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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
Romain Berardozzi, Virgile Adam, Alexandre Martins, Dominique Bourgeois
Photoactivated localization microscopy (PALM) is a powerful technique to investigate cellular nanostructures quantitatively and dynamically. However, the use of PALM for molecular counting or single-particle tracking remains limited by the propensity of photoconvertible fluorescent protein markers (PCFPs) to repeatedly enter dark states. By designing the single mutants mEos2-A69T and Dendra2-T69A, we completely swapped the blinking behaviors of mEos2 and Dendra2, two popular PCFPs. We combined X-ray crystallography and single-molecule microscopy to show that blinking in mEos2 and Dendra2 is largely controlled by the orientation of arginine 66, a highly conserved residue in Anthozoan PCFPs...
January 20, 2016: Journal of the American Chemical Society
Wenting Li, Emmanuelle Bouveret, Yan Zhang, Kuanqing Liu, Jue D Wang, James C Weisshaar
During amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re-programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three Escherichia coli strains, each expressing a different RelA-fluorescent protein (RelA-FP) construct: RelA-YFP, RelA-mEos2 and RelA-Dendra2...
February 2016: Molecular Microbiology
Frédéric Cassé, Stéphane Martin
Spines are small protrusions on dendritic membranes receiving inputs from axonal termini. They consist in a head connected to the dendritic shaft by a narrow neck and contain multiple synaptic proteins that interact in a coordinated manner to allow for synaptic communication. This process involves many proteins that are moving in and out spines. However, comparing this synaptodendritic movement in basal and stimulated conditions is very challenging. Here we describe an elegant method to measure the activity-dependent diffusion of synaptic proteins using Dendra2 photoconversion...
2015: Frontiers in Cellular Neuroscience
Toh Hean Ch'ng, Martina DeSalvo, Peter Lin, Ajay Vashisht, James A Wohlschlegel, Kelsey C Martin
Previous studies have revealed a critical role for CREB-regulated transcriptional coactivator (CRTC1) in regulating neuronal gene expression during learning and memory. CRTC1 localizes to synapses but undergoes activity-dependent nuclear translocation to regulate the transcription of CREB target genes. Here we investigate the long-distance retrograde transport of CRTC1 in hippocampal neurons. We show that local elevations in calcium, triggered by activation of glutamate receptors and L-type voltage-gated calcium channels, initiate active, dynein-mediated retrograde transport of CRTC1 along microtubules...
2015: Frontiers in Molecular Neuroscience
Erika Herrero-Garcia, Elixabet Perez-de-Nanclares-Arregi, Marc S Cortese, Ane Markina-Iñarrairaegui, Elixabet Oiartzabal-Arano, Oier Etxebeste, Unai Ugalde, Eduardo A Espeso
In Aspergillus nidulans, asexual differentiation requires the presence of the transcription factor FlbB at the cell tip and apical nuclei. Understanding the relationship between these two pools is crucial for elucidating the biochemical processes mediating conidia production. Tip-to-nucleus communication was demonstrated by photo-convertible FlbB::Dendra2 visualization. Tip localization of FlbB depends on Cys382 in the C-terminus and the bZIP DNA-binding domain in the N-terminus. FlbE, a critical FlbB interactor, binds the bZIP domain...
November 2015: Molecular Microbiology
Hugo R Caires, Maria Gomez-Lazaro, Carla M Oliveira, David Gomes, Denisa D Mateus, Carla Oliveira, Cristina C Barrias, Mário A Barbosa, Catarina R Almeida
Mesenchymal Stem/Stromal Cells (MSC) are a promising cell type for cell-based therapies - from tissue regeneration to treatment of autoimmune diseases - due to their capacity to migrate to damaged tissues, to differentiate in different lineages and to their immunomodulatory and paracrine properties. Here, a simple and reliable imaging technique was developed to study MSC dynamical behavior in natural and bioengineered 3D matrices. Human MSC were transfected to express a fluorescent photoswitchable protein, Dendra2, which was used to highlight and follow the same group of cells for more than seven days, even if removed from the microscope to the incubator...
2015: Scientific Reports
Adam J Mellott, Heather E Shinogle, David S Moore, Michael S Detamore
Not all cells behave uniformly after treatment in tissue engineering studies. In fact, some treated cells display no signs of treatment or show unique characteristics not consistent with other treated cells. What if the "unique" cells could be isolated from a treated population, and further studied? Photo-convertible reporter proteins, such as Dendra2, allow for the ability to selectively identify unique cells with a secondary label within a primary labeled treated population. In the current study, select cells were identified and labeled through photo-conversion of Dendra2-transfected human Wharton's Jelly cells (hWJCs) for the first time...
March 1, 2015: Cellular and Molecular Bioengineering
Meghan C Drummond, Melanie Barzik, Jonathan E Bird, Duan-Sun Zhang, Claude P Lechene, David P Corey, Lisa L Cunningham, Thomas B Friedman
The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24-48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips...
April 21, 2015: Nature Communications
Kerrie H Lodowski, Yoshikazu Imanishi
Outer segment (OS) directed trafficking is required for accomplishing the extremely high concentration of rhodopsin and explicitly high photon sensitivity of rod photoreceptor cells. Aberrant targeting of rhodopsin often leads to blinding disorders, due to various mechanisms causing rhodopsin mislocalization. Until recently, it has been challenging to monitor the dynamics of rhodopsin biogenesis and trafficking. Here, we describe a new method to visualize rhodopsin trafficking in living and unfixed Xenopus laevis rod photoreceptors...
2015: Methods in Molecular Biology
Munenori Kitagawa, Tomomichi Fujita
Plant growth, development, and environmental responses require the proper regulation of intercellular movement of signals and nutrients. For this, plants have specialized cytoplasmic channels, the plasmodesmata (PD), which allow the symplasmic movement of micro- and macromolecules between neighboring cells. Internal and external signals spatio-temporally regulate the movement of molecules through the PD to control plant development and environmental responses. Although some aspects of targeted movement of molecules have been revealed, the mechanisms of non-targeted, diffusible flow of molecules through PD, and its regulation and function, remain poorly understood, particularly at the cellular level...
January 2015: Journal of Plant Research
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