Angewandte
Chemie
DOI: 10.1002/anie.201403349
Visualizing the Golgi Apparatus
Hot Paper
Super-Resolution Imaging of the Golgi in Live Cells with
a Bioorthogonal Ceramide Probe**
Roman S. Erdmann, Hideo Takakura, Alexander D. Thompson, Felix Rivera-Molina,
Edward S. Allgeyer, Joerg Bewersdorf, Derek Toomre,* and Alanna Schepartz*
Abstract: We report a lipid-based strategy to visualize Golgi
structure and dynamics at super-resolution in live cells. The
method is based on two novel reagents: a trans-cyclooctene-
containing ceramide lipid (Cer-TCO) and a highly reactive,
tetrazine-tagged near-IR dye (SiR-Tz). These reagents assem-
ble via an extremely rapid “tetrazine-click” reaction into Cer-
SiR, a highly photostable “vital dye” that enables prolonged
live-cell imaging of the Golgi apparatus by 3D confocal and
STED microscopy. Cer-SiR is nontoxic at concentrations as
high as 2 mm and does not perturb the mobility of Golgi-
resident enzymes or the traffic of cargo from the endoplasmic
reticulum through the Golgi and to the plasma membrane.
widely used to label the Golgi, but they bleach too rapidly for
prolonged imaging or super-resolution methods. Photostable
dyes typically used for stimulated emission depletion (STED)
microscopy, such as Atto 647N and STAR 635,[8] suffer from
nonspecific binding[9] and lack of cell permeability and are ill
suited for live-cell STED imaging of intracellular structures.
Herein we report a strategy to visualize Golgi structure
and dynamics at super-resolution in live cells using a novel
lipid-based fluorescent probe as a contrast agent. The labeling
logic is based on two novel reagents: a trans-cyclooctene-
containing ceramide lipid (Cer-TCO) and a highly reactive,
tetrazine-tagged, near-IR dye (SiR-Tz). These reagents
rapidly assemble via a “tetrazine-click” reaction[10] into Cer-
SiR, a nontoxic “vital dye” whose extreme photostability
enables prolonged live-cell imaging by 3D confocal and
STED microscopy (Figure 1). Cer-TCO was synthesized in six
chemical steps; SiR-Tz was synthesized via a route modeled
Super-resolution “nanoscopes” greatly increase the resolv-
ing power of light microscopes, revealing new details of
organelle structure, function, and dynamics.[1] However, the
complex requirements for nanoscopy pose real challenges for
fluorophore design and labeling: the fluorophore must be
bright, photostable, and live-cell-compatible, and the labeling
must yield a high fluorophore density that is benign to
organelle function. As nanoscopes push the resolution to tens
of nanometers[2] there is a critical need for high-density yet
photostable probes[3] to demark organelle boundaries and
study their dynamics.[4] While most nanoscopes image labeled
proteins,[5] lipids are a complementary attractive target,[6] as
they are present at approximately a hundredfold higher
density and their organization defines the boundary of the
organelle. Commercial fluorescent lipids, such as BODI-
PYFL C5-ceramide (BODIPY-Cer)[7] are cell permeable, and
[*] Dr. R. S. Erdmann,[+] A. D. Thompson, Prof. Dr. A. Schepartz
Department of Chemistry, Yale University
225 Prospect Street, New Haven CT 06511 (USA)
E-mail: alanna.schepartz@yale.edu
Dr. R. S. Erdmann,[+] Dr. H. Takakura,[+] Dr. E. S. Allgeyer,
Prof. Dr. J. Bewersdorf, Prof. Dr. D. Toomre
Department of Cell Biology
Yale University School of Medicine
333 Cedar Street, New Haven, CT 06520 (USA)
E-mail: derek.toomre@yale.edu
[+] R.S.E. and H.T. contributed equally to this work.
[**] This study was supported by the Wellcome Trust Foundation and by
the National Institutes of Health (GM83257 to A.D.T. and A.S.).
R.S.E. was supported by a postdoctoral fellowship from the Swiss
National Science Foundation. H.T. is supported by a JSPS post-
doctoral fellowship for research abroad. We are grateful to Kai
Johnsson (EPFL) for a generous gift of SiR-OH and SiR-BG and to
Brian Storrie (UAMS) for the GalNAcT2-GFP cell line.
Figure 1. Two-step procedure for high-density labeling of the Golgi in
live cells. Cells are treated first with Cer-TCO, a trans-cyclooctene-
containing ceramide lipid, and then reacted with SiR-Tz, a tetrazine
derivative of a highly photostable silicon rhodamine dye. The product
of this reaction, Cer-SiR (only one isomer shown), allows extensive
live-cell imaging by 3D confocal and STED super-resolution micros-
copy.
Supporting information for this article is available on the WWW
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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