Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging

AbstractReversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

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Super-resolution imaging of fluorescent dipoles via polarized structured illumination microscopy

Nature Communications ◽

10.1038/s41467-019-12681-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Karl Zhanghao ◽

Xingye Chen ◽

Wenhui Liu ◽

Meiqi Li ◽

Yiqiong Liu ◽

...

Keyword(s):

Hippocampal Neurons ◽

Fluorescent Protein ◽

Imaging Modality ◽

Super Resolution ◽

Vital Role ◽

Molecular Structures ◽

Polarization Microscopy ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Resolution Imaging

Abstract Fluorescence polarization microscopy images both the intensity and orientation of fluorescent dipoles and plays a vital role in studying molecular structures and dynamics of bio-complexes. However, current techniques remain difficult to resolve the dipole assemblies on subcellular structures and their dynamics in living cells at super-resolution level. Here we report polarized structured illumination microscopy (pSIM), which achieves super-resolution imaging of dipoles by interpreting the dipoles in spatio-angular hyperspace. We demonstrate the application of pSIM on a series of biological filamentous systems, such as cytoskeleton networks and λ-DNA, and report the dynamics of short actin sliding across a myosin-coated surface. Further, pSIM reveals the side-by-side organization of the actin ring structures in the membrane-associated periodic skeleton of hippocampal neurons and images the dipole dynamics of green fluorescent protein-labeled microtubules in live U2OS cells. pSIM applies directly to a large variety of commercial and home-built SIM systems with various imaging modality.

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A comprehensive dataset of image sequences covering 20 fluorescent protein labels and 12 imaging conditions for use in super-resolution imaging

Data in Brief ◽

10.1016/j.dib.2020.105273 ◽

2020 ◽

Vol 29 ◽

pp. 105273 ◽

Cited By ~ 1

Author(s):

Benjamien Moeyaert ◽

Peter Dedecker

Keyword(s):

Fluorescent Protein ◽

Super Resolution ◽

Image Sequences ◽

Resolution Imaging ◽

Imaging Conditions

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EYE ON CHINA

Asia-Pacific Biotech News ◽

10.1142/s0219030317000477 ◽

2017 ◽

Vol 21 (07) ◽

pp. 4-11

Keyword(s):

G Protein ◽

Oral Delivery ◽

Cell Activation ◽

Super Resolution ◽

Academic Paper ◽

Conjugated Polymer Nanoparticles ◽

Resolution Imaging ◽

Academy Of Sciences ◽

G Protein Coupled ◽

Intestinal Remodeling

Chinese Researchers Develop 3D Super-Resolution Imaging at Unprecedented Depth. Scientists Advance Photosynthesis Using Conjugated Polymer Nanoparticles. Bayer Partners with Chinese Academy of Sciences to Study Photosynthetic Efficiency. Scientists Reveal How G protein-coupled Receptor Kinases Recognize and Phosphorylate G protein-coupled Receptors. China Fights Academic Paper Fraud After Peer Review Scandal. Beijing 4P Health Research Institute Initiates China’s “100K Wellness Pioneer Project” Using UniteGen and Sapientia™ Integrated Platform. SGS Expands Extractables and Leachables Testing Capabilities at its Shanghai Facility. DeltaHealth Cooperates with Cardiologists & Partners for Better Patient Care. Scientists Demonstrate RNAi as an Antiviral Immunity in Mammals. Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell Activation and Intestinal Remodeling in DrosophilaShare.

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Fast Super-Resolution Imaging Technique and Immediate Early Nanostructure Capturing by a Photoconvertible Fluorescent Protein

Nano Letters ◽

10.1021/acs.nanolett.9b02855 ◽

2019 ◽

Vol 20 (4) ◽

pp. 2197-2208 ◽

Cited By ~ 3

Author(s):

Mingshu Zhang ◽

Zhifei Fu ◽

Changqing Li ◽

Anyuan Liu ◽

Dingming Peng ◽

...

Keyword(s):

Fluorescent Protein ◽

Imaging Technique ◽

Super Resolution ◽

Immediate Early ◽

Resolution Imaging

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A V0-ATPase-dependent apical trafficking pathway maintains the polarity of the intestinal absorptive membrane

10.1101/412122 ◽

2018 ◽

Cited By ~ 1

Author(s):

Aurélien Bidaud-Meynard ◽

Ophélie Nicolle ◽

Markus Heck ◽

Grégoire Michaux

Keyword(s):

Brush Border ◽

Molecular Mechanisms ◽

Super Resolution ◽

Structural Defects ◽

Intestinal Epithelial ◽

Intestinal Membrane ◽

Apical Trafficking ◽

Resolution Imaging ◽

Trafficking Defects

AbstractIntestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining genetic RNAi screen and in vivo super-resolution imaging in the C. elegans intestine, we uncovered that the V0 sector of the V-ATPase (V0-ATPase) controls a late apical trafficking step, involving RAB-11 endosomes and the SNARE SNAP-29, necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids in enterocyte polarity maintenance. Finally, we demonstrate that depletion of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from patients with Microvillus inclusion disease (MVID) and used this new in vivo MVID model to follow the dynamics of microvillus inclusions. Hence, we describe a new function for the V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of C. elegans intestine as an in vivo model to better understand the molecular mechanisms of rare genetic enteropathies.Summary statementV0-ATPase controls a late apical trafficking step involved in the maintenance of the apical absorptive intestinal membrane and its depletion phenocopies the trafficking and structural defects of MVID in C. elegans.

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Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents.

10.1101/2020.02.27.968578 ◽

2020 ◽

Author(s):

Juan Wang ◽

Avtar Singh ◽

Abdullah Ozer ◽

Warren R Zipfel

Keyword(s):

Fluorescent Protein ◽

Super Resolution ◽

Rna Aptamers ◽

Cellular Structures ◽

Intracellular Proteins ◽

Resolution Imaging ◽

Green Fluorescent ◽

Protein Tags ◽

Super Resolution Microscopy ◽

Conventional Antibody

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins.

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A new probe for super-resolution imaging of membranes elucidates trafficking pathways

The Journal of Cell Biology ◽

10.1083/jcb.201402066 ◽

2014 ◽

Vol 205 (4) ◽

pp. 591-606 ◽

Cited By ~ 82

Author(s):

Natalia H. Revelo ◽

Dirk Kamin ◽

Sven Truckenbrodt ◽

Aaron B. Wong ◽

Kirsten Reuter-Jessen ◽

...

Keyword(s):

Synaptic Vesicles ◽

Cultured Cells ◽

Neuromuscular Junctions ◽

Organ Of Corti ◽

Super Resolution ◽

Molecular Composition ◽

Cultured Neurons ◽

Vesicle Recycling ◽

Resolution Imaging ◽

Super Resolution Microscopy

The molecular composition of the organelles involved in membrane recycling is difficult to establish as a result of the absence of suitable labeling tools. We introduce in this paper a novel probe, named membrane-binding fluorophore-cysteine-lysine-palmitoyl group (mCLING), which labels the plasma membrane and is taken up during endocytosis. It remains attached to membranes after fixation and permeabilization and can therefore be used in combination with immunostaining and super-resolution microscopy. We applied mCLING to mammalian-cultured cells, yeast, bacteria, primary cultured neurons, Drosophila melanogaster larval neuromuscular junctions, and mammalian tissue. mCLING enabled us to study the molecular composition of different trafficking organelles. We used it to address several questions related to synaptic vesicle recycling in the auditory inner hair cells from the organ of Corti and to investigate molecular differences between synaptic vesicles that recycle actively or spontaneously in cultured neurons. We conclude that mCLING enables the investigation of trafficking membranes in a broad range of preparations.

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Mechanistic insights into GLUT1 activation and clustering revealed by super-resolution imaging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803859115 ◽

2018 ◽

Vol 115 (27) ◽

pp. 7033-7038 ◽

Cited By ~ 18

Author(s):

Qiuyan Yan ◽

Yanting Lu ◽

Lulu Zhou ◽

Junling Chen ◽

Haijiao Xu ◽

...

Keyword(s):

Mammalian Cells ◽

Cell Membranes ◽

Molecular Mechanisms ◽

Glucose Transporter ◽

Super Resolution ◽

Average Diameter ◽

Activation Mechanism ◽

Membrane Expression ◽

Close Relationship ◽

Resolution Imaging

The glucose transporter GLUT1, a plasma membrane protein that mediates glucose homeostasis in mammalian cells, is responsible for constitutive uptake of glucose into many tissues and organs. Many studies have focused on its vital physiological functions and close relationship with diseases. However, the molecular mechanisms of its activation and transport are not clear, and its detailed distribution pattern on cell membranes also remains unknown. To address these, we first investigated the distribution and assembly of GLUT1 at a nanometer resolution by super-resolution imaging. On HeLa cell membranes, the transporter formed clusters with an average diameter of ∼250 nm, the majority of which were regulated by lipid rafts, as well as being restricted in size by both the cytoskeleton and glycosylation. More importantly, we found that the activation of GLUT1 by azide or MβCD did not increase its membrane expression but induced the decrease of the large clusters. The results suggested that sporadic distribution of GLUT1 may facilitate the transport of glucose, implying a potential association between the distribution and activation. Collectively, our work characterized the clustering distribution of GLUT1 and linked its spatial structural organization to the functions, which would provide insights into the activation mechanism of the transporter.

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Membrane-associated periodic skeleton is a signaling platform for RTK transactivation in neurons

Science ◽

10.1126/science.aaw5937 ◽

2019 ◽

Vol 365 (6456) ◽

pp. 929-934 ◽

Cited By ~ 23

Author(s):

Ruobo Zhou ◽

Boran Han ◽

Chenglong Xia ◽

Xiaowei Zhuang

Keyword(s):

Tyrosine Kinases ◽

Super Resolution ◽

Erk Signaling ◽

Extracellular Signal Regulated Kinase ◽

Rtk Signaling ◽

Extracellular Signal ◽

Extracellular Stimuli ◽

Resolution Imaging ◽

G Protein Coupled

Actin, spectrin, and related molecules form a membrane-associated periodic skeleton (MPS) in neurons. The function of the MPS, however, remains poorly understood. Using super-resolution imaging, we observed that G protein–coupled receptors (GPCRs), cell adhesion molecules (CAMs), receptor tyrosine kinases (RTKs), and related signaling molecules were recruited to the MPS in response to extracellular stimuli, resulting in colocalization of these molecules and RTK transactivation by GPCRs and CAMs, giving rise to extracellular signal–regulated kinase (ERK) signaling. Disruption of the MPS prevented such molecular colocalizations and downstream ERK signaling. ERK signaling in turn caused calpain-dependent MPS degradation, providing a negative feedback that modulates signaling strength. These results reveal an important functional role of the MPS and establish it as a dynamically regulated platform for GPCR- and CAM-mediated RTK signaling.

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