Live-cell STED microscopy of mitochondrial cristae

Mitochondria are highly dynamic organelles that exhibit a complex inner architecture. They exhibit a smooth outer membrane and a highly convoluted inner membrane that forms invaginations called cristae. Imaging cristae in living cells poses a formidable challenge for light microscopy. Relying on a cell line stably expressing the mitochondrial protein Cox8A fused to the SNAP-tag and using STED (stimulated emission depletion) super-resolution microscopy, we demonstrate the visualization of cristae dynamics in cultivated human cells.


Introduction
Mitochondria form tubular and highly dynamic networks in mammalian cells that constantly undergo fusion and fission events 1,2 . They are double-membrane organelles that exhibit a smooth outer membrane and a highly convoluted inner membrane. Cristae are invaginations of the inner membrane that generally adopt tubular or lamellar shapes and project into the matrix space. The cristae architecture adapts to different cellular conditions and is changed upon various processes 3,4 .
However, the actual cristae dynamics in these processes are poorly understood.
A challenge for studying cristae dynamics is the small size of mitochondria. The diameter of mitochondrial tubules is generally between 200 and 700 nm, and in many mammalian cell types the crista-to-crista distance is below 100 nm. Hence, because of the diffraction limit in optical microscopy (~250 nm, depending on the wavelength), visualization of cristae dynamics has been a notorious challenge 5 .
Electron microscopy provides the required resolution, but it is restricted to fixed samples. So far, the overall mitochondrial dynamics and the cristae movements, difficulties in labelling and concerns on light induced photo-toxicity have hampered the visualization of single cristae dynamics using live-cell nanoscopy 6,7 . In fixed cells, cristae were first visualized using isoSTED nanoscopy 8 . Using structured illumination microscopy providing a resolution of around 100 nm, mitochondrial substructures were resolved in living cells [9][10][11] . However, the discrimination between individual cristae and groups of cristae remained difficult because of the limited resolution.
We overcome this problem by using diffraction-unlimited STED super-resolution microscopy (STED nanoscopy) in combination with a genome edited human cell line that enables labeling of the cristae with a silicon rhodamine dye. Thereby, we unequivocally visualize the dynamics of individual cristae using nanoscopy.

Visualization of the cristae architecture in living cells
We have generated a human HeLa cell line that stably expresses the full length Cytochrome C Oxidase Subunit 8A (Cox8A), an integral protein of the mitochondrial inner membrane, fused to the SNAP-tag 12 . As Cox8A is a subunit of complex IV of the respiratory chain, it is expected to be preferentially localized in the crista membrane 13,14 . The fusion construct was targeted to the chromosomal AAVS1 (adeno-associated virus integration site 1) Safe Harbor Locus using a CRISPR/Cas9 based genome editing strategy, ensuring largely constant expression levels 15,16 .
Labeling of living cells by adding the cell permeant dye SNAP-Cell SiR to the medium 17 resulted in brightly fluorescent mitochondria. As expected, diffraction-limited confocal recordings did not reveal any sub-mitochondrial structures, whereas with STED microscopy (excitation: 640 nm, STED: 775 nm), we were able to record cristae in mitochondria of living HeLa cells with ~50 nm resolution ( Fig. 1). Individual cristae could be resolved, typically exhibiting a crista-to-crista distance between 70 and 90 nm, which is fully in line with electron micrographs recorded from the same cell line (Fig.1, Fig. 2A). We were able to record entire cells, thereby getting an overview on the cristae architecture of mitochondria with different shapes or with different subcellular positions ( Fig. 1).

Mitochondrial nucleoids occupy the voids between groups of cristae
The cristae frequently occurred in groups, separated by voids of several hundred nanometers size.
Labeling of the mitochondrial DNA with the cell-permeable dye PicoGreen allowed us to analyze the location of mitochondrial DNA with respect to the cristae in live mitochondria. Unexpectedly, we found that in this cell line most free spaces in the mitochondrial matrix that were devoid of cristae were occupied by mitochondrial nucleoids (Fig. 2B). We note that mitochondrial nucleoids, because of their low contrast, are invisible in conventional EM and hence the detailed localization of nucleoids has not been shown previously.

Time lapse imaging of cristae dynamics
Using our labeling strategy, image sequences with 10-20 frames could be recorded. This allowed us to capture individual cristae and their dynamics across larger fields of view. By this we could, for example, capture the dynamics of cristae apparently moving in groups during the fission of a mitochondrial tubule ( Fig. 2C; Suppl. Movie 1). Due to the flexibility of the beam-scanning STED imaging scheme, the size of the recorded fields of view can be adapted, facilitating video sequences with frame rates in the second range. Even at this temporal resolution we observed an unexpected level of cristae movements (Suppl. Movie 2 and 3).

Discussion
The intricate mitochondrial membrane architecture is vital for the functioning of mitochondria as cellular powerhouses. It is widely accepted that the mitochondrial cristae are dynamic structures that are remodeled upon various cellular stimuli, but also upon apoptosis and during ageing 4,18-20 .
The studies demonstrating cristae adaptations relied on electron microscopy that can visualize the membrane architecture of mitochondria in fixed cells. However, very little is known about the actual dynamics of these processes, as a clear visualization of the cristae structure in living cells has been a notorious challenge in the past.
The mechanisms of mitochondrial cristae biogenesis are debated and different mechanisms for this process have been suggested [21][22][23][24][25][26][27][28] . To better analyze the formation of cristae and their structure and dynamics under different cellular conditions, a new approach for high-resolution live cell imaging of mitochondrial cristae is urgently needed. We suggest that the Cox8A-SNAP cell line in combination with nanoscopy will be a valuable resource to study cristae dynamics and cristae biogenesis. We note that our images have been recorded with a commercial STED microscope which will allow also non-specialized laboratories to record cristae dynamics. The SNAP-tag allows to change the fluorophore according to the requirements of any light microscopy technique. This versatility will facilitate an immediate comparison of different fluorophores, labelling strategies and imaging modalities on a dynamic and challenging live cell sample. Moreover, the chemical fixation of the double membraned mitochondria is often difficult and we expect this cell line will be a valuable resource for evaluating and systematically improving fixation conditions for optical microscopy. The cell line will be freely available to the scientific community after publication of the manuscript in a peer-reviewed journal.

Cloning of plasmids
To generate the donor plasmid AAVS1-Blasticidin-CAG-Cox8A-SNAP, the plasmid AAVS1-Basticidin-CAG-Flpe-ERT2 was linearized by using the restriction endonucleases SalI and EcoRV. Cox8A-SNAP was amplified by PCR from pSNAPf-Cox8A (New England Biolabs, Ipswich, MA, USA) and subsequently integrated into the linearized plasmid by Gibson assembly.

Generation of a stable cell line
To generate the stable cell line, HeLa cells were co-transfected with the plasmids AAVS1-

Staining of live cells for STED nanoscopy
Cells were seeded in glass bottom dishes (ibidi GmbH, Martinsried, Germany) one day before the Generally, dwell times of 7-10 µs were used. For STED images, each line was scanned 4 to 8 times and the signal was accumulated. For the confocal images, each line was scanned once.