scholarly journals Role of Ubiquitin and Proteasomes in Phagosome Maturation

2005 ◽  
Vol 16 (4) ◽  
pp. 2077-2090 ◽  
Author(s):  
Warren L. Lee ◽  
Moo-Kyung Kim ◽  
Alan D. Schreiber ◽  
Sergio Grinstein
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Continuous Distribution ◽  
Endocytic Pathway ◽  
Phagosome Maturation ◽  
Fcγ Receptors ◽  
Acidic Compartment

Phagosomes undergo multiple rounds of fusion with compartments of the endocytic pathway during the course of maturation. Despite the insertion of vast amounts of additional membrane, the phagosomal surface area remains approximately constant, implying active ongoing fission. To investigate the mechanisms underlying phagosomal fission we monitored the fate of Fcγ receptors (FcγR), which are known to be cleared from the phagosome during maturation. FcγR, which show a continuous distribution throughout the membrane of nascent phagosomes were found at later times to cluster into punctate, vesicular structures, before disappearing. In situ photoactivation of receptors tagged with a light-sensitive fluorescent protein revealed that some of these vesicles detach, whereas others remain associated with the phagosome. By fusing FcγR to pH-sensitive fluorescent proteins, we observed that the cytoplasmic domain of the receptors enters an acidic compartment, indicative of inward budding and formation of multivesicular structures. The topology of the receptor was confirmed by flow cytometry of purified phagosomes. Phagosomal proteins are ubiquitylated, and ubiquitylation was found to be required for formation of acidic multivesicular structures. Remarkably, proteasomal function is also involved in the vesiculation process. Preventing the generation of multivesicular structures did not impair the acquisition of late endosomal and lysosomal markers, indicating that phagosomal fusion and fission are controlled separately.

Possibile ruolo della citofluorometria (CFM) nel monitoraggio della risposta alla terapia endocavitaria con BCG nel carcinoma in situ della vescica: Monitoring the response to intravesical BCG in the treatment of cis: A possible role of flow cytometry

1995 ◽  
Vol 62 (2) ◽  
pp. 245-247
Author(s):  
M. De Siati ◽  
D. Grassi ◽  
N. Franzolin ◽  
L.S. Azzolina
Keyword(s):  
Flow Cytometry ◽  
Carcinoma In Situ ◽  
Recurrent Disease ◽  
Intravesical Therapy ◽  
Urinary Cytology ◽  
Bacillus Calmette Guerin ◽  
Intravesical Bcg

We report our experience on the treatment of carcinoma in situ (CIS) using intravesical therapy with the Bacillus Calmette-Guerin (BCG). From November 1992 to September 1994, 18 patients received treatment: 6 had associated CIS and 12 secondary CIS. Ploidy of each tumour was determined by flow cytometry. Aneuploidy was found in 12 cases, diploidy in 6 cases. After treatment, a standard bladder mapping was performed: 14 patients showed no evidence of cystoscopic and histological disease and if previously aneuploid, became diploid. 4 patients has recurrent disease after therapy; they were all aneuploid before treatment. One of these showed a persistent aneuploidy, although both voided urinary cytology and histological samples were negative. Six months later, a recurrent CIS was seen at the time of cystoscopy. These results enhance the interest in flow cytometry as a possible predictor of response to BCG in the treatment of CIS.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

Autofluorescence and Apoptosis in Flow Cytometry: Correlative Methods for Fluorescence and Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 618-619
Author(s):  
R. W. Smith
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Statistical Evaluation ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Apoptotic Cells ◽  
Chemical Probes ◽  
Clear Separation ◽  
Dying Cells

Flow cytometry offers several advantages in evaluating cellular fluorescence and separating values from chemical probes or transfected fluorescent proteins from autofluorescence. Autofluorescence can introduce artificial values that cannot be separated by examination of the fluorescence histogram. Often a comparison of fluorescence in two detectors can separate autofluorescence from positive fluorescence. A region can be defined around the cells of interest for further statistical evaluation or for subsequent sorting.Autofluorescence is also correlated with apoptosis in that apoptotic cells often increase autofluorescence values. A clear separation of living cells from dying cells can be achieved by the introduction of a DNA stain, such as 7AAD, which will penetrate cells with disrupted or damaged cellular membranes. Intact living cells will not show the 7AAD fluorescence.This separation is important in transfected fluorescent protein studies, as the transfection process is often toxic to cells.

Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

2016 ◽  
Vol 26 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Lin Liu ◽  
Xiangyan Zhang ◽  
Siyuan Yang ◽  
Yao Zhai ◽  
Weijia Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Qrt Pcr ◽  
Key Factor ◽  
Green Fluorescent

<b><i>Aims:</i></b> The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. <b><i>Methods:</i></b> The <i>bla</i><sub>CTX-M</sub> promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of <i>bla</i><sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. <b><i>Results:</i></b> The promoters of all the <i>bla</i><sub>CTX-M</sub> genes were provided by IS<i>Ecp1 </i>and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of <i>bla</i><sub>CTX-M</sub> was influenced. <b><i>Conclusion:</i></b> The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the <i>bla</i><sub>CTX-M</sub> gene does not specifically enhance the expression of<i> bla</i><sub>CTX-M</sub>, contrary to previous reports. The expression of <i>bla</i><sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.

Imaging pancreatic β-cells in the intact pancreas

2006 ◽  
Vol 290 (5) ◽  
pp. E1041-E1047 ◽  
Author(s):  
Manami Hara ◽  
Restituto F. Dizon ◽  
Benjamin S. Glick ◽  
Catherine S. Lee ◽  
Klaus H. Kaestner ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Confocal Imaging ◽  
Β Cells ◽  
Β Cell ◽  
Disease States ◽  
Adult Mice ◽  
And Function

We have developed a method to visualize fluorescent protein-labeled β-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the β-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, β-cell clusters, and single β-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary β-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red β-cells. The imaging technique that we have developed, coupled with the transgenic mice in which β-cells and β-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.

scholarly journals Crystal structure of the fluorescent protein fromDendronephthyasp. in both green and photoconverted red forms

2016 ◽  
Vol 72 (8) ◽  
pp. 922-932 ◽  
Author(s):  
Nadya V. Pletneva ◽  
Sergei Pletnev ◽  
Alexey A. Pakhomov ◽  
Rita V. Chertkova ◽  
Vladimir I. Martynov ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Blue Light ◽  
Considerable Increase ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Three Dimensional ◽  
Red Shift ◽  
Peptide Backbone

The fluorescent protein fromDendronephthyasp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

scholarly journals A Validated Set of Fluorescent-Protein-Based Markers for Major Organelles in Yeast (Saccharomyces cerevisiae)

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Jing Zhu ◽  
Zheng-Tan Zhang ◽  
Shi-Wei Tang ◽  
Bo-Song Zhao ◽  
Hui Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Budding Yeast ◽  
Endocytic Pathway ◽  
Target Protein ◽  
Eukaryotic Cells ◽  
Internal Space ◽  
Early Endosomes ◽  
The Impact

ABSTRACT Eukaryotic cells share a basic scheme of internal organization featuring membrane-based organelles. The use of fluorescent proteins (FPs) greatly facilitated live-cell imaging of organelle dynamics and protein trafficking. One major limitation of this approach is that the fusion of an FP to a target protein can and often does compromise the function of the target protein and alter its subcellular localization. The optimization process to obtain a desirable fusion construct can be time-consuming or even unsuccessful. In this work, we set out to provide a validated set of FP-based markers for major organelles in the budding yeast (Saccharomyces cerevisiae). Out of over 160 plasmids constructed, we present a final set of 42 plasmids, the recommendations for which are backed up by meticulous evaluations. The tool set includes three colors (green, red, and blue) and covers the endoplasmic reticulum (ER), nucleus, Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, and lipid droplets. The fidelity of the markers was established by systematic cross-comparison and quantification. Functional assays were performed to examine the impact of marker expression on the secretory pathway, endocytic pathway, and metabolic activities of mitochondria and peroxisomes. Concomitantly, our work constitutes a reassessment of organelle identities in this model organism. Our data support the recognition that “late Golgi” and “early endosomes,” two seemingly distinct terms, denote the same compartment in yeast. Conversely, all other organelles can be visually separated from each other at the resolution of conventional light microscopy, and quantification results justify their classification as distinct entities. IMPORTANCE Cells contain elaborate internal structures. For eukaryotic cells, like those in our bodies, the internal space is compartmentalized into membrane-bound organelles, each tasked with specialized functions. Oftentimes, one needs to visualize organelles to understand a complex cellular process. Here, we provide a validated set of fluorescent protein-based markers for major organelles in budding yeast. Yeast is a commonly used model when investigating basic mechanisms shared among eukaryotes. Fluorescent proteins are produced by cells themselves, avoiding the need for expensive chemical dyes. Through extensive cross-comparison, we make sure that each of our markers labels and only labels the intended organelle. We also carefully examined if the presence of our markers has any negative impact on the functionality of the cells and found none. Our work also helps answer a related question: are the structures we see really what we think they are?

scholarly journals Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

2003 ◽  
Vol 69 (11) ◽  
pp. 6848-6855 ◽  
Author(s):  
Yutao Fu ◽  
Charles O'Kelly ◽  
Michael Sieracki ◽  
Daniel L. Distel
Keyword(s):  
Flow Cytometry ◽  
Prey Species ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Heterotrophic Flagellate ◽  
Broad Host Range ◽  
Bacterial Cells ◽  
Protein Markers ◽  
Significant Difference ◽  
Paraphysomonas Imperforata

ABSTRACT Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

scholarly journals Generation and Characterization of a New FRET-Based Ca2+ Sensor Targeted to the Nucleus

2021 ◽  
Vol 22 (18) ◽  
pp. 9945
Author(s):  
Luisa Galla ◽  
Nicola Vajente ◽  
Diana Pendin ◽  
Paola Pizzo ◽  
Tullio Pozzan ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Processes ◽  
A Cell ◽  
Dynamic Concentration ◽  
Subcellular Compartmentalization

Calcium (Ca2+) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca2+]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca2+] changes. To this aim, as nucleoplasm Ca2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca2+ probe. In particular, we modified the previously described nuclear Ca2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca2+ measurements. The affinity for Ca2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

scholarly journals TGF-β signaling is essential for joint morphogenesis

2007 ◽  
Vol 177 (6) ◽  
pp. 1105-1117 ◽  
Author(s):  
Anna Spagnoli ◽  
Lynda O'Rear ◽  
Ronald L. Chandler ◽  
Froilan Granero-Molto ◽  
Douglas P. Mortlock ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Receptor Gene ◽  
Artificial Chromosome ◽  
Transforming Growth Factor Β ◽  
Gene Expressions ◽  
Β Receptor ◽  
Green Fluorescent

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor β (TGF-β) signaling in mice lacking the TGF-β type II receptor gene (Tgfbr2) in their limbs (Tgfbr2PRX-1KO). In Tgfbr2PRX-1KO mice, the loss of TGF-β responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2Prx1KO joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2–green fluorescent protein–β–GEO–bacterial artificial chromosome β-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2PRX-1KO mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-β receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2PRX-1KO growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-β signaling represents a means of entry to initiate the process.

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