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Atsushi Taniguchi, Yukiko Kimura, Ikue Mori, Shigenori Nonaka, Shin-Ichi Higashijima
Green-to-red photoconvertible fluorescent proteins have been found to undergo efficient photoconversion by a new method termed primed conversion that uses dual wave-length illumination with blue and red/near-infrared light. By modifying a confocal laser-scanning microscope (CLSM) such that two laser beams only meet at the focal plane, confined photoconversion at the axial dimension has been achieved. The necessity of this custom modification to the CLSM, however, has precluded the wide-spread use of this method...
December 13, 2017: Development, Growth & Differentiation
Francesca Pennacchietti, Travis J Gould, Samuel T Hess
Fluorescent proteins are used extensively for biological imaging applications; photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used widely in superresolution localization microscopy methods such as fluorescence photoactivation localization microscopy and photoactivated localization microscopy. However, their optimal use depends on knowledge of not only their bulk fluorescence properties, but also their photophysical properties at the single molecule level. We have used fluorescence correlation spectroscopy and cross-correlation spectroscopy to quantify the diffusion, photobleaching, fluorescence intermittency, and photoconversion dynamics of Dendra2, a well-known PAFP used in localization microscopy...
November 7, 2017: Biophysical Journal
Alexey A Pakhomov, Vladimir I Martynov, Alexander N Orsa, Alena A Bondarenko, Rita V Chertkova, Konstantin A Lukyanov, Alexander G Petrenko, Igor E Deyev
Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets...
December 2, 2017: Biochemical and Biophysical Research Communications
Ignacio Íñigo-Marco, Miguel Valencia, Laura Larrea, Ricardo Bugallo, Mikel Martínez-Goikoetxea, Iker Zuriguel, Montserrat Arrasate
α-Synuclein (aSyn) is the main driver of neurodegenerative diseases known as "synucleinopathies," but the mechanisms underlying this toxicity remain poorly understood. To investigate aSyn toxic mechanisms, we have developed a primary neuronal model in which a longitudinal survival analysis can be performed by following the overexpression of fluorescently tagged WT or pathologically mutant aSyn constructs. Most aSyn mutations linked to neurodegenerative disease hindered neuronal survival in this model; of these mutations, the E46K mutation proved to be the most toxic...
September 26, 2017: Proceedings of the National Academy of Sciences of the United States of America
Manuel Alexander Mohr, Andrei Yu Kobitski, Lluc Rullan Sabater, Karin Nienhaus, Christopher John Obara, Jennifer Lippincott-Schwartz, Gerd Ulrich Nienhaus, Periklis Pantazis
Green-to-red photoconvertible fluorescent proteins (pcFPs) are powerful tools for super-resolution localization microscopy and protein tagging. Recently, they have been found to undergo efficient photoconversion not only by the traditional 400-nm illumination but also by an alternative method termed primed conversion, employing dual wavelength illumination with blue and far-red/near-infrared light. Primed conversion has been reported only for Dendra2 and its mechanism has remained elusive. Here, we uncover the molecular mechanism of primed conversion by reporting the intermediate "primed" state to be a triplet dark state formed by intersystem crossing...
September 11, 2017: Angewandte Chemie
Jie Gao, Sean X Naughton, Wayne D Beck, Caterina M Hernandez, Guangyu Wu, Zhe Wei, Xiangkun Yang, Michael G Bartlett, Alvin V Terry
Chlorpyrifos (CPF) is an extensively used organophosphorus pesticide that has recently come under increasing scrutiny due to environmental health concerns particularly its association with neurodevelopmental defects. While the insecticidal actions and acute toxicity of CPF are attributed to its oxon metabolite (CPO) which potently inhibits the cholinergic enzyme acetylcholinesterase (AChE), there is significant evidence that CPF, CPO, and other organophosphates may affect a variety of neuronal targets and processes that are not directly related to AChE...
September 2017: Neurotoxicology
Bartosz Turkowyd, Alexander Balinovic, David Virant, Haruko G Gölz Carnero, Fabienne Caldana, Marc Endesfelder, Dominique Bourgeois, Ulrike Endesfelder
Photoconversion of fluorescent proteins by blue and complementary near-infrared light, termed primed conversion (PC), is a mechanism recently discovered for Dendra2. We demonstrate that controlling the conformation of arginine at residue 66 by threonine at residue 69 of fluorescent proteins from Anthozoan families (Dendra2, mMaple, Eos, mKikGR, pcDronpa protein families) represents a general route to facilitate PC. Mutations of alanine 159 or serine 173, which are known to influence chromophore flexibility and allow for reversible photoswitching, prevent PC...
September 11, 2017: Angewandte Chemie
Xin Hui, Benjamin Sauer, Lars Kaestner, Karsten Kruse, Peter Lipp
Translocation of cytosolic cPKC to the plasma membrane is a key event in their activation process but its exact nature is still unclear with particular dispute whether sole diffusion or additional active transport along the cell's cytoskeleton contributes to cPKC's dynamics. This was addressed by analyzing the recruitment behavior of PKCα while manipulating the cytoskeleton. Photolytic Ca(2+) uncaging allowed us to quantify the kinetics of PKCα redistribution to the plasma membrane when fused to monomeric, dimeric and tetrameric fluorescence proteins...
March 28, 2017: Scientific Reports
David K Heidary, Ashley Fox, Chris I Richards, Edith C Glazer
Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins...
April 2017: SLAS Discovery
Juri N Bach, Giacomo Giacomelli, Marc Bramkamp
Photo-activated localization microscopy (PALM) is one of the light microscopy techniques providing highest resolution. Single photo-activatable or photo-switchable fluorescent molecules are stochastically excited. The point spread function of this event is recorded and the exact fluorophore position is calculated. This chapter describes how bacterial samples can be prepared for PALM to achieve routinely a resolution of ≤30 nm using fluorophores such as mNeonGreen, Dendra2, and PAmCherry. It is also explained how to perform multicolor PALM and combine it with total internal reflection (TIRF) microscopy to increase resolution...
2017: Methods in Molecular Biology
Sarah Kube, Nils Hersch, Elena Naumovska, Thomas Gensch, Johnny Hendriks, Arne Franzen, Lisa Landvogt, Jan-Peter Siebrasse, Ulrich Kubitscheck, Bernd Hoffmann, Rudolf Merkel, Agnes Csiszár
Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation...
January 17, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
Manuel Alexander Mohr, Paul Argast, Periklis Pantazis
The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2...
December 2016: Nature Protocols
N V Klementieva, K A Lukyanov, N M Markina, S A Lukyanov, E V Zagaynova, A S Mishin
Recently, an unusual phenomenon of primed conversion of fluorescent protein Dendra2 by combined action of blue (488 nm) and near-infrared (700-780 nm) lasers was discovered. Here we demonstrate that primed conversion can be induced by red lasers (630-650 nm) common for most confocal and single molecule detection microscopes.
October 20, 2016: Chemical Communications: Chem Comm
Jacob I Ayers, Benjamin McMahon, Sabrina Gill, Herman L Lelie, Susan Fromholt, Hilda Brown, Joan Selverstone Valentine, Julian P Whitelegge, David R Borchelt
A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates...
January 2017: Journal of Neurochemistry
Olli Matilainen, Sweta Jha, Carina I Holmberg
The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates...
2016: Methods in Molecular Biology
Ján Jásik, Boris Bokor, Stanislav Stuchlík, Karol Mičieta, Ján Turňa, Elmon Schmelzer
By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells...
October 2016: Plant Physiology
Jitka Bolková, Christian Lanctôt
The parental genomes are initially spatially separated in each pronucleus after fertilization. Here we have used green-to-red photoconversion of Dendra2-H2B-labeled pronuclei to distinguish maternal and paternal chromatin domains and to track their spatial distribution in living Caenorhabditis elegans embryos starting shortly after fertilization. Intermingling of the parental chromatin did not occur until after the division of the AB and P1 blastomeres, at the 4-cell stage. Unexpectedly, we observed that the intermingling of chromatin did not take place during mitosis or during chromatin decondensation, but rather ∼ 3-5 minutes into the cell cycle...
2016: International Journal of Developmental Biology
Nan Luo, An Yan, Zhenbiao Yang
Exocytosis plays crucial roles in regulating the distribution and function of plasma membrane (PM) and extracellular matrix proteins. However, measuring the exocytosis rate of a specific protein by conventional methods is very difficult because of exocytosis-independent trafficking such as endocytosis, which also affects membrane protein distribution. Here, we describe a novel method, corrected fluorescence recovery after photoconversion, in which exocytosis-dependent and -independent trafficking events are measured simultaneously to accurately determine exocytosis rate...
May 2016: Traffic
N Griffiths, E-A Jaipargas, M R Wozny, K A Barton, N Mathur, K Delfosse, J Mathur
Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development...
August 2016: Journal of Microscopy
Jie Gao, Sean X Naughton, Heike Wulff, Vikrant Singh, Wayne D Beck, Jordi Magrane, Bobby Thomas, Navneet Ammal Kaidery, Caterina M Hernandez, Alvin V Terry
The extensive use of organophosphates (OPs) is an ongoing environmental health concern due to multiple reports of OP-related neurologic abnormalities. The mechanism of the acute toxicity of OPs has been attributed to inhibition of acetylcholinesterase (AChE), but there is growing evidence that this may not account for all the long-term neurotoxic effects of OPs. In previous experiments (using ex vivo and in vitro model systems) we observed that the insecticide OP chlorpyrifos impaired the movements of vesicles and mitochondria in axons...
March 2016: Journal of Pharmacology and Experimental Therapeutics
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