Super-resolution mapping of glutamate receptors in <i>C. elegans</i> by confocal correlated PALM

Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. PALM is therefore a powerful tool in resolving structures and putative interactions of biomolecules at the ultimate analytical detection limit. However, its limited imaging depth restricts PALM mostly to in vitro applications. Considering the additional need for anatomical context when imaging a multicellular organism, these limitations render the use of PALM in whole animals difficult. Here we integrated PALM with confocal microscopy for correlated imaging of the C. elegans nervous system, a technique we termed confocal correlated PALM (ccPALM). The neurons, lying below several tissue layers, could be visualized up to 10 μm deep inside the animal. By ccPALM, we visualized ionotropic glutamate receptor distributions in C. elegans with an accuracy of 20 nm, revealing super-resolution structure of receptor clusters that we mapped onto annotated neurons in the animal. Pivotal to our results was the TIRF-independent detection of single molecules, achieved by genetic regulation of labeled receptor expression and localization to effectively reduce the background fluorescence. By correlating PALM with confocal microscopy, this platform enables dissecting biological structures with single molecule resolution in the physiologically relevant context of whole animals

Super-resolution mapping of glutamate receptors in C. elegans by confocal correlated PALM (vol 5, 13532, 2016)

status: publishe

Super-resolution mapping of glutamate receptors in C. elegans by confocal correlated PALM

Photoactivated localization microscopy (PALM) is a super-resolution imaging technique based on the detection and subsequent localization of single fluorescent molecules. PALM is therefore a powerful tool in resolving structures and putative interactions of biomolecules at the ultimate analytical detection limit. However, its limited imaging depth restricts PALM mostly to in vitro applications. Considering the additional need for anatomical context when imaging a multicellular organism, these limitations render the use of PALM in whole animals difficult. Here we integrated PALM with confocal microscopy for correlated imaging of the C. elegans nervous system, a technique we termed confocal correlated PALM (ccPALM). The neurons, lying below several tissue layers, could be visualized up to 10 μm deep inside the animal. By ccPALM, we visualized ionotropic glutamate receptor distributions in C. elegans with an accuracy of 20 nm, revealing super-resolution structure of receptor clusters that we mapped onto annotated neurons in the animal. Pivotal to our results was the TIRF-independent detection of single molecules, achieved by genetic regulation of labeled receptor expression and localization to effectively reduce the background fluorescence. By correlating PALM with confocal microscopy, this platform enables dissecting biological structures with single molecule resolution in the physiologically relevant context of whole animals.status: publishe

A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible. SeqA-PAmCherry was imaged in wild type E. coli, expressed from plasmid or genetically engineered into the bacterial genome, replacing the native seqA gene. Unsynchronized cells as well as cells with a synchronized cell cycle were imaged at various time points, in order to investigate the evolution of SeqA localization during the cell cycle. We found that SeqA indeed localized into discrete foci but these were not the only subcellular localizations of the protein. A significant amount of SeqA-PAmCherry molecules was localized outside the foci and in a fraction of cells we saw patterns indicating localization at the membrane. Using quantitative PALM, we counted protein copy numbers per cell, protein copy numbers per focus, the numbers of foci per cell and the sizes of the SeqA clusters. The data showed broad cell-to-cell variation and we did not observe a correlation between SeqA-PAmCherry protein numbers and the cell cycle under the experimental conditions of this study. The numbers of SeqA-PAmCherry molecules per focus as well as the foci sizes also showed broad distributions indicating that the foci are likely not characterized by a fixed number of molecules. We also imaged an E. coli strain devoid of the dam methylase (Δdam) and observed that SeqA-PAmCherry no longer formed foci, and was dispersed throughout the cell and localized to the plasma membrane more readily. We discuss our results in the context of the limitations of the technique.crosscheck: This document is CrossCheck deposited related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_article: http://dx.doi.org/10.1039/C5FD90093J related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: This article is freely available. This article is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported Licence (CC BY-NC 3.0) history: Received 4 May 2015; Accepted 10 July 2015; Accepted Manuscript published 13 July 2015; Advance Article published 9 October 2015; Version of Record published 12 December 2015status: publishe

Membrane distribution of the glycine receptor alpha 3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.status: publishe

Membrane distribution of the glycine receptor alpha 3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) {alpha}3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both {alpha}3K and {alpha}3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for {alpha}3L compared to {alpha}3K (mean radius 92 ± 4 and 56 +/- 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 +/- 200 {mu}m(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 +/- 6 nm). These results demonstrate that RNA splicing determines GlyR {alpha}3 membrane distribution, which has consequences for neuronal GlyR physiology and function

The ER Stress Sensor PERK Coordinates ER-Plasma Membrane Contact Site Formation through Interaction with Filamin-A and F-Actin Remodeling

Loss of ER Ca2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca2+. Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton.publisher: Elsevier articletitle: The ER Stress Sensor PERK Coordinates ER-Plasma Membrane Contact Site Formation through Interaction with Filamin-A and F-Actin Remodeling journaltitle: Molecular Cell articlelink: http://dx.doi.org/10.1016/j.molcel.2017.01.020 content_type: article copyright: © 2017 Elsevier Inc.status: publishe

The ER stress sensor PERK coordinates ER-plasma membrane contact site formation through interaction with filamin-A and F-actin remodeling

Loss of ER Ca2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca2+. Recent studies suggest that the ER stress sensor and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton