Mitochondrial dynamics and quantification of mitochondria-derived vesicles in cardiomyoblasts using structured illumination microscopy

Mitochondria are essential energy-providing organelles of particular importance in energy-demanding tissue such as the heart. The production of mitochondria-derived vesicles (MDVs) is a cellular mechanism by which cells ensure a healthy pool of mitochondria. These vesicles are small and fast-moving objects not easily captured by imaging. In this work, we have tested the ability of the optical super-resolution technique 3DSIM to capture high-resolution images of MDVs. We optimized the imaging conditions both for high-speed video microscopy and fixed-cell imaging and analysis. From the 3DSIM videos, we observed an abundance of MDVs and many dynamic mitochondrial tubules. The density of MDVs in cells was compared for cells under normal growth conditions and cells during metabolic perturbation. Our results indicate a higher abundance of MDVs in H9c2 cells during glucose deprivation compared with cells under normal growth conditions. Furthermore, the results reveal a large untapped potential of 3DSIM in MDV research

Structured Illumination Microscopy of Biological Structures

Abstract of presentation held at Norwegian Electro-Optics Meeting, Henningsvær, Norway, 2-4 May 2018.Resolution in optical microscopy has long been limited to the Abbe diffraction limit, i.e. about 250 nm laterally for visible wavelengths on a very good microscope. In the last two decades several techniques have been devised to circumvent this limit: an achievement which was recognized with the 2014 Nobel Prize in Chemistry. Structured Illumination Microscopy (SIM) was the first of these techniques to become commercially available, and continues to be the only super-resolution technique which is practically compatible with living cells, while also requiring the least modification to conventional sample-labeling protocols. SIM utilizes Moiré patterns and frequency shifting to improve resolution 2X in each dimension, as well as significantly improve the contrast for the mid-range spatial frequencies. These advances have unlocked a new realm of biological inquiry: the combination of the high biochemical specificity of fluorescent probes with resolution previously only possible with electron microscopy now enables the direct study of sub-organelle colocalization and the dynamics of living cells. Here, we will present both the basics of the SIM technique as well as a sampling of its biological applications from our lab at UiT, including sub-mitochondrial localization and dynamics, sieve-like nanostructures in liver cells, and large-scale visualization of super-resolved cardiac tissue sections, as well as discuss the practical limitations and implications of this work

High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts

Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy

Multifocus microscopy with optical sectioning and high axial resolution

Multifocus microscopy enables recording of entire volumes in a single camera 11 exposure. In dense samples, multifocus microscopy is severely hampered by background haze. 12 Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers 13 improved axial resolution capabilities. In our method, a dithered oblique light-sheet scans the 14 sample volume during a single exposure, while fluorescence from each illuminated plane in the 15 sample is mapped onto a line on the camera with a multifocus optical element. A synchronized 16 rolling shutter readout realizes optical sectioning. We describe the technique theoretically and 17 verify its optical sectioning and resolution improvement capabilities. We demonstrate a 18 prototype system with a multifocus beam splitter cascade and record monolayers of endothelial 19 cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue 20 and visualize the distribution of mitochondria at high axial resolution. Our method manages to 21 capture sub-diffraction sized mitochondria-derived vesicles up to 30 µm deep into the tissue

Multifocus microscopy with optically sectioned axial superresolution

Multifocus microscopy enables recording of entire volumes in a single camera exposure. In dense samples, multifocus microscopy is severely hampered by background haze. Here, we introduce a scalable multifocus method that incorporates optical sectioning and offers axial superresolution capabilities. In our method, a dithered oblique light-sheet scans the sample volume during a single exposure, while generated fluorescence is linearised onto the camera with a multifocus optical element. A synchronised rolling shutter readout realised optical sectioning. We describe the technique theoretically and verify its optical sectioning and superresolution capabilities. We demonstrate a prototype system with a multifocus beam splitter cascade and record monolayers of endothelial cells at 35 volumes per second. We furthermore image uncleared engineered human heart tissue and visualise the distribution of mitochondria at axial superresolution. Our method manages to capture sub-diffraction sized mitochondria-derived vesicles up to 30 um deep into the tissue

The ability to form full-length intron RNA circles is a general property of nuclear group I introns

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3′ splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5′ splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility

Three-dimensional structured illumination microscopy data of mitochondria and lysosomes in cardiomyoblasts under normal and galactose-adapted conditions

This three-dimensional structured illumination microscopy (3DSIM) dataset was generated to highlight the suitability of 3DSIM to investigate mitochondria-derived vesicles (MDVs) in H9c2 cardiomyoblasts in living or fxed cells. MDVs act as a mitochondria quality control mechanism. The cells were stably expressing the tandem-tag eGFP-mCherry-OMP25-TM (outer mitochondrial membrane) which can be used as a sensor for acidity. A part of the dataset is showing correlative imaging of lysosomes labeled using LysoTracker in fxed and living cells. The cells were cultivated in either normal or glucose-deprived medium containing galactose. The resulting 3DSIM data were of high quality and can be used to undertake a variety of studies. Interestingly, many dynamic tubules derived from mitochondria are visible in the 3DSIM videos under both glucose and galactose-adapted growth conditions. As the raw 3DSIM data, optical parameters, and reconstructed 3DSIM images are provided, the data is especially suitable for use in the development of SIM reconstruction algorithms, bioimage analysis methods, and for biological studies of mitochondria