scholarly journals Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

2021 ◽  
Vol 22 (16) ◽  
pp. 8930
Author(s):  
Thomas K. Smylla ◽  
Krystina Wagner ◽  
Armin Huber
Keyword(s):  
Protein Transport ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Expression Patterns ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Visual Signal ◽  
Genetic Screens ◽  
Drosophila Eye

The Drosophila eye has been used extensively to study numerous aspects of biological systems, for example, spatio-temporal regulation of differentiation, visual signal transduction, protein trafficking and neurodegeneration. Right from the advent of fluorescent proteins (FPs) near the end of the millennium, heterologously expressed fusion proteins comprising FPs have been applied in Drosophila vision research not only for subcellular localization of proteins but also for genetic screens and analysis of photoreceptor function. Here, we summarize applications for FPs used in the Drosophila eye as part of genetic screens, to study rhodopsin expression patterns, subcellular protein localization, membrane protein transport or as genetically encoded biosensors for Ca2+ and phospholipids in vivo. We also discuss recently developed FPs that are suitable for super-resolution or correlative light and electron microscopy (CLEM) approaches. Illustrating the possibilities provided by using FPs in Drosophila photoreceptors may aid research in other sensory or neuronal systems that have not yet been studied as well as the Drosophila eye.

scholarly journals RIM-Binding Protein 2 organizes Ca2+ channel topography and regulates release probability and vesicle replenishment at a fast central synapse

2021 ◽  
Author(s):  
Tanvi Butola ◽  
Theocharis Alvanos ◽  
Anika Hintze ◽  
Peter Koppensteiner ◽  
David Kleindienst ◽  
...  
Keyword(s):  
Binding Protein ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Auditory Pathway ◽  
Nerve Fibers ◽  
Release Probability ◽  
High Release ◽  
Train Stimulation ◽  
Bushy Cells

RIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Augmenting Ca2+ influx by adding extracellular Ca2+ restored release probability but not EPSC amplitude, and uncovered an impairment of SV replenishment during train stimulation. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient mice in vivo.

scholarly journals Efficient generation of endogenous protein reporters for mouse preimplantation embryos

2020 ◽  
Author(s):  
Dan O’Hagan ◽  
Amy Ralston
Keyword(s):  
High Efficiency ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Preimplantation Embryos ◽  
List Type ◽  
Gene Coding ◽  
Endogenous Protein ◽  
Mouse Lines

SummaryFluorescent proteins and epitope tags can reveal protein localization in cells and animals. However, the large size of many tags hinders efficient genome targeting. Accordingly, many studies have relied on characterizing overexpressed proteins, which might not recapitulate endogenous protein activities. We present two approaches for higher throughput production of endogenous protein reporters. Our first approach makes use of a split fluorescent protein mNeonGreen2 (mNG2). Knock-in of a small portion of the mNG2 gene, in frame with gene coding regions of interest was highly efficient in embryos, eliminating the need to establish mouse lines. When complemented by the larger portion of the mNG2 gene, fluorescence was reconstituted and endogenous protein localization faithfully reported in living embryos. However, we report a threshold of detection using this approach. By contrast, the V5 epitope enabled high efficiency and higher sensitivity protein reporting. We describe complementary advantages and prospective applications of these two approaches.HighlightsSplit fluorescent protein for in vivo protein localization in living embryosV5 tagging for in vivo localization of low abundance proteinsBypassing the need for founder mouse lines for preimplantation studiesGuidelines and strategies for implementation and prospective applications

scholarly journals Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maarten W. Tuijtel ◽  
Abraham J. Koster ◽  
Stefan Jakobs ◽  
Frank G. A. Faas ◽  
Thomas H. Sharp
Keyword(s):  
Electron Microscopy ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution

scholarly journals In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Li He ◽  
Richard Binari ◽  
Jiuhong Huang ◽  
Julia Falo-Sanjuan ◽  
Norbert Perrimon
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Patterns ◽  
Cell Type ◽  
Dynamic Information ◽  
New Genes ◽  
Dual Color ◽  
Dynamic Expression ◽  
Spatio Temporal

Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

scholarly journals In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans

2020 ◽  
Vol 22 (1) ◽  
pp. 119
Author(s):  
Motomichi Doi ◽  
Megumi Sato ◽  
Yoshihiro Ohmiya
Keyword(s):  
Gene Expression ◽  
Fluorescent Proteins ◽  
Morphological Changes ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Gene Expression Profiles ◽  
Simultaneous Analysis ◽  
Soil Nematode ◽  
Living Organisms

Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.

scholarly journals Improved Split Fluorescent Proteins for Endogenous Protein Labeling

2017 ◽  
Author(s):  
Siyu Feng ◽  
Sayaka Sekine ◽  
Veronica Pessino ◽  
Han Li ◽  
Manuel D. Leonetti ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Protein Localization ◽  
Super Resolution ◽  
Screening Strategy ◽  
Protein Labeling ◽  
Cell Contact ◽  
Endogenous Protein ◽  
Endogenous Proteins

ABSTRACTSelf-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen21-10/11 that improves the ratio of complemented signal to the background of FP1-10-expressing cells compared to the commonly used split-GFP1-10/11, as well as a 10-fold brighter red-colored split-sfCherry21-10/11. Based on split-sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry211 and GFP11, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.

scholarly journals Regulation of beta-amyloid production in neurons by astrocyte-derived cholesterol

2020 ◽  
Author(s):  
Hao Wang ◽  
Joshua A. Kulas ◽  
Heather A. Ferris ◽  
Scott B. Hansen
Keyword(s):  
Amyloid Precursor Protein ◽  
Lipid Rafts ◽  
Cholesterol Synthesis ◽  
Protein Localization ◽  
Super Resolution ◽  
Precursor Protein ◽  
Targeted Deletion ◽  
Amyloid Aβ

ABSTRACTAlzheimer’s Disease (AD) is characterized by the presence of β-Amyloid (Aβ) plaques, tau tangles, inflammation, and loss of cognitive function. Genetic variation in a cholesterol transport protein, apolipoprotein E (apoE), is the most common genetic marker for sporadic AD. In vitro evidence suggests apoE links to Aβ production through nanoscale lipid compartments (also called lipid rafts), but its regulation in vivo is unclear. Here we use super-resolution imaging in mouse brain to show apoE utilizes astrocyte-derived cholesterol to specifically traffic neuronal amyloid precursor protein (APP) into lipid rafts where it interacts with β- and γ-secretases to generate Aβ-peptide. We find that targeted deletion of astrocyte cholesterol synthesis robustly reduces amyloid and tau burden in a mouse model of AD. Treatment with cholesterol-free apoE or knockdown of cholesterol synthesis in astrocytes decreases cholesterol levels in cultured neurons and causes APP to traffic out of lipid rafts where it interacts with α-secretase and gives rise to soluble APPα (sAPPα), a neuronal protective product of APP. Changes in cellular cholesterol have no effect on α-, β-, and γ-secretase trafficking, suggesting the ratio of Aβ to sAPPα is regulated by the trafficking of the substrate, not the enzymes. Treatment of astrocytes with inflammatory cytokines IL-1β, IL-6 and TNF-α upregulates the synthesis of cholesterol in the astrocytes. We conclude that cholesterol is kept low in neurons to inhibit Aβ formation and enable astrocyte regulation of Aβ formation by cholesterol regulation.HighlightsApoE regulates amyloid precursor protein localization to rafts and its exposure to α-vs. β-secretase.α-, β-, and γ-Secretases are activated by substrate presentation.ApoE specifically transports astrocyte cholesterol to neurons.Astrocyte cholesterol synthesis disruption prevents Alzheimer’s-associated amyloid pathology in mice.

scholarly journals Precision of fiducial marker alignment for correlative super‐resolution fluorescence and transmission electron microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Adeeba Fathima ◽  
César Augusto Quintana-Cataño ◽  
Christoph Heintze ◽  
Michael Schlierf
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Specific Protein ◽  
Fiducial Markers ◽  
Transmission Electron ◽  
Microscopy Techniques

AbstractRecent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

scholarly journals In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer

2019 ◽  
Author(s):  
Li He ◽  
Richard Binari ◽  
Jiuhong Huang ◽  
Julia Falo-Sanjuan ◽  
Norbert Perrimon
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Expression Patterns ◽  
Cell Type ◽  
Dynamic Information ◽  
New Genes ◽  
Dual Color ◽  
Dynamic Expression ◽  
Spatio Temporal

AbstractFluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach in vivo are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in Drosophila. In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns.

Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues

2010 ◽  
Vol 90 (3) ◽  
pp. 1103-1163 ◽  
Author(s):  
Dmitriy M. Chudakov ◽  
Mikhail V. Matz ◽  
Sergey Lukyanov ◽  
Konstantin A. Lukyanov
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Visible Spectrum ◽  
Living Cells ◽  
Alternative Possibilities ◽  
Structure Evolution ◽  
Marine Animals

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

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