Mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells

Wollman, Adam J. M.; Hedlund, Erik; Shashkova, Sviatlana; Leake, Mark C.

doi:10.1101/568675

Cited by 2 publications

References 45 publications

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Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

Shepherd

Lecinski

Wragg

et al. 2020

Preprint

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The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

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Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

Shepherd

Lecinski

Wragg

et al. 2020

Preprint

View full text Add to dashboard Cite

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Impact of Saccharomyces cerevisiae on the Field of Single-Molecule Biophysics

Jalloh

Karpova

2022

IJMS

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Cellular functions depend on the dynamic assembly of protein regulator complexes at specific cellular locations. Single Molecule Tracking (SMT) is a method of choice for the biochemical characterization of protein dynamics in vitro and in vivo. SMT follows individual molecules in live cells and provides direct information about their behavior. SMT was successfully applied to mammalian models. However, mammalian cells provide a complex environment where protein mobility depends on numerous factors that are difficult to control experimentally. Therefore, yeast cells, which are unicellular and well-studied with a small and completely sequenced genome, provide an attractive alternative for SMT. The simplicity of organization, ease of genetic manipulation, and tolerance to gene fusions all make yeast a great model for quantifying the kinetics of major enzymes, membrane proteins, and nuclear and cellular bodies. However, very few researchers apply SMT techniques to yeast. Our goal is to promote SMT in yeast to a wider research community. Our review serves a dual purpose. We explain how SMT is conducted in yeast cells, and we discuss the latest insights from yeast SMT while putting them in perspective with SMT of higher eukaryotes.

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Mapping the 3D genome through high speed single-molecule tracking of functional transcription factors in single living cells

Cited by 2 publications

References 45 publications

Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

Molecular crowding in single eukaryotic cells: using cell environment biosensing and single-molecule optical microscopy to probe dependence on extracellular ionic strength, local glucose conditions, and sensor copy number

Impact of Saccharomyces cerevisiae on the Field of Single-Molecule Biophysics

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