On-cell coordination chemistry: Chemogenetic activation of membrane-bound glutamate receptors in living cells

Scaffold, anchoring, and adaptor proteins coordinate the assembly and localization of signaling complexes providing efficiency and specificity in signal transduction. The PKA, PKC, and protein phosphatase-2B/calcineurin (CaN) scaffold protein A–kinase anchoring protein (AKAP) 79 is localized to excitatory neuronal synapses where it is recruited to glutamate receptors by interactions with membrane-associated guanylate kinase (MAGUK) scaffold proteins. Anchored PKA and CaN in these complexes could have important functions in regulating glutamate receptors in synaptic plasticity. However, direct evidence for the assembly of complexes containing PKA, CaN, AKAP79, and MAGUKs in intact cells has not been available. In this report, we use immunofluorescence and fluorescence resonance energy transfer (FRET) microscopy to demonstrate membrane cytoskeleton–localized assembly of this complex. Using FRET, we directly observed binding of CaN catalytic A subunit (CaNA) and PKA-RII subunits to membrane-targeted AKAP79. We also detected FRET between CaNA and PKA-RII bound simultaneously to AKAP79 within 50 Å of each other, thus providing the first direct evidence of a ternary kinase–scaffold–phosphatase complex in living cells. This finding of AKAP-mediated PKA and CaN colocalization on a nanometer scale gives new appreciation to the level of compartmentalized signal transduction possible within scaffolds. Finally, we demonstrated AKAP79-regulated membrane localization of the MAGUK synapse-associated protein 97 (SAP97), suggesting that AKAP79 functions to organize even larger signaling complexes.

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Resonance energy transfer microscopy: observations of membrane-bound fluorescent probes in model membranes and in living cells.

The Journal of Cell Biology ◽

10.1083/jcb.103.4.1221 ◽

1986 ◽

Vol 103 (4) ◽

pp. 1221-1234 ◽

Cited By ~ 74

Author(s):

P S Uster ◽

R E Pagano

Keyword(s):

Energy Transfer ◽

Fluorescent Probes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Living Cells ◽

Model Membranes ◽

Energy Donor ◽

Membrane Bound ◽

Energy Acceptor ◽

In Cells

A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C10). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C10 and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can co-localize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.

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Video microscopic studies of membrane dynamics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100085460 ◽

1991 ◽

Vol 49 ◽

pp. 232-233

Author(s):

Ken Jacobson ◽

Greta Lee ◽

Juliet Lee

Keyword(s):

Living Cells ◽

Model System ◽

Random Motion ◽

Membrane Dynamics ◽

Two Dimensional ◽

Planar Bilayer ◽

Microscopic Studies ◽

Membrane Bound ◽

Random Patterns ◽

Membrane Components

The application of several imaging technologies and photobleaching to problems in membrane dynamics will be presented. These techniques emphasize different aspects of the measurement of translational transport: (1) tracing the trajectories of individual molecules undergoing a two dimensional random walk, and (2) investigating bulk changes in the distribution of membrane components in the plane of the membrane in single, locomoting cells.Nanovid microscopy, which uses 30-40 nm colloidal gold probes combined with video-enhanced contrast, allows investigation of random and directed movements of individual molecules in artificial membranes or in the plasma membrane of living cells. The movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-phosphatidylethanolamine (Fl-PE) labelled with 30 ran gold anti-fluorescein (anti-Fl) to validate the technique in a model system. The membrane-bound gold particles moved in random patterns which were indistinguishable from those produced by computer simulations of two dimensional random motion.

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The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0344 ◽

2008 ◽

Vol 19 (9) ◽

pp. 3944-3955 ◽

Cited By ~ 21

Author(s):

Li Wang ◽

Mary A. Bittner ◽

Daniel Axelrod ◽

Ronald W. Holz

Keyword(s):

Plasma Membrane ◽

Chromaffin Cells ◽

Close Association ◽

Living Cells ◽

Full Length ◽

Intact Cells ◽

Permeabilized Cells ◽

Functional Implications ◽

Membrane Bound

We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

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Mechanisms and regulation underlying membraneless organelle plasticity control

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjab028 ◽

2021 ◽

Author(s):

Hazrat Ismail ◽

Xu Liu ◽

Fengrui Yang ◽

Junying Li ◽

Ayesha Zahid ◽

...

Keyword(s):

Phase Separation ◽

Neurodegenerative Disorders ◽

Living Cells ◽

Cell Plasticity ◽

Chemical Modifications ◽

Membrane Bound ◽

Functional Complexity ◽

Cellular Compartments ◽

Reversible Phase ◽

Separation Results

Abstract Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments, such as membrane-bound and membraneless organelles, for spatiotemporal catalysis of physiochemical reactions essential for cell plasticity control. Emerging evidence and view support the notion that membraneless organelles are built by multivalent interactions of biomolecules via phase separation and transition mechanisms. In healthy cells, dynamic chemical modifications regulate membraneless organelle plasticity, and reversible phase separation is essential for cell homeostasis. Emerging evidence revealed that aberrant phase separation results in numerous neurodegenerative disorders, cancer, and other diseases. In this review, we provide molecular underpinnings on (i) mechanistic understanding of phase separation, (ii) unifying structural and mechanistic principles that underlie this phenomenon, (iii) various mechanisms that are used by cells for the regulation of phase separation, and (iv) emerging therapeutic and other applications.

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Imaging the electrical activity of organelles in living cells

Communications Biology ◽

10.1038/s42003-021-01916-6 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Ella Matamala ◽

Cristian Castillo ◽

Juan P. Vivar ◽

Patricio A. Rojas ◽

Sebastian E. Brauchi

Keyword(s):

Electrical Activity ◽

Living Cells ◽

Eukaryotic Cells ◽

Subcellular Targeting ◽

Spatiotemporal Resolution ◽

Non Invasive ◽

Optical Multiplexing ◽

Voltage Dependent ◽

Membrane Bound ◽

Fret Pair

AbstractEukaryotic cells are complex systems compartmentalized in membrane-bound organelles. Visualization of organellar electrical activity in living cells requires both a suitable reporter and non-invasive imaging at high spatiotemporal resolution. Here we present hVoSorg, an optical method to monitor changes in the membrane potential of subcellular membranes. This method takes advantage of a FRET pair consisting of a membrane-bound voltage-insensitive fluorescent donor and a non-fluorescent voltage-dependent acceptor that rapidly moves across the membrane in response to changes in polarity. Compared to the currently available techniques, hVoSorg has advantages including simple and precise subcellular targeting, the ability to record from individual organelles, and the potential for optical multiplexing of organellar activity.

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The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

Biochemical Society Transactions ◽

10.1042/bst20150285 ◽

2016 ◽

Vol 44 (2) ◽

pp. 624-629 ◽

Cited By ~ 8

Author(s):

Laura E. Kilpatrick ◽

Stephen J. Hill

Keyword(s):

G Protein ◽

Fluorescence Correlation Spectroscopy ◽

Photon Counting ◽

Living Cells ◽

Correlation Spectroscopy ◽

G Protein Coupled Receptors ◽

Fluorescence Correlation ◽

Membrane Bound ◽

Molecular Brightness ◽

G Protein Coupled

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

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Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology

eLife ◽

10.7554/elife.39578 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 46

Author(s):

Peipei Zhang ◽

Baochang Fan ◽

Peiguo Yang ◽

Jamshid Temirov ◽

James Messing ◽

...

Keyword(s):

Material Properties ◽

Stress Granules ◽

Living Cells ◽

Editorial Note ◽

Confounding Variable ◽

Cytoplasmic Inclusions ◽

Primary Driver ◽

Membrane Bound ◽

Protein Granules ◽

Lateral Sclerosis

Stress granules (SGs) are non-membrane-bound RNA-protein granules that assemble through phase separation in response to cellular stress. Disturbances in SG dynamics have been implicated as a primary driver of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), suggesting the hypothesis that these diseases reflect an underlying disturbance in the dynamics and material properties of SGs. However, this concept has remained largely untestable in available models of SG assembly, which require the confounding variable of exogenous stressors. Here we introduce a light-inducible SG system, termed OptoGranules, based on optogenetic multimerization of G3BP1, which is an essential scaffold protein for SG assembly. In this system, which permits experimental control of SGs in living cells in the absence of exogenous stressors, we demonstrate that persistent or repetitive assembly of SGs is cytotoxic and is accompanied by the evolution of SGs to cytoplasmic inclusions that recapitulate the pathology of ALS-FTD.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (<xref ref-type="decision-letter" rid="SA1">see decision letter</xref>).

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Harnessing biomolecular condensates in living cells

The Journal of Biochemistry ◽

10.1093/jb/mvz028 ◽

2019 ◽

Vol 166 (1) ◽

pp. 13-27 ◽

Cited By ~ 4

Author(s):

Hideki Nakamura ◽

Robert DeRose ◽

Takanari Inoue

Keyword(s):

Living Cells ◽

Technological Advance ◽

Liquid Droplets ◽

Molecular Tools ◽

Rna Molecules ◽

Cell Functions ◽

Pathological Conditions ◽

A Cell ◽

Membrane Bound

Abstract As part of the ‘Central Dogma’ of molecular biology, the function of proteins and nucleic acids within a cell is determined by their primary sequence. Recent work, however, has shown that within living cells the role of many proteins and RNA molecules can be influenced by the physical state in which the molecule is found. Within living cells, both protein and RNA molecules are observed to condense into non-membrane-bound yet distinct structures such as liquid droplets, hydrogels and insoluble aggregates. These unique intracellular organizations, collectively termed biomolecular condensates, have been found to be vital in both normal and pathological conditions. Here, we review the latest studies that have developed molecular tools attempting to recreate artificial biomolecular condensates in living cells. We will describe their design principles, implementation and unique characteristics, along with limitations. We will also introduce how these tools can be used to probe and perturb normal and pathological cell functions, which will then be complemented with discussions of remaining areas for technological advance under this exciting theme.

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