Dynamic Imaging of Homo-FRET in Live Cells by Fluorescence Anisotropy Microscopy

Ghosh, Subhasri; Saha, Suvrajit; Goswami, Debanjan; Bilgrami, Sameera; Mayor, Satyajit

doi:10.1016/b978-0-12-388448-0.00024-3

Cited by 55 publications

(90 citation statements)

References 35 publications

(52 reference statements)

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“…Proximity measurements based on fl uorescence (or Förster) resonance energy transfer (FRET) [reviewed in (67)(68)(69)] and, more recently, super-resolution imaging ( 71,72 ) have been utilized to explore the nanoscale organization of the GPI-anchored proteins at the cell surface.…”

Section: Diffusional Dynamics Of Gpi-anchored Proteinsmentioning

confidence: 99%

“…More recently, the implementation of homo-FRET measurement on several microscopy modalities/platforms allowed studying GPI-anchored protein clustering at superior spatiotemporal resolution ( 68,83 ). Homo-FRET imaging at a higher spatial resolution ( ‫ف‬ 300 nm) showed that GPIanchored protein nanoclusters exhibit spatially nonrandom distributions ( Fig.…”

Section: Gpi-anchored Protein Nanoclustering Requires Transbilayer Acmentioning

confidence: 99%

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GPI-anchored protein organization and dynamics at the cell surface

Saha

Anilkumar

Mayor

2016

Journal of Lipid Research

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of the protein called glycosylphosphatidylinositol (GPI)-anchored proteins. The basic structure of a GPI-anchored protein consists of phosphatidylinositol (PI) linked to an unusual non-N -acetyl glucosamine, which, in turn, is linked to three mannose residues followed by an ethanolamine covalently linked to the protein via an amide linkage (EtNP-6Man ␣ 1-2Man ␣ 1-6Man ␣ 1-4GlcN ␣ 1-6 myo inositolphospholipid, Fig. 1A ). Depending on the species and functional context, there may exist variations in the side chain associated with the glycan core. These have been summarized in ( 1 ). The lipid moiety is necessary for the incorporation of GPI-anchored proteins into so-called lipid rafts/microdomains ( 2, 3 ), which can serve as a sorting station for a number of cell signaling molecules, thereby functioning as a reaction center. GPI-anchored proteins can exist in different forms depending on the context and the tissue in which they are expressed. Alternate splicing can cause the same protein to exhibit TM, soluble, or GPI-anchored forms; for example, neural cell adhesion molecule (NCAM) can exist in its GPI-anchored and soluble form when expressed in muscles; whereas, it takes up a TM form instead of the soluble form in brain. GPI anchoring of proteins occurs at the luminal face of The plasma membrane of the cell is comprised of a diverse set of proteins, many of which are transmembrane (TM) proteins spanning the entire bilayer, but a significant proportion of proteins are also lipid tethered, containing a complex glycan core attached to the C terminus

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Section: Diffusional Dynamics Of Gpi-anchored Proteinsmentioning

confidence: 99%

Section: Gpi-anchored Protein Nanoclustering Requires Transbilayer Acmentioning

confidence: 99%

GPI-anchored protein organization and dynamics at the cell surface

Saha

Anilkumar

Mayor

2016

Journal of Lipid Research

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“…The excitation and emission dipoles of GFP are highly aligned to one another and may be referred to collectively as the transition dipole 28,29 . Furthermore, the difference in time between excitation and emission is short relative to tumbling time for GFP 27 (and integrin-GFP fusions). The high NA objectives used in TIRF somewhat lower the absolute values of anisotropy 27 ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…1). For GFP in solution, these effects result in a drop of the observed anisotropy from the typical value of 0.32 50 to~0.18-0.20 27,51 . As these depolarization effects are similar across the various experimental systems used in this article, the relative changes in anisotropy and not the absolute values of anisotropy are significant.…”

Section: Methodsmentioning

confidence: 99%

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Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

Nordenfelt

Moore

Mehta

et al. 2016

Preprint

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Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

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Multiplexed imaging of intracellular protein networks

2016

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Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. V C 2016 International Society for Advancement of Cytometry

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Dynamic Imaging of Homo-FRET in Live Cells by Fluorescence Anisotropy Microscopy

Cited by 55 publications

References 35 publications

GPI-anchored protein organization and dynamics at the cell surface

GPI-anchored protein organization and dynamics at the cell surface

Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

Multiplexed imaging of intracellular protein networks

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