ACS Chemical Neuroscience
Letter
the cytosol and can be successfully used to visualize
intracellular inclusions of αSyn.
CONCLUSIONS
■
To the best of our knowledge, we present the first dye that
allows selective imaging of αSyn amyloid fibrils in the
cytoplasm of living cells. RB1 is a water-soluble rotor-like
dye that strongly increases the fluorescence intensity upon
interaction with amyloid fibrils. It shows a strong red-shift of
absorption spectra (up to 76 nm) upon binding to amyloids
(Kd = 30 nM) that allows selective excitation of its bound
form. Altogether, the superior optical behavior of RB1 and
strong affinity to αSyn fibrils make it an attractive noninvasive
tool for monitoring of αSyn aggregates in living cells that could
facilitate studies of Parkinson’s disease mechanisms.
METHODS
■
Synthesis and characterization of the dyes are presented in SI.
Measurements of Spectra in Solvents. Absorption and
emission spectra of dyes in solvents were recorded on Duetta
spectrometer (Horiba Scientific) in a 1 mL quartz cuvette at room
temperature. Stock solutions of the probes (1 mM) were prepared in
DMSO. λexc was 525 nm for both RB1 and RB2. Excitation and
emission slits were 5 nm.
Figure 5. Visualization of αSyn fibrils with RB1 in living cells. (a)
Staining of SH-SY5Y cells incubated with labeled fibrils: Green,
ConA-FITC (λexc = 488 nm, λem = 500−550 nm); red, RB1 (λexc
=
561 nm, λem = 580−625 nm); (b) blue, fibrils labeled with Atto 647
(λexc = 633 nm, λem = 650−700 nm). (c) Merged image of panels (a)
and (b). (d) Co-localization plot between Atto 647 and RB1 signal in
the cytosol (Mander’s coefficient = 80%, measured from three
independent samples).
Fluorescence quantum yield of the probes was determined using
Rhodamine B (Φ = 0.68 in 94% ethanol)37 as the reference.
Preparation of Fibrils. The purification of monomeric αSyn from
E. coli was done following the published protocol.8
αSyn-WT and αSyn-A53T fibrils were prepared by the incubation
of 200 μM corresponding αSyn monomer solution in buffer (10 mM
Tris-HCl, pH 7.4, 140 mM NaCl, 10 mM EDTA, and 10 mM NaN3)
at 37 °C with constant shaking (200 rpm) for 5 days.
Lysozyme fibrils were prepared by incubation 200 μM hen egg-
white lysozyme (Sigma, L6876) in 140 mM NaCl and 10 mM NaN3,
pH 2.0 (adjusted with HCl) at 65 °C with shaking for 48 h.38
Insulin fibrils were prepared accordingly to standard procedures.39
Namely, the procedures used 200 μM human insulin (Sigma, I2643)
in 140 mM NaCl and 10 mM NaN3, pH 1.6. The solution was
incubated for 15 min at 60 °C and then 3 days at 37 °C with constant
shaking (200 rpm).
The recombinant production and His-tag purification of mono-
meric Amyloid-β(1−40) and Amyloid-β(1−42) from E. coli were
performed according to standard protocols.40 Aβ(1−40) and
Aβ(1−42) fibrils were prepared by incubating 100 μM corresponding
peptide in 10 mM Tris-HCl, pH 7.4, 140 mM NaCl and 10 mM
NaN3 at 37 °C with shaking for 48 h.
Spectral Measurements in the Presence of Amyloid Fibrils.
Absorption and emission spectra were recorded on a Tecan SPARK
plate reader at room temperature. DMSO stock solutions of the
probes were diluted in aqueous buffer. Measurements were performed
in a black 384-well plate with transparent bottom (cat. no. 242764;
Nunc/Thermo Fisher Scientific). Fluorescence quantum yields were
determined using Rhodamine B (ΦR = 0.68 in 94% ethanol)37 as the
reference, in the same way as in solvents.
Preparation of αSyn Fibrils for Cell Experiments. Labeled
αSyn fibrils for cell experiments were prepared by incubation of a
mixture of 80 μM of αSyn-WT and 20 μM αSyn-S9C-Atto 647 in 10
mM Tris-HCl pH 7.4, 140 mM NaCl, 10 mM EDTA, and 10 mM
NaN3 buffer. The aggregation was initiated by the addition of 0.5 μM
WT αSyn fibrils (prepared previously), and the solution was shaken at
37 °C for 5 days. Then, the fibrils were precipitated by 15 min ×
12 000g centrifugation. The supernatant was removed, and the
precipitated fibrils were resuspended in Krebs-Ringer solution (buffer
suitable for cell experiments). The concentration of the fibrils was
determined by Atto 647 absorption (ε = 12 0000 M−1 cm−1). The
fibrils were sonicated in bath sonicator for 15 min to break them into
50−100 nm long fragments (“seeds”).
also observed RB1 staining of cellular nucleoli. It probably
occurred because of the intercalation of the positively charged
RB1 into RNA, which was also observed in the solution
(Figure S4). However, since nucleoli are morphologically
distinct from fibrils, their staining can be easily distinguished
from αSyn aggregates and even used as an internal control of
dye performance.
Then we checked RB1 staining of the intracellular fibrils that
were not labeled with Atto 647 dye. Namely, we stained cells
that were incubated with nonlabeled fibrils, and as a control,
we used cells that were not treated with fibrils (Figure S8). As
expected, untreated cells showed nucleolus RB1 staining with
negligible cytoplasmic signal, while in the presence of fibrils,
we observed clearly distinguishable bright cytosolic aggregates.
To exclude any possible artifacts related to the fibril uptake
by cells, we decided to visualize αSyn fibrils formed inside the
cells. For that, we incubated the cells with Atto 647 labeled
monomeric αSyn and stimulated its aggregation by inhibiting
cell proteasomes with MG-132.36 The fluorescence pattern of
RB1 staining in the cells with induced fibrillization was very
similar to the staining in the cells incubated with αSyn fibrils
(Figure S9). Namely, RB1 signal from cytosol always
overlapped with the signal of Atto 647 label on αSyn.
However, Atto 647 signal often did not colocalize with RB1
signal showing that part of αSyn remains nonfibrillized.
To verify RB1 applicability to other cellular systems, we
performed the same type of fibril staining in HeLa cells, where
RB1 showed a similar pattern of staining (Figure S10). We also
found that RB1 can be used in cellular systems without
harming them. Indeed, cell viability assay showed that RB1 was
not cytotoxic at 5 μM concentrations; namely, the cell viability
was greater than 95% after 24 h (Figure S11). Altogether, our
studies demonstrated that RB1 selectively stains αSyn fibrils in
1296
ACS Chem. Neurosci. 2021, 12, 1293−1298