scholarly journals Single-strand DNA Aptamers as Probes for Protein Localization in Cells

2003 ◽  
Vol 51 (6) ◽  
pp. 797-808 ◽  
Author(s):  
Kristi K.H. Stanlis ◽  
J. Richard McIntosh
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Freeze Substitution ◽  
Accurate Localization ◽  
Cell Structures ◽  
Specific Proteins ◽  
General Protein ◽  
Green Fluorescent ◽  
Very High

The accurate localization of proteins in fixed cells is important for many studies in cell biology, but good fixation is often antagonistic to good immunolabeling, given the density of well-preserved cells and the size of most labeled antibody probes. We therefore explored the use of single-stranded oligonucleotides (aptamers), which can bind to proteins with very high affinity and specificity but which are only ∼10 kD. To evaluate these probes for general protein localization, we sought an aptamer that binds to a widely used protein tag, the green fluorescent protein (GFP). Although this quest was not successful, we were able to solve several practical problems that will confront any such labeling effort, e.g., the rates at which oligonucleotides enter fixed cells of different kinds and the extent of nonspecific oligonucleotide binding to both mammalian and yeast cell structures. Because such localization methods would be of particular value for electron microscopy of optimally fixed material, we also explored the solubility of aptamers under conditions suitable for freeze-substitution fixation. We found that aptamers are sufficiently soluble in cold organic solvents to encourage the view that this approach may be useful for the localization of specific proteins in context of cellular fine structure.

scholarly journals Benchmarking Various Green Fluorescent Protein Variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for Live Cell Imaging

2013 ◽  
Vol 79 (20) ◽  
pp. 6481-6490 ◽  
Author(s):  
Wout Overkamp ◽  
Katrin Beilharz ◽  
Ruud Detert Oude Weme ◽  
Ana Solopova ◽  
Harma Karsens ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Streptococcus Pneumoniae ◽  
Lactococcus Lactis ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Experimental Conditions ◽  
Content Type ◽  
Green Fluorescent

ABSTRACTGreen fluorescent protein (GFP) offers efficient ways of visualizing promoter activity and protein localizationin vivo, and many different variants are currently available to study bacterial cell biology. Which of these variants is best suited for a certain bacterial strain, goal, or experimental condition is not clear. Here, we have designed and constructed two “superfolder” GFPs with codon adaptation specifically forBacillus subtilisandStreptococcus pneumoniaeand have benchmarked them against five other previously available variants of GFP inB. subtilis,S. pneumoniae, andLactococcus lactis, using promoter-gfpfusions. Surprisingly, the best-performing GFP under our experimental conditions inB. subtiliswas the one codon optimized forS. pneumoniaeandvice versa. The data and tools described in this study will be useful for cell biology studies in low-GC-rich Gram-positive bacteria.

scholarly journals Better Protein Localization

2011 ◽  
Vol 19 (5) ◽  
pp. 8-10
Author(s):  
Stephen W. Carmichael ◽  
Philip Oshel
Keyword(s):  
Electron Microscopy ◽  
Green Fluorescent Protein ◽  
Cell Membrane ◽  
Fluorescence Microscopy ◽  
Light Microscopy ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
On Line ◽  
Specific Proteins ◽  
Green Fluorescent

Localizing specific proteins within cells, tissues, and organisms has been a goal of microscopists for generations. In the early 1990s, a breakthrough was made when a molecule originally derived from a jellyfish was introduced as a probe for fluorescence microscopy. This molecule is green fluorescent protein (GFP), and it has become well known for its usefulness in localizing proteins at the level of the light microscope. It is also well known that electron microscopy (EM) offers far superior spatial resolution over light microscopy, but the application of probes to localize specific proteins has required antibodies conjugated with colloidal metals (such as gold). Delivery of antibodies into the cell commonly requires detergents to permeabilize the cell membrane, which compromises the ultrastructural detail. Another breakthrough was recently published on-line by Xiaokun Shu, Varda Lev-Ram, Thomas Deerinck, Yingchuan Qi, Ericka Ramko, Michael Davidson, Yishi Jin, Mark Ellisman, and Roger Tsien: they have developed a method similar to using GFP for light microscopy, but for specifically tagging proteins at the EM level.

scholarly journals Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant-adapted rhabdovirus

2007 ◽  
Vol 88 (6) ◽  
pp. 1810-1820 ◽  
Author(s):  
Michael M. Goodin ◽  
Romit Chakrabarty ◽  
Sharon Yelton ◽  
Kathleen Martin ◽  
Anthony Clark ◽  
...  
Keyword(s):  
Protein Function ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
M Protein ◽  
N Protein ◽  
Glycoprotein G ◽  
Putative Site ◽  
Infected Cells ◽  
Green Fluorescent

Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells.

scholarly journals A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

2009 ◽  
Vol 9 (1) ◽  
pp. 224-226 ◽  
Author(s):  
Chengda Zhang ◽  
James B. Konopka
Keyword(s):  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Codon Usage ◽  
Signal Intensity ◽  
Fluorescent Protein ◽  
Protein Localization ◽  
Protein Variant ◽  
Green Fluorescent ◽  
Green Fluorescent Protein Variant

ABSTRACT Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.

Green Fluorescent Protein: A Tool for Gene Expression and Cell Biology in Mycobacteria

2003 ◽  
pp. 245-260
Author(s):  
Laura E. Via ◽  
Subramanian Dhandayuthapani ◽  
Dusanka Deretic ◽  
V. Deretic
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein A ◽  
Green Fluorescent

scholarly journals Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology

2019 ◽  
Vol 20 (14) ◽  
pp. 3479 ◽  
Author(s):  
Jean-Denis Pedelacq ◽  
Stéphanie Cabantous
Keyword(s):  
Protein Interactions ◽  
Self Assembly ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Protein Detection ◽  
Molecular Engineering ◽  
Fluorescent Markers ◽  
Diverse Area ◽  
Green Fluorescent

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

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