scholarly journals Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Prasath Paramasivam ◽  
Christian Franke ◽  
Martin Stöter ◽  
Andreas Höijer ◽  
Stefano Bartesaghi ◽  
...  
Keyword(s):  
Super Resolution ◽  
Lipid Nanoparticles ◽  
Endosomal Escape ◽  
Human Adipocytes ◽  
Promising Strategy ◽  
Cargo Transport ◽  
Endosomal Recycling ◽  
Endosomal Acidification ◽  
Poor Understanding ◽  
Super Resolution Microscopy

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.

scholarly journals Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

2020 ◽  
Author(s):  
Prasath Paramasivam ◽  
Christian Franke ◽  
Martin Stöter ◽  
Andreas Höijer ◽  
Stefano Bartesaghi ◽  
...  
Keyword(s):  
Super Resolution ◽  
Microscopic Analysis ◽  
Endosomal Escape ◽  
Mrna Accumulation ◽  
Cargo Transport ◽  
Endosomal Recycling ◽  
Endosomal Acidification ◽  
Low Cytotoxicity ◽  
Super Resolution Microscopy ◽  
Shed Light

AbstractDelivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the endosomal parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy in primary human adipocytes. Quantitative multi-parametric microscopic analysis revealed that total LNP uptake is a poor predictor of delivery. Prolonged uptake impaired endosomal acidification causing mRNA accumulation in compartments defective in cargo transport and unproductive for delivery, indicating parameters of cytotoxicity. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By super-resolution microscopy we could resolve single fluorescently labelled mRNA within sub-endosomal compartments and capture events of mRNA escape from Transferrin-positive recycling tubules. Our results shed light into the mechanisms of endosomal escape and define quantitative parameters that can guide the development of mRNA formulations towards high efficacy and low cytotoxicity.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

Novel organic dyes for multicolor localization-based super-resolution microscopy

2015 ◽  
Vol 9 (1-2) ◽  
pp. 161-170 ◽  
Author(s):  
Martin Lehmann ◽  
Gregor Lichtner ◽  
Haider Klenz ◽  
Jan Schmoranzer
Keyword(s):  
Organic Dyes ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Bio-orthogonal Red and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-resolution Microscopy

2021 ◽  
Author(s):  
Philipp Werther ◽  
Klaus Yserentant ◽  
Felix Braun ◽  
Kristin Grußmayer ◽  
Vytautas Navikas ◽  
...  
Keyword(s):  
Super Resolution ◽  
Live Cell ◽  
Fluorogenic Probes ◽  
Super Resolution Microscopy

scholarly journals Super-resolution microscopy reveals that Na+/K+-ATPase signaling protects against glucose-induced apoptosis by deactivating Bad

2021 ◽  
Vol 12 (8) ◽  
Author(s):  
Kristoffer Bernhem ◽  
Jacopo M. Fontana ◽  
Daniel Svensson ◽  
Liang Zhang ◽  
Linnéa M. Nilsson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Chronic Diseases ◽  
Kidney Diseases ◽  
Super Resolution ◽  
Diabetic Patients ◽  
Diabetic Kidney ◽  
Renal Epithelial Cells ◽  
Apoptotic Process ◽  
Induced Apoptosis ◽  
Super Resolution Microscopy

AbstractActivation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na+,K+-ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca2+/calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

scholarly journals Simultaneous spatiotemporal super-resolution and multi-parametric fluorescence microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jagadish Sankaran ◽  
Harikrushnan Balasubramanian ◽  
Wai Hoh Tang ◽  
Xue Wen Ng ◽  
Adrian Röllin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Temporal Dynamics ◽  
Growth Factor Receptor ◽  
Super Resolution ◽  
Actin Binding ◽  
Biological Knowledge ◽  
Data Set ◽  
Epidermal Growth ◽  
Molecular Brightness ◽  
Super Resolution Microscopy

AbstractSuper-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either temporal or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~100 nm), temporal dynamics (~2 ms), and molecular brightness from the exact same data set. Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of the actin binding polypeptide Lifeact and corrects structural artefacts. Multi-parametric analysis of epidermal growth factor receptor (EGFR) and Lifeact suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides biological knowledge that cannot be obtained from sequential measurements.

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