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(STED). The result is an isotropic optical
resolution (therefore the name isoSTED).
This nanoscope, which in essence can
be operated like a conventional confocal
microscope, allowed us to arbitrarily address
any plane inside the cell. We first
imaged mammalian (PtK2) cells labeled
for a subunit (Tom20) of the translocase of
the mitochondrial outer membrane (TOM)
complex. The TOM complex, residing in
the outer membrane, serves as the mitochondrial
entry gate for the vast majority
of nuclear-encoded protein precursors.
We took several optical sections, each only
40 nm thick, from a single mitochondrion.
This allowed us to visualize the distributions
of individual TOM complexes on the
mitochondrial surface (Fig. 4a) .
In the next step, we used the isoSTED
nanoscope to image the spatial relationship
of two differently labeled mitochondrial
proteins. We decided to image in
addition to Tom20 also the matrix protein
mtHsp70 (also referred to as mortalin
or GRP 75). mtHsp70 is a component
of the protein import motor located at the
matrix side of the inner membrane. Previous
biochemical evidence demonstrated
that mtHsp70 is involved in protein import
by binding to the transported precursor
proteins when they reach the matrix side
of the translocase. The intermittent association
of mtHsp70 with the import machinery
may suggest that a sizeable fraction of
the mtHsp70 pool is at any time associated
with the inner membrane of the mitochondrion.
However, we did not find a
noticeable enrichment of mtHsp70 at the
mitochondrial rim, indicating that the majority
of mtHsp70 is located in the mitochondrial
matrix (Fig 4b).
These data demonstrate that with iso-
STED nanoscopy we are able to analyze
the distribution and co-localization of proteins
within mitochondria, which would
be impossible using conventional light
microscopy due to its diffraction limited
resolution.
Seeing cristae with focused light
But is it possible at all to visualize the
folds of the inner membrane just relying
on focused light? Clearly, from a light
microscopists' point of view, the visualization
of the cristae is a major challenge
because of the pronounced convolution
of the inner membrane in a very confined
space. In fact, arguably, it is one of the most
challenging structural elements in a cell to
image with far field optical microscopy,
Fig. 5: Light microscopic analysis of the arrangement of cristae inside mitochondria of intact PtK2
cells using the isoSTED nanoscope. The mitochondrial inner membrane was labeled with antibodies
directed against an abundant protein complex of the inner membrane, the F 1F 0-ATPase. (a) Overview
and (b) close-up of an optical section of mitochondria recorded at their equatorial plane (thickness
~ 30 nm). The brackets indicate regions in which the cristae are perpendicularly oriented to the longitudinal
axis of the organelle, whereas the arrowheads point to inner-mitochondrial regions devoid
of cristae. Scale bars: 500 nm.
Abb. 5: Lichtmikroskopische Untersuchung der Anordnung der Cristae im Inneren von Mitochondrien
intakter PtK2-Zellen. Die mitochondriale Innenmembran wurde mit Antikörpern gegen die
F 1F 0-ATPase, eines häufigen Proteinkomplexes der Innenmembran, dekoriert. (a) Übersicht und
(b) Detailvergrößerung eines optischen Schnittes (Dicke ~ 30 nm) durch die Mitte eines Mitochondriums.
Die Klammern weisen auf Bereiche hin, in denen die Cristae senkrecht zur Längsachse
des Organells ausgerichtet sind, wohingegen die Pfeilspitzen auf Bereiche zeigen, in denen keine
Cristae vorkommen. Größenstandard: 500 nm.
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and its imaging remained elusive. We
solved this problem with isoSTED nanoscopy
in combination with meticulously
optimized labeling and fixation conditions
(Schmidt et al., 2009).
In general, the images revealed heterogeneous
cristae arrangements even within
a single mitochondrial tubule (Fig. 5).
Regions of stacked cristae alternated with
relatively large regions of up to ~10 5 nm 2 ,
which were devoid of cristae. These images
demonstrate that it is indeed possible to
visualize the intricate foldings of the mitochondrial
membrane with focused light.
Altogether, we have shown that the inner
membrane of mitochondria is subcompartmentalized.
The data suggest that the
functional differences of the inner boundary
membrane and the cristae membrane
are more significant than previously anticipated.
Using isoSTED nanoscopy we are
now able to visualize sub-mitochondrial
protein distributions, even individual cristae
arrangements, in intact cells. Together
with new GFP-based probes (Andresen
et al., 2008), we strongly anticipate that
these findings will facilitate the analysis
of the molecular mechanisms determining
the subcompartmentalization of the inner
membrane of mitochondria and beyond.
References:
Andresen, M., A.C. Stiel, J. Folling, D. Wenzel,
A. Schonle, A. Egner, C. Eggeling, S.W. Hell,
and S. Jakobs. 2008. Photoswitchable fluorescent
proteins enable monochromatic multi-
label imaging and dual color fluorescence nano-
scopy. Nature Biotechnol. 26:1035-1040.
Schmidt, R., C.A. Wurm, S. Jakobs, J. Engelhardt,
A. Egner, and S.W. Hell. 2008. Spherical nanosized
focal spot unravels the interior of cells.
Nature Methods. 5:539-544.
Schmidt, R., C.A. Wurm, A. Punge, A. Egner, S.
Jakobs, and S.W. Hell. 2009. Mitochondrial
cristae revealed with focused light. Nano Lett.
9:2508-2510.
Suppanz, I.E., C.A. Wurm, D. Wenzel, and S.
Jakobs. 2009. The m-AAA protease processes
cytochrome c peroxidase preferentially at
the inner boundary membrane of mitochondria.
Mol Biol Cell. 20:572-580.
Vogel, F., C. Bornhovd, W. Neupert, and A.S.
Reichert. 2006. Dynamic subcompartmentalization
of the mitochondrial inner membrane.
J Cell Biol. 175:237-247.
Wurm, C.A., and S. Jakobs. 2006. Differential
protein distributions define two subcompartments
of the mitochondrial inner membrane
in yeast. FEBS Lett. 580:5628-5634.