scholarly journals ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

2019 ◽  
Author(s):  
Jelmer Willems ◽  
Arthur P.H. de Jong ◽  
Nicky Scheefhals ◽  
Harold D. MacGillavry
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Protein Complexes ◽  
Neuronal Cell ◽  
Super Resolution ◽  
Protein Distribution ◽  
Neuronal Processes ◽  
Endogenous Proteins ◽  
Fluorescent Tags ◽  
Super Resolution Microscopy

ABSTRACTThe correct subcellular distribution of protein complexes establishes the complex morphology of neurons and is fundamental to their functioning. Thus, determining the dynamic distribution of proteins is essential to understand neuronal processes. Fluorescence imaging, in particular super-resolution microscopy, has become invaluable to investigate subcellular protein distribution. However, these approaches suffer from the limited ability to efficiently and reliably label endogenous proteins. We developed ORANGE: an Open Resource for the Application of Neuronal Genome Editing, that mediates targeted genomic integration of fluorescent tags in neurons. This toolbox includes a knock-in library for in-depth investigation of endogenous protein distribution, and a detailed protocol explaining how knock-in can be developed for novel targets. In combination with super-resolution microscopy, ORANGE revealed the dynamic nanoscale organization of endogenous neuronal signaling molecules, synaptic scaffolding proteins, and neurotransmitter receptors. Thus, ORANGE enables quantitation of expression and distribution for virtually any protein in neurons at high resolution and will significantly further our understanding of neuronal cell biology.

scholarly journals Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

2017 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Genome Editing ◽  
Cell Biology ◽  
Cellular Structures ◽  
Human Stem Cells ◽  
Alpha Tubulin ◽  
Junction Protein ◽  
Endogenous Proteins ◽  
Clonal Lines

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

scholarly journals A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization

2021 ◽  
Author(s):  
Yiran Chang ◽  
Danie J Dickinson
Keyword(s):  
Cell Biology ◽  
Protein Complexes ◽  
Quantitative Model ◽  
Cell Polarization ◽  
Protein Distribution ◽  
Cell Stage ◽  
Viscous Forces ◽  
Dependent Protein ◽  
Regulate Protein ◽  
Size Threshold

Regulation of subcellular components' localization and motion is a critical theme in cell biology. Cells use the actomyosin cortex to regulate protein distribution on the plasma membrane, but the interplay between membrane binding, cortical movements and protein distribution remains poorly understood. In a polarizing one-cell stage Caenorhabditis elegans embryo, actomyosin flows transport PAR protein complexes into an anterior cortical domain to establish the anterior-posterior axis of the animal. Oligomerization of a key scaffold protein, PAR-3, is required for aPAR cortical localization and segregation. Although PAR-3 oligomerization is essential for polarization, it remains unclear how oligomer size contributes to aPAR segregation because PAR-3 oligomers are a heterogeneous population of many different sizes. To address this question, we engineered PAR-3 to defined sizes. We report that PAR-3 trimers are necessary and sufficient for PAR-3 function during polarization and later embryo development, while larger PAR-3 clusters are dispensable. Quantitative analysis of PAR-3 diffusion showed that PAR-3 clusters larger than a trimer are transported by viscous forces without being physically captured by the actomyosin cortex. Our study provides a quantitative model for size-dependent protein transportation of membrane proteins by cortical flow.

scholarly journals Super-resolution microscopy can identify specific protein distribution patterns in platelets incubated with cancer cells

Nanoscale ◽  
2019 ◽  
Vol 11 (20) ◽  
pp. 10023-10033 ◽  
Author(s):  
Jan Bergstrand ◽  
Lei Xu ◽  
Xinyan Miao ◽  
Nailin Li ◽  
Ozan Öktem ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Dictionary Learning ◽  
Super Resolution ◽  
Distribution Patterns ◽  
Specific Protein ◽  
Protein Distribution ◽  
Activated Platelets ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Super-resolution imaging of P-selectin in platelets together with dictionary learning allow specifically activated platelets to be identified in an automatic objective manner.

Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging

Science ◽  
2013 ◽  
Vol 341 (6146) ◽  
pp. 655-658 ◽  
Author(s):  
Anna Szymborska ◽  
Alex de Marco ◽  
Nathalie Daigle ◽  
Volker C. Cordes ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Super Resolution ◽  
Molecular Organization ◽  
Nuclear Pore ◽  
Protein Assemblies ◽  
Large Protein ◽  
Whole Cells ◽  
Molecular Machinery ◽  
Scaffold Structure ◽  
Super Resolution Microscopy

Much of life’s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

scholarly journals Translation Microscopy (TRAM) for super-resolution imaging

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhen Qiu ◽  
Rhodri S Wilson ◽  
Yuewei Liu ◽  
Alison R Dun ◽  
Rebecca S Saleeb ◽  
...  
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Ground Truth ◽  
Image Plane ◽  
Ease Of Use ◽  
Stimulated Emission ◽  
Multiple Diffraction ◽  
Microscopy Technique ◽  
Resolution Imaging ◽  
Super Resolution Microscopy

Abstract Super-resolution microscopy is transforming our understanding of biology but accessibility is limited by its technical complexity, high costs and the requirement for bespoke sample preparation. We present a novel, simple and multi-color super-resolution microscopy technique, called translation microscopy (TRAM), in which a super-resolution image is restored from multiple diffraction-limited resolution observations using a conventional microscope whilst translating the sample in the image plane. TRAM can be implemented using any microscope, delivering up to 7-fold resolution improvement. We compare TRAM with other super-resolution imaging modalities, including gated stimulated emission deletion (gSTED) microscopy and atomic force microscopy (AFM). We further developed novel ‘ground-truth’ DNA origami nano-structures to characterize TRAM, as well as applying it to a multi-color dye-stained cellular sample to demonstrate its fidelity, ease of use and utility for cell biology.

Optical super-resolution microscopy unravels the molecular composition of functional protein complexes

Nanoscale ◽  
2019 ◽  
Vol 11 (39) ◽  
pp. 17981-17991 ◽  
Author(s):  
Marina S. Dietz ◽  
Mike Heilemann
Keyword(s):  
Single Molecule ◽  
Protein Complexes ◽  
Super Resolution ◽  
Molecular Composition ◽  
Functional Protein ◽  
Super Resolution Microscopy

The molecular composition of functional protein complexes can be determined from single-molecule super-resolution images.

scholarly journals ClusterViSu, a method for clustering of protein complexes by Voronoi tessellation in super-resolution microscopy

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Leonid Andronov ◽  
Igor Orlov ◽  
Yves Lutz ◽  
Jean-Luc Vonesch ◽  
Bruno P. Klaholz
Keyword(s):  
Protein Complexes ◽  
Voronoi Tessellation ◽  
Super Resolution ◽  
Super Resolution Microscopy
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