scholarly journals Single-molecule 3D orientation imaging reveals nanoscale compositional heterogeneity in lipid membranes

2020 ◽  
Author(s):  
Jin Lu ◽  
Hesam Mazidi ◽  
Tianben Ding ◽  
Oumeng Zhang ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Lipid Membranes ◽  
Soft Matter ◽  
Spatial Organization ◽  
Imaging Spectroscopy ◽  
Polar Angle ◽  
Compositional Heterogeneity ◽  
Orientation Imaging ◽  
Functional Dynamics ◽  
And Function

AbstractIn soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules/μm2) of single molecules with nanoscale resolution remains elusive. We present SMOLM, single-molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red’s (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme-induced compositional heterogeneity within membranes, where NR within liquid-ordered vs. liquid-disordered domains shows a ~4° difference in polar angle and a ~0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid-lipid, lipid-protein, and lipid-dye interactions with single-molecule, nanoscale resolution.

scholarly journals Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

2020 ◽  
Vol 59 (40) ◽  
pp. 17572-17579
Author(s):  
Jin Lu ◽  
Hesam Mazidi ◽  
Tianben Ding ◽  
Oumeng Zhang ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Lipid Membranes ◽  
Compositional Heterogeneity ◽  
Orientation Imaging

scholarly journals Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

2020 ◽  
Vol 132 (40) ◽  
pp. 17725-17732
Author(s):  
Jin Lu ◽  
Hesam Mazidi ◽  
Tianben Ding ◽  
Oumeng Zhang ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Lipid Membranes ◽  
Compositional Heterogeneity ◽  
Orientation Imaging

scholarly journals Rücktitelbild: Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes (Angew. Chem. 40/2020)

2020 ◽  
Vol 132 (40) ◽  
pp. 17912-17912
Author(s):  
Jin Lu ◽  
Hesam Mazidi ◽  
Tianben Ding ◽  
Oumeng Zhang ◽  
Matthew D. Lew
Keyword(s):  
Single Molecule ◽  
Lipid Membranes ◽  
Compositional Heterogeneity ◽  
Orientation Imaging

scholarly journals Superresolution 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

2020 ◽  
Vol 118 (3) ◽  
pp. 21a
Author(s):  
Jin Lu ◽  
Hesam Mazidi ◽  
Tianben Ding ◽  
Oumeng Zhang ◽  
Matthew D. Lew
Keyword(s):  
Lipid Membranes ◽  
Compositional Heterogeneity ◽  
Orientation Imaging

Protein clustering and spatial organization in T-cells

2015 ◽  
Vol 43 (3) ◽  
pp. 315-321 ◽  
Author(s):  
Michael J. Shannon ◽  
Garth Burn ◽  
Andrew Cope ◽  
Georgina Cornish ◽  
Dylan M. Owen
Keyword(s):  
T Cell ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Cell Protein ◽  
Single Molecule Level ◽  
Composition And Structure ◽  
Resolution Analysis ◽  
And Function ◽  
Super Resolution Microscopy

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

scholarly journals Aggregation-related quenching of LHCII in liposomes revealed by single-molecule spectroscopy

2020 ◽  
Author(s):  
Marijonas Tutkus ◽  
Jevgenij Chmeliov ◽  
Gediminas Trinkunas ◽  
Parveen Akhtar ◽  
Petar H. Lambrev ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Lipid Membranes ◽  
Photochemical Quenching ◽  
Liposome Size ◽  
Function Relationship ◽  
Structure And Function Relationship ◽  
Protein Density ◽  
And Function

AbstractIncorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data

2016 ◽  
Vol 27 (22) ◽  
pp. 3627-3636 ◽  
Author(s):  
Sophie V. Pageon ◽  
Philip R. Nicovich ◽  
Mahdie Mollazade ◽  
Thibault Tabarin ◽  
Katharina Gaus
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Single Molecule ◽  
Cell Activation ◽  
Spatial Organization ◽  
Focal Adhesions ◽  
Cell Receptor ◽  
Intact Cells ◽  
Analysis Strategy ◽  
Signaling Activity

Advances in fluorescence microscopy are providing increasing evidence that the spatial organization of proteins in cell membranes may facilitate signal initiation and integration for appropriate cellular responses. Our understanding of how changes in spatial organization are linked to function has been hampered by the inability to directly measure signaling activity or protein association at the level of individual proteins in intact cells. Here we solve this measurement challenge by developing Clus-DoC, an analysis strategy that quantifies both the spatial distribution of a protein and its colocalization status. We apply this approach to the triggering of the T-cell receptor during T-cell activation, as well as to the functionality of focal adhesions in fibroblasts, thereby demonstrating an experimental and analytical workflow that can be used to quantify signaling activity and protein colocalization at the level of individual proteins.

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