Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

Protein transport in the plant secretory pathwayThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 523-530 ◽  
Author(s):  
Sally L. Hanton ◽  
Federica Brandizzi
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Secretory Pathway ◽  
Protein Transport ◽  
New Technologies ◽  
Special Issue ◽  
Recent Developments ◽  
Selection Of ◽  
Made In

The study of the plant secretory pathway is a relatively new field, developing rapidly over the last 30 years. Many exciting discoveries have already been made in this area, but as old questions are answered new ones become apparent. Our understanding of the functions and mechanisms of the plant secretory pathway is constantly expanding, in part because of the development of new technologies, mainly in bioimaging. The increasing accessibility of these new tools in combination with more established methods provides an ideal way to increase knowledge of the secretory pathway in plants. In this review we discuss recent developments in understanding protein transport between organelles in the plant secretory pathway.

scholarly journals Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell)

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
Aris Haryanto ◽  
Michael Kann
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Intracellular Localization ◽  
Living Cells ◽  
Marker Protein ◽  
Dna Encoding ◽  
Protein Fusion ◽  
Fluorescent Marker ◽  
Localization Study

The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

scholarly journals Microfluidics-based selection of red-fluorescent proteins with decreased rates of photobleaching

2015 ◽  
Vol 7 (2) ◽  
pp. 263-273 ◽  
Author(s):  
Kevin M. Dean ◽  
Jennifer L. Lubbeck ◽  
Lloyd M. Davis ◽  
Chola K. Regmi ◽  
Prem P. Chapagain ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Microfluidic Platform ◽  
Red Fluorescent Proteins ◽  
New Variant ◽  
Selection Of

We use a high-throughput microfluidic platform that sorts cells on the basis of fluorescent protein photostability to identify a new variant with improved photon output.

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.

The nuclear tubular invaginations are dynamic structures inside the nucleus of HeLa cellsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

2006 ◽  
Vol 84 (3-4) ◽  
pp. 477-486 ◽  
Author(s):  
Rebecca K.Y. Lee ◽  
Pauline P.Y. Lui ◽  
Erika K.S. Ngan ◽  
Julian C.K. Lui ◽  
Y.K. Suen ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Membrane Protrusion ◽  
Special Issue ◽  
A Cell ◽  
Dynamic Structures ◽  
Protein Transfection ◽  
Confocal And Electron Microscopy ◽  
Selection Of

Nuclear tubules (NTs) were found in the nucleus of HeLa cells. Although no function has been ascribed to these structures, our previous data has shown that they are the sites of Ca2+ release with mitochondria shuttled around. In the present study, we further characterized these NTs through different fluorescent dye-labeling and red fluorescent protein transfection experiments. We found that doxorubicin (Dox) is a good indicator to demonstrate the NTs since Dox is fluorescent and DNA is able to quench its fluorescence. By using confocal and electron microscopy, we show that the number and nature of the NTs in HeLa vary from cell to cell, ranging from tubular to intricately branched structures. Additionally, these NTs are double-membrane invaginations of the nuclear envelope and usually lie close to nucleolus. At rest, NTs appeared to be stable and their mouths are always closed. Upon Ca2+ ionomycin stimulation, various forms of dynamism, including membrane protrusion to the nucleus, enlargement and shrinkage of the NTs, and distortion of the nuclear envelope and NTs were observed over a time scale of minutes. These observations suggest that the NT represents a specialized and dynamic compartment inside the nucleus under the control of Ca2+.

scholarly journals Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy

2017 ◽  
Author(s):  
Alessia Lepore ◽  
Hannah Taylor ◽  
Dirk Landgraf ◽  
Burak Okumus ◽  
Sebastián Jaramillo-Riveri ◽  
...  
Keyword(s):  
Cell Biology ◽  
Quantitative Methods ◽  
Fluorescent Protein ◽  
Detection Efficiency ◽  
Fluorescent Proteins ◽  
Attractive Alternative ◽  
Highly Sensitive ◽  
Endogenous Proteins ◽  
Photo Stability ◽  
Better Than

ABSTRACTCell biology is increasingly dependent on quantitative methods resulting in the need for microscopic labelling technologies that are highly sensitive and specific. Whilst the use of fluorescent proteins has led to major advances, they also suffer from their relatively low brightness and photo-stability, making the detection of very low abundance proteins using fluorescent protein-based methods challenging. Here, we characterize the use of the self-labelling protein tag called HaloTag, in conjunction with an organic fluorescent dye, to label and accurately count endogenous proteins present in very low numbers (<7) in individualEscherichia colicells. This procedure can be used to detect single molecules in fixed cells with conventional epifluorescence illumination and a standard microscope. We show that the detection efficiency of proteins labelled with the HaloTag is ≥80%, which is on par or better than previous techniques. Therefore, this method offers a simple and attractive alternative to current procedures to detect low abundance molecules.

scholarly journals mCherry contains a fluorescent protein isoform that interferes with its reporter function

2021 ◽  
Author(s):  
Maxime Fages-Lartaud ◽  
Lisa Tietze ◽  
Florence Elie ◽  
Rahmi Lale ◽  
Martin Frank Hohmann-Marriott
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Initiation Site ◽  
Translation Initiation Site ◽  
Red Fluorescent Protein ◽  
Functional Protein ◽  
Red Fluorescent Proteins ◽  
Expression Studies ◽  
Protein Isoform

AbstractFluorescent proteins are essential reporters in cell biology and molecular biology. Here, we reveal that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform. The short isoform creates significant background fluorescence that biases the outcome of expression studies. Our investigation identifies the short protein isoform, traces its origin, and determines the extent of the issue within the family of red fluorescent protein. Our analysis shows that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. Finally, we provide a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

1997 ◽  
Vol 3 (S2) ◽  
pp. 133-134 ◽  
Author(s):  
Ammasi Periasamy ◽  
Richard N. Day
Keyword(s):  
Transcription Factors ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Resonance Energy ◽  
Living Cells ◽  
Specific Gene ◽  
Green Fluorescent ◽  
Prl Gene

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

Masterminds or minions? Cysteine proteinases in plant programmed cell deathThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 651-667 ◽  
Author(s):  
Christopher P. Trobacher ◽  
Adriano Senatore ◽  
John S. Greenwood
Keyword(s):  
Cell Death ◽  
Cell Biology ◽  
Apoptotic Cell ◽  
Gene Families ◽  
Cysteine Proteinases ◽  
Special Issue ◽  
Multicellular Organisms ◽  
Protease Expression ◽  
Signalling Cascade ◽  
Selection Of

Cysteine proteinases are ubiquitously involved in programmed cell death (PCD) in multicellular organisms. In animals, one group of cysteine proteinases, the cysteine-dependent aspartate-specific proteinases (caspases), are involved in a proteolytic signalling cascade that controls apoptosis, the most studied form of PCD. The enzymes act as both masterminds and executioners in apoptotic cell death. In plants, members of the metacaspase family, as well as those of the papain-like and legumain families, of cysteine proteinases have all been implicated in PCD. These enzymes often belong to sizeable gene families, with Arabidopsis having 9 metacaspase, 32 papain-like, and 4 legumain genes. This redundancy has made it difficult to ascertain the functional importance of any particular enzyme in plant PCD, as many are often expressed in a given tissue undergoing PCD. As yet, mechanisms similar to the apoptotic caspase cascade in animals have not been uncovered in plants and, indeed, may not exist. Are the various cysteine proteinases, so often implicated in plant PCD, merely acting as minions in the process? This review will outline reports of cysteine proteinases associated with plant PCD, discuss problems in determining the function of specific proteases, and suggest avenues for determining how these enzymes might be regulated and how PCD pathways upstream of protease expression and activation might operate.

Protein import into chloroplasts: an ever-evolving storyThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 531-542 ◽  
Author(s):  
Matthew D. Smith
Keyword(s):  
Plant Growth ◽  
Cell Biology ◽  
Growth And Development ◽  
Protein Import ◽  
Special Issue ◽  
Plant Growth And Development ◽  
Core Components ◽  
Envelope Membranes ◽  
Inner Envelope ◽  
Selection Of

Chloroplasts are but one type of a diverse group of essential organelles that distinguish plant cells and house many critical biochemical pathways, including photosynthesis. The biogenesis of plastids is essential to plant growth and development and relies on the targeting and import of thousands of nuclear-encoded proteins from the cytoplasm. The import of the vast majority of these proteins is dependent on translocons located in the outer and inner envelope membranes of the chloroplast, termed the Toc and Tic complexes, respectively. The core components of the Toc and Tic complexes have been identified within the last 12 years; however, the precise functions of many components are still being elucidated, and new components are still being identified. In Arabidopsis thaliana (and other species), many of the components are encoded by more than one gene, and it appears that the isoforms differentially associate with structurally distinct import complexes. Furthermore, it appears that these complexes represent functionally distinct targeting pathways, and the regulation of import by these separate pathways may play a role in the differentiation and specific functions of distinct plastid types during plant growth and development. This review summarizes these recent discoveries and emphasizes the mechanisms of differential Toc complex assembly and substrate recognition.

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