT-enhanced yellow fluorescent protein (eYFP) and SSTR2DRY-eYFP internalized on octreotide treatment. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, increased GH synthesis in wild-type GC cells and primary pituitary cultures. GpSSTR2WT cells induced GH synthesis more strongly on SAHA treatment, evident by both higher GH peptide and mRNA levels compared with the moderate but similar GH increase observed in GpCon and GpSSTR2DRY cells. In vivo SAHA also increased GH release from GpSSTR2WT but not from control xenografts. Endogenous rat GH promoter chromatin immunoprecipitation showed decreased baseline acetylation of the GH promoter with exacerbated acetylation after SAHA treatment in GpSSTR2WT compared with that of either GpSSTR2DRY or control cells, the latter 2 transfectants exhibiting similar GH promoter acetylation levels. In conclusion, modestly increased SSTR2 expression constitutively decreases GH synthesis, an effect partially mediated by GH promoter histone deacetylation.

Functional studies of the kidney of living animals using multicolor two-photon microscopy

2002 ◽  
Vol 283 (3) ◽  
pp. C905-C916 ◽  
Author(s):  
Kenneth W. Dunn ◽  
Ruben M. Sandoval ◽  
Katherine J. Kelly ◽  
Pierre C. Dagher ◽  
George A. Tanner ◽  
...  
Keyword(s):  
Intravital Microscopy ◽  
Cell Biology ◽  
Fluid Transport ◽  
Biological Tissues ◽  
Two Photon Microscopy ◽  
Functional Studies ◽  
Two Photon ◽  
Cell Vitality ◽  
The Way

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.

scholarly journals Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

2015 ◽  
Vol 81 (24) ◽  
pp. 8277-8293 ◽  
Author(s):  
Tony Tumlirsch ◽  
Anna Sznajder ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Proteome Analysis ◽  
Enhanced Yellow Fluorescent Protein ◽  
Granule Formation ◽  
Content Type ◽  
Cell Extracts ◽  
C And N

ABSTRACTA protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion ofphaXresulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs.In vivocolocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of theRalstonia eutropha ppk(ppkReu) genes in anEscherichia coliΔppkbackground and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

2020 ◽  
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Biological Studies ◽  
Functionally Impaired ◽  
Z Ring

AbstractThe Escherichia coli Min system plays an important role in the proper placement of the septum ring (Z-ring) at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator together with the membrane-bound ATPase MinD, which results in MinD having a concentration gradient with maxima at the poles and minimum at mid-cell. MinC, the direct inhibitor of the Z-ring initiator protein FtsZ, forms a complex with MinD at the membrane, thus mirroring MinD polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. The existence of the oscillations was revealed by performing time-lapse microscopy with fluorescently-labeled Min proteins. These fusion proteins have been since then widely used to study properties of the Min system. Here we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that the eYFP compromises MinE ability to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. Taken together, our results indicate that this fusion is functionally impaired and should be used with caution in cell biological studies.

scholarly journals FnCas12a/crRNA-Mediated Genome Editing in Eimeria tenella

2021 ◽  
Vol 12 ◽  
Author(s):  
Peipei Cheng ◽  
Zhihao Zhang ◽  
Fayu Yang ◽  
Shuo Cai ◽  
Lina Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Yellow Fluorescent Protein ◽  
Ectopic Expression ◽  
Eimeria Tenella ◽  
Selection Marker ◽  
Dna Double Strand Breaks ◽  
Enhanced Yellow Fluorescent Protein

Eimeria species are intracellular parasites residing inside the intestinal epithelial cell, which cause poultry coccidiosis and result in significant financial losses in the poultry industry. Genome editing of Eimeria is of immense importance for the development of vaccines and drugs. CRISPR/Cas9 has been utilized for manipulating the genome of Eimeria tenella (E. tenella). Ectopic expression of Cas9, i.e., via plasmids, would introduce transgene, which substantially limits its application, especially for vaccine development. In this study, we initially optimized the condition of the transfection protocol. We demonstrated that with the optimized condition, the transfection of FnCas12a (also known as “FnCpf1”) protein and crRNA targeting EtHistone H4 triggered DNA double-strand breaks in vivo. We then used this strategy to knock-in a coding cassette for an enhanced yellow fluorescent protein (EYFP) and dihydrofolate reductase–thymidylate synthase gene (DHFR) as a selection marker to tag endogenous EtActin. The engineered E. tenella parasite possesses EYFP expression in its entire life cycle. Our results demonstrated that FnCas12a could trigger genome editing in E. tenella, which augments the applicability of the dissection of gene function and the development of anticoccidial drugs and vaccines for Eimeria species.

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