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ChemComm
with the structural connection of these two organelles.28 To our 40 F. Beltram for his scientific advices, Dr. F. Signori for DLS
knowledge these are the first available quantitative data on the
dielectric properties of ER and NE in living cells.
measurements, and Dr. F. Cardarelli for useful discussions.
Notes and references
a
Center for Nanotechnology Innovation @ NEST, Istituto Italiano di
Tecnologia, Pisa, Italy
45 b NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa,
Italy; E-mail: r.bizzarri@sns.it
c Nanobiophysics, Faculty of Science and Technology, University of
Twente, The Netherlands
d Istituto di Biofisica –CNR, via Moruzzi 1, 56124, Pisa, Italy
50 § G. Signore and G. Abbandonato contributed equally to this work
† Electronic Supplementary Information (ESI) available: Materials and
methods; synthesis of compounds; Table S1, Table S2, Table S3;
Schemes S1, S2, S3; Figures S1, S2, S3, S4, S5, S6, and S7;
chromatographic analysis and mass spectra. See DOI: 10.1039/b000000x/
55
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Fig. 2 Fluorescence images (a, c) and ε maps (b, d) of subcellular regions
of CHO cells treated with 1 or 3 (Relevant wholeꢀcell images and maps
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localizes mostly in the endoplasmic reticulum (ER) and nuclear envelope
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10 3 stains the plasma membrane and vesicles budding therefrom (arrows);
(d) ε map referring to (c) from pixel by pixel GP measurement. GP was
measured by eq. 1 by considering λ1=480ꢀ525 and λ2=540ꢀ580 nm.
As expected from its lipid chain, 3 localizes prevalently into
the plasma membrane (PM) and, with time, also in vesicles
15 budding therefrom (Fig. 2 and S6). We found <ε>=10.6±1.8 for
PM (25 CHO cells), in agreement with the value obtained from
capacitance studies in excitable cells.29 Comparison of <ε> with
the values detected for Lo and Ld in LUVs suggests intermediate
order of PM’s lipid bilayer as also reported by lifetime studies.14
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20 More,
ε maps of PM showed significant heterogeneity
characterized by clusters of similar ε values. This observation
was confirmed by the comparison of the spatial autocorrelation of
ε (spCε) along the PM (average of 25 cells) with the spCε
measured in homogeneous solutions with ε=8ꢀ11. The spCε vs.
25 distance plot (Fig. S7) showed a residual correlation up to 1 ꢀm
in cells, while in homogeneous solutions spCε disappeared at
250ꢀ300 nm (the lateral resolution of the confocal microscope).
PM clusters with similar ε may be early budding vesicles, but
further experiments are needed to clarify this interesting
30 phenomenon. Finally, cholesterol depletion of CHO PM by
methylꢀβꢀcyclodextrin led to a statistically significant decrease of
<ε> (Table S3), in keeping with the higher ε seen in cholesterolꢀ
enriched LUVs (Table 1). Thus 3 may find use to study structural
perturbations of PM induced by cholesterol depletion.30
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35
In conclusion we demonstrated the use of 1 and its
bioconjugates 2 and 3 for reporting on ε in biological specimens
such as proteins and living cells. Studies to correlate membrane ε
and protein localization are under way. This work was supported
by FIRB project RBPR05JH2P by MIUR. We acknowledge Prof.
105
467.
28 S.H. Park and C. Blackstone, EMBO Rep, 2010, 11, 515.
29 R. Glaser, Biophysics, SpringerꢀVerlag, Berlin, 2000.
30 M. Hao, S. Mukherjee and F.R. Maxfield, Proc Natl Acad Sci U S A,
2001, 98, 13072.
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