Fluorescence-Based Detection of Proteins and Their Interactions in Live Cells

Stoneman, Michael R.; Raicu, Valerică

doi:10.1021/acs.jpcb.3c01419

Cited by 9 publications

(4 citation statements)

References 65 publications

(114 reference statements)

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“…To effectively detect and characterize the fluorescence of leaf compressions, we utilized a previously described two-photon optical microspectroscopy technique 15 , 16 , 24 . This technique involves scanning a focused laser beam across a fossil sample and capturing fluorescence spectra at the pixel level for each scanned location.…”

Section: Resultsmentioning

confidence: 99%

Two-photon excitation fluorescence microspectroscopy protocols for examining fluorophores in fossil plants

Stoneman,

McCoy,

Gee

et al. 2024

Commun Biol

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Fluorescence emission is common in plants. While fluorescence microscopy has been widely used to study living plants, its application in quantifying the fluorescence of fossil plants has been limited. Fossil plant fluorescence, from original fluorophores or formed during fossilization, can offer valuable insights into fluorescence in ancient plants and fossilization processes. In this work, we utilize two-photon fluorescence microspectroscopy to spatially and spectrally resolve the fluorescence emitted by amber-embedded plants, leaf compressions, and silicified wood. The advanced micro-spectroscope utilized, with its pixel-level spectral resolution and line-scan excitation capabilities, allows us to collect comprehensive excitation and emission spectra with high sensitivity and minimal laser damage to the specimens. By applying linear spectral unmixing to the spectrally resolved fluorescence images, we can differentiate between (a) the matrix and (b) the materials that comprise the fossil. Our analysis suggests that the latter correspond to durable tissues such as lignin and cellulose. Additionally, we observe potential signals from chlorophyll derivatives/tannins, although minerals may have contributed to this. This research opens doors to exploring ancient ecosystems and understanding the ecological roles of fluorescence in plants throughout time. Furthermore, the protocols developed herein can also be applied to analyze non-plant fossils and biological specimens.

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Section: Resultsmentioning

confidence: 99%

Two-photon excitation fluorescence microspectroscopy protocols for examining fluorophores in fossil plants

Stoneman,

McCoy,

Gee

et al. 2024

Commun Biol

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“…Several fluorescence-based methods have been developed to quantify protein interactions in live cells (Martin-Fernandez 2023;Sankaran and Wohland 2023;Stoneman and Raicu 2023).…”

Section: Introductionmentioning

confidence: 99%

HER4 is a high affinity dimerization partner for all EGFR/HER/ErbB-family proteins

Singh,

Kim,

Smith

2024

Preprint

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Human epidermal growth factor receptors (HER), also known as EGFR or ErbB receptors, are a subfamily of receptor tyrosine kinases (RTKs) that play crucial roles in cell growth, division, and differentiation. HER4 (ErbB4) is the least studied member of this family, partly because its expression is lower in later stages of development. Recent work has suggested that HER4 can play a role in metastasis through cell migration and invasiveness; however, unlike EGFR and HER2, the precise role that HER4 plays in tumorigenesis is still unresolved. Early work on HER family proteins suggested that there are direct interactions between the four members, but to date, there has been no single study of all four receptors in the same cell line studied with the same biophysical method. Here, we quantitatively measure the degree of association between HER4 and the other HER-family proteins in live cells with a time-resolved fluorescence technique called pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). PIE-FCCS is sensitive to the oligomerization state of membrane proteins in live cells, while simultaneously measuring protein expression levels and diffusion coefficients. Our PIE-FCCS results demonstrate that HER4 interacts directly with all HER family members in the cell plasma membrane. The interaction between HER4 and other HER family members intensified in the presence of a HER4-specific ligand. Our work suggests that HER4 is a preferred dimerization partner for all HER family proteins, even in the absence of ligands.

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“…Fluorescence techniques (e.g., FRET) are usually relatively easy to implement and have the potential to cleverly probe many different aspects of the cytoplasm and dynamics without much disruption, but these methods lack high spatial resolution, limiting their translation to the protein domain scale . A recent Perspective on fluorescence-based methods as applied to in-cell studies just appeared in this journal . Cryo-EM has the advantage of capturing (almost) native snapshots of the cellular environment but suffers from sample preparation heterogeneity.…”

Section: Introductionmentioning

confidence: 99%

In-Cell Dynamics: The Next Focus of All-Atom Simulations

Samuel Russell,

Alaeen,

Pogorelov

2023

J. Phys. Chem. B

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The cell is a crowded space where large biomolecules and metabolites are in continuous motion. Great strides have been made in in vitro studies of protein dynamics, folding, and protein−protein interactions, and much new data are emerging of how they differ in the cell. In this Perspective, we highlight the current progress in atomistic modeling of in-cell environments, both bacteria and mammals, with emphasis on classical all-atom molecular dynamics simulations. These simulations have been recently used to capture and characterize functional and non-functional protein−protein interactions, protein folding dynamics of small proteins with varied topologies, and dynamics of metabolites. We further discuss the challenges and efforts for updating modern force fields critical to the progress of cellular environment simulations. We also briefly summarize developments in relevant state-of-the-art experimental techniques. As computational and experimental methodologies continue to progress and produce more directly comparable data, we are poised to capture the complex atomistic picture of the cell.

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Fluorescence-Based Detection of Proteins and Their Interactions in Live Cells

Cited by 9 publications

References 65 publications

Two-photon excitation fluorescence microspectroscopy protocols for examining fluorophores in fossil plants

Two-photon excitation fluorescence microspectroscopy protocols for examining fluorophores in fossil plants

HER4 is a high affinity dimerization partner for all EGFR/HER/ErbB-family proteins

In-Cell Dynamics: The Next Focus of All-Atom Simulations

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