A) Illustration of the approach. A C. elegans embryo, staged
based on morphology, is placed into a flow chamber and trapped in the center by
a small constriction. The embryo is crushed to generate a lysate, and proteins
of interest are captured using antibodies bound to the coverglass floor of the
chamber. The device is placed directly on a TIRF microscope to interrogate
molecular complexes via single-molecule imaging.
B) Images of mNG::HaloTag molecules pulled down from a single embryo labeled with
JF646 HaloTag ligand. The mNG channel is shown in green and the JF646 (far red)
channel is shown in red. Scale bars represent 5 μm.
C) Quantification of the number of green and far-red spots per image as a
function of position along the length of the chamber.
D) Quantification of the fraction of colocalized spots for an mNG::HaloTag fusion
protein labeled with JF646 (left graph), an mNG::mKate2 fusion protein (center
graph) or an mScarlet-I::HaloTag fusion protein labeled with JF646 (right
graph).
E) Images of mNG::AraD tetramers pulled down from a single embryo. Scale bars
represent 5 μm.
F) Example of a photobleaching trace from a single mNG::AraD complex, showing
four photobleaching steps.
G) Blue bars: histogram showing the distribution of photobleaching step counts in
a population of molecules (data from four single-embryo experiments are
combined). Red line: fit of the data to the binomial distribution , where PN is the
probability of detecting N photobleaching steps given the
fraction d of mNG molecules detected in this assay.
See also Figures S1, S2 and
S3.