buffer three times on ice. Cells were subsequently treated with 50 mM glycine
to quench unreacted aldehydes, followed by 10 mM potassium cyanide and
5 mM aminotriazole in the cacodylate buffer for 20 min on ice to suppress
background. Photooxidation was performed using a Leica SPI II inverted
confocal microscope equipped with a 150-W xenon light source. Labeled
cells were located by epifluorescence and treated with a fresh solution of
diaminobenzidine (prepared by dissolving 5.4 mg diaminobenzidine in 1 mL
0.1 N HCl and diluting to 10 mL total volume using cacodylate buffer;
Sigma). A stream of oxygen gas was blown over the surface of the so-
lution and the cells were illuminated using a 562/40 filter (Semrock) at full
intensity for 10–20 min until a brown reaction product appeared. These illu-
mination conditions are much more intense than for live-cell imaging, such
that most fluorescence was photobleached after the 10- to 20-min treatment.
The cells were then rinsed in cold cacodylate buffer and postfixed with 1%
osmium tetroxide on ice. After rinses in cold water, cells were either stained
with 2% (wt/vol) aqueous uranyl acetate at 4 °C overnight en bloc or directly
dehydrated in cold ethanol series [20, 50, 70, 90, and 100% (vol/vol)] for 3 min
each on ice, followed by a room temperature rinse in 100% ethanol. Cells were
embedded in Durcupan ACM resin (Electron Microscopy Sciences). Standard
80-nm-thick sections were imaged using a JEOL 1200 EX transmission electron
microscope operated at 80 kV. Cells in Fig. 4B were treated with 1 μg/mL
colcemid (Sigma) for 4 h before addition of resorufin-AM2.
ACKNOWLEDGMENTS. Birka Hein [Max Planck Institute (MPI) for Biophysical
Chemistry] performed preliminary STED imaging experiments. Stefan Hell (MPI
for Biophysical Chemistry) provided experimental advice and performed
critical reading of the manuscript. Chayasith Uttamapinant [Massachusetts
Institute of Technology (MIT)] provided the coumarin compounds and the
LAP–β-actin, vimentin-LAP, LAP-MAP2, and HaloTag–β-actin plasmids. Caro-
lyn Kwa (MIT) and Ken Loh (MIT) provided assistance with neuron cultures.
Justin Cohen (MIT) and Samuel Thompson (MIT) provided LplA protein.
Katharine White (MIT) provided the LAP-CaaX, LAP-NLS, and LAP-NES plas-
mids. Michael Davidson (Florida State University) provided the mApple–
β-actin plasmid. We thank Kyle Gee (Life Technologies) for helpful discus-
sions about the resorufin fluorophore. Diffraction data were obtained at the
Advanced Photon Source on the Northeastern Collaborative Access Team
beamlines, which are supported by Award RR-15301 from the National Center
for Research Resources at the National Institutes of Health (NIH). Use of the
Advanced Photon Source, an Office of Science User Facility operated for the US
Department of Energy (DOE) Office of Science by Argonne National Labora-
tory, was supported by the US DOE under Contract DE-AC02-06CH11357.
This work was funded by NIH Grants DP1 OD003961 and R01 GM072670
(to A.Y.T.), NIH Grant GM103412 (to M.H.E.), and the American Chemical Soci-
ety (A.Y.T.). C.L.D. is a Howard Hughes Medical Institute (HHMI) Investigator.
L.G.N. acknowledges support from a National Science Foundation Minority
Post-Doctoral Fellowship. A.Z.Y. acknowledges support from the Lord Founda-
tion and the HHMI-Massachusetts Institute of Technology Summer Research
Program in Chemical Biology.
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PNAS
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Published online October 13, 2014
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E4559