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diffraction-limited resolution of fluorescence
microscopy of around 250 nm.
Hence conventional light microscopy
cannot be used to study sub-mitochondrial
protein distributions in wild-type cells.
Nonetheless, recently two studies using
the budding yeast Saccharomyces cerevisiae
as a model organism demonstrated
conclusively that the IBM and the CM
have different protein compositions.
Using live cell fluorescence microscopy
in conjunction with genetically enlarged
mitochondria (a detour to circumvent the
problems associated with the diffraction
barrier), we (Wurm and Jakobs, 2006)
and Andreas Reichert and colleagues
with quantitative immunogold electron
microscopy (Vogel et al., 2006) demonstrated
different protein compositions
of the IBM and the CM. However, these
initial studies primarily concentrated on
proteins involved in protein import and
oxidative phosphorylation. Hence we
next focused on other cellular processes
to analyze differences in the functions of
both domains of the inner membrane.
The m-AAA protease processes the cytochrome
c peroxidase preferentially at
the IBM
Many proteins of the inner membrane
assemble into large protein complexes.
Such assembly processes require extensive
quality control mechanisms to avoid
the accumulation of deleterious malfolded
or misassembled proteins. We determined
Fig. 4: isoSTED nanoscopy enables the visualization of protein distributions
within mitochondria of intact mammalian cells. (a) Distribution
of the TOM complex. Shown is a confocal image (at the
sides) and the corresponding isoSTED image (middle) taken at the
center of the mitochondria of a PtK2 cell. The cell was decorated
with antibodies against Tom20. The isoSTED image reveals that the
TOM complexes line the envelope of the organelles (b) Two-color
isoSTED imaging. The cells were decorated with antisera against
Tom20 and mtHsp70. The proteins have distinct distributions. Displayed
is an optical section (thickness ~50 nm) at the equatorial
planes of the mitochondria. Scale bars, 500 nm (a) and 1 µm (b).
Abb. 4: isoSTED-Nanoskopie ermöglicht die Abbildung von Proteinverteilungen
innerhalb der Mitochondrien von Säugerzellen. (a)
Verteilung des TOM-Komplexes. Gezeigt ist eine konfokale Abbildung
(an den Seiten) und die entsprechende isoSTED-Abbildung (in
der Mitte) der Mitochondrien einer PtK2-Zelle. Die Zelle wurde mit
Antikörpern gegen das Protein Tom20 markiert. Das isoSTED-Bild
zeigt, dass die TOM-Komplexe in der äußeren Membran aufgereiht
sind. (b) Zweifarben-isoSTED-Nanoskopie. Die Zellen wurden mit
Antikörpern gegen Tom20 und mtHsp70 markiert. Die isoSTED-
Bilder belegen, dass beide Proteine unterschiedliche Verteilungen
haben. Abgebildet ist ein optischer Schnitt (Dicke ~ 50 nm) in der
Mitte der Mitochondrien. Größenstandards, 500 nm (a) und 1 µm (b).
the inner membrane distribution of a central
component of the proteolytic control
system, namely the m-AAA protease,
and its substrate cytochrome c peroxidase
(Ccp1) within budding yeast mitochondria
(Suppanz et al., 2009). For this study, we
used the approaches mentioned above,
namely fluorescence microscopy in conjunction
with genetically enlarged mitochondria
and quantitative immunogold
electron microscopy.
The m-AAA protease is a hetero-oligomeric
complex composed of the homologous
subunits AFG3L2 and paraplegin in
humans and Yta10 (Afg3) and Yta12 (Rca1)
in yeast. We found that both subunits of
the m-AAA protease are preferentially,
but not exclusively, localized in the IBM
(Fig. 3a). Likewise, the membrane-anchored
precursor form of Ccp1 accumulates in the
IBM of mitochondria lacking a functional
m-AAA protease (Fig. 3b, top). Only upon
proteolytic cleavage the mature form
mCcp1 moves into the cristae space
(Fig. 3b, bottom).
The preferential localization of the
m-AAA protease in the IBM points to the
intriguing idea that the insertion, assembly,
and quality control of the proteins of
the mitochondrial inner membrane may
predominantly take place in the IBM,
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suggesting that the functional differences
of the IBM and the CM are more significant
than previously anticipated.
Concomitantly this study clearly demonstrated
the limitations of the utilized
methods for analyzing submitochondrial
structures, most notably the time-consuming
and painstaking sample preparation
for electron microscopy, the difficulties
when using two labels and the lack of a
possibility to image submitochondrial dynamics.
Many of these problems would be
alleviated by light microscopy; however,
wild-type mitochondria are too small,
even for the best conventional fluorescence
microscopes.
Resolving inner-mitochondrial protein
distributions using isoSTED nanoscopy
To facilitate a high (diffraction-unlimited)
optical resolution in all three room
dimensions at the same time, recently an
isoSTED fluorescence nanoscope enabling
a nearly spherical focal spot of 40-45 nm
(l/16) in diameter was introduced (Schmidt
et al., 2008). In the isoSTED nanoscope,
the effective focus is produced by overlapping
the excitation spot of two opposing
oil immersion lenses with a hollow
sphere of light for switching off the fluorescence
by Stimulated Emission Depletion