3 research outputs found
Light Sheet Microscopy for Tracking Single Molecules on the Apical Surface of Living Cells
Single particle tracking is a powerful
tool to study single molecule
dynamics in living biological samples. However, current tracking techniques,
which are based mainly on epifluorescence, confocal, or TIRF microscopy,
have difficulties in tracking single molecules on the apical surface
of a cell. We present here a three-dimensional (3D) single particle
tracking technique that is based on prism coupled light-sheet microscopy
(PCLSM). This novel design provides a signal-to-noise ratio comparable
to confocal microscopy while it has the capability of illuminating
at arbitrary depth. We demonstrate tracking of single EGF molcules
on the apical surface of live cell membranes from their binding to
EGF receptors until they are internalized or photobleached. We found
that EGF exhibits multiple diffusion behaviors on live A549 cell membranes.
At room temperature, the average diffusion coefficient of EGF on A549
cells was measured to be 0.13 Ī¼m<sup>2</sup>/s. Depletion of
cellular cholesterol with methyl-Ī²-cyclodextrin leads to a broader
distribution of diffusion coefficients and an increase of the average
diffusion coefficient at room temperature. This light-sheet based
3D single particle tracking technique solves the technique difficulty
of tracking single particles on apical membranes and is able to document
the whole ālifetimeā of a particle from binding till
photobleaching or internalization
Light Sheet Microscopy for Tracking Single Molecules on the Apical Surface of Living Cells
Single particle tracking is a powerful
tool to study single molecule
dynamics in living biological samples. However, current tracking techniques,
which are based mainly on epifluorescence, confocal, or TIRF microscopy,
have difficulties in tracking single molecules on the apical surface
of a cell. We present here a three-dimensional (3D) single particle
tracking technique that is based on prism coupled light-sheet microscopy
(PCLSM). This novel design provides a signal-to-noise ratio comparable
to confocal microscopy while it has the capability of illuminating
at arbitrary depth. We demonstrate tracking of single EGF molcules
on the apical surface of live cell membranes from their binding to
EGF receptors until they are internalized or photobleached. We found
that EGF exhibits multiple diffusion behaviors on live A549 cell membranes.
At room temperature, the average diffusion coefficient of EGF on A549
cells was measured to be 0.13 Ī¼m<sup>2</sup>/s. Depletion of
cellular cholesterol with methyl-Ī²-cyclodextrin leads to a broader
distribution of diffusion coefficients and an increase of the average
diffusion coefficient at room temperature. This light-sheet based
3D single particle tracking technique solves the technique difficulty
of tracking single particles on apical membranes and is able to document
the whole ālifetimeā of a particle from binding till
photobleaching or internalization
Genetically Encoding an Electrophilic Amino Acid for Protein Stapling and Covalent Binding to Native Receptors
Covalent bonds can be generated within
and between proteins by
an unnatural amino acid (Uaa) reacting with a natural residue through
proximity-enabled bioreactivity. Until now, Uaas have been developed
to react mainly with cysteine in proteins. Here we genetically encoded
an electrophilic Uaa capable of reacting with histidine and lysine,
thereby expanding the diversity of target proteins and the scope of
the proximity-enabled protein cross-linking technology. In addition
to efficient cross-linking of proteins inter- and intramolecularly,
this Uaa permits direct stapling of a protein Ī±-helix in a recombinant
manner and covalent binding of native membrane receptors in live cells.
The target diversity, recombinant stapling, and covalent targeting
of endogenous proteins enabled by this versatile Uaa should prove
valuable in developing novel research tools, biological diagnostics,
and therapeutics by exploiting covalent protein linkages for specificity,
irreversibility, and stability