This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.
While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.
Astrocytes are critical players in the maintenance of brain homeostasis. They are perhaps most well known to have significant structural roles in the brain, as part of the blood-brain barrier1 and by supporting neurons and synapses throughout the brain2. Astrocyte support of neurons is both structural3 and metabolic4,5, with astrocytes promoting neurogenesis and synaptogenesis while also providing key metabolites like lactate to active neurons4,6,7. Beyond the role of structural support, astrocytes are active cells that take part in Ca2+ signaling and buffering (including spontaneous mitochondrial Ca2+ influxes)8,9, K+ buffering10, and can adapt and react to the needs of the brain in times of injury11,12. Being such dynamic cells, astrocytes have robust energy requirements, which necessitate an efficient mitochondrial network. These mitochondria also have a crucial role in buffering excessive reactive oxygen species (ROS)13. In addition to their individual or local roles of energy generation and ROS buffering, mitochondria function as a network14. In this sense, they maintain equilibrium between fissioning and fusioning mitochondria, representing new/reduced mitochondria and older/oxidized mitochondria, respectively15. The overall redox state of a cell can be gauged by the redox state of the mitochondrial network. In pathology, this is a critical piece of information that can shed light on which cells may not be functioning optimally.
In recent years, many sensors have been developed to study the dynamics and functions of mitochondria in cells. For example, sensors measuring energy exchange (ATP), redox state (NADH/NAD+, ROS), and enzymatic functionality (cAMP, Ca2+, Zn2+) are currently used in the study of mitochondrial function16. Among them, MitoTimer permits to follow the changes in mitochondrial morphology (size, shape, surface area), mobility (speed, displacement), and dynamics (fusioning and fissioning events), as well as the overall mitochondrial turnover rate and redox state. MitoTimer is a mutant red fluorescent protein, drFP58317, with a mitochondrial signal from subunit VIII of human cytochrome c oxidase18,19 to visualize newly synthesized mitochondria in green (488 nm) and oxidized mitochondria in red (555 nm). Using the green (488 nm) and red (555 nm) fluorescence ratio permits simultaneous evaluation of individual mitochondria, their morphology analysis, fusion/fission events, and redox state history20,21. This unique property can be used to investigate many scientific questions regarding mitochondria's physiological and pathological roles and is therefore very promising for unveiling the underlying mechanisms of mitochondrial dynamics within many different cell types.
We recently developed a new lentiviral vector (LV-G1-MitoTimer-MiR124T, hereafter called LV-G1-MitoTimer) to study mitochondria's dynamic and functions specifically in astrocytes in vitro and in vivo22. LV-G1-MitoTimer uses a truncated version of the glial fibrillary acidic protein (GFAP) promoter gfaABC1D, with a B3 enhancer (gfaABC1D(B3), hereafter called G1) combined with the previously described miR124T neuronal detargeting system23. It allows exclusive expression of the mitochondrial biosensor in astrocytes in vitro and in vivo22. Presented here are the different steps to perform a culture of rat hippocampal astrocytes and equip them with the LV-G1-MitoTimer biosensor, as well as the different microscopy steps to follow the behavior of astrocyte mitochondria during several consecutive hours/days.
Here, a novel method to longitudinally follow the dynamics and turnover of the mitochondrial system in a cultured astrocyte is proposed. Unlike a time-lapse approach on a fixed group of cells or one individual cell at a time (most often used in the literature)24,25, researchers can follow the evolution of the mitochondrial system during several days on the same individual cells. In contrast to single well live imaging where high levels of light exposure are required, and the selection of many individual cells is less feasible, the proposed method takes advantage of this microscope's ability to image several different cells in different areas of a well and to come back to those same cells at various time points to re-image them. Thanks to normalization to a baseline carried out for each measured criterion on each cell of interest, it takes the mitochondrial system's complexity into account and investigates the effect of treatment on each cell relative to its own baseline image. The microscope's ability to autonomously carry out this type of imaging on up to 16 wells at a time (imaging 5 cells per well) allows for the heterogeneity of the mitochondrial system to be properly taken into account during analysis without the experimental variability that comes with imaging various conditions on different days.
The quality of the cultures, the levels of viral infections expressing the LV-G1-MitoTimer biosensor, the type of microscope and objectives, and the selection of suitable cells are critical variables that must remain as consistent as possible in this protocol. The cell densities, the type of vector, and the viral titers can be adapted according to the question. Although previous work shows that LV-G1-MitoTimer expression has no deleterious consequences for mitochondrial function and dynamics21,22,26,27, it is essential to verify that the concentration is not toxic for the cells (for example, checking the total number of cells in control well). As a single focal plane is used, astrocytes should be: (1) as flat as possible, (2) isolated from other labeled cells (to simplify the analysis in case of displacement in the dish), and (3) possessing high fluorescence levels. As cells in culture can be highly variable in morphology, the mitochondrial system can be highly heterogeneous. In this context, analyzing ROIs (and not the whole cell) compensates for some problematic regions, such as the perinuclear regions, and decreases variability. It is essential to do the baseline on relatively similar cells and sample as many cells as possible. Consequently, high content acquisition and analysis microscopes are ideally suited. During this longitudinal monitoring, it is also important not to overexpose the cells to light to avoid biosensor bleaching.
This imaging method is not without its complexities, and throughout the protocol, several notes are included, which take into account troubleshooting done during previous tests with the microscope. For example, the choice of plate coating used depends on the intended assay, but recommendations for the most suitable choices for astrocyte primary cultures have been included. Additionally, image acquisition should be performed on at least 5 cells per condition due to intercellular variability. More specifically, some cells selected at baseline imaging will die, some will move out of the frame of the assigned image acquisition area, and some will change their morphology, making the mitochondria very difficult to individualize in analysis. Imaging many cells from the beginning increase the likelihood of a large enough sample size of cells to analyze at the end of the experiment. In addition to the more complex aspects of this imaging technique, there are some outright limitations as far as who can benefit from this type of imaging and analysis. In order to take full advantage of the automatization of image acquisition, the microscope used must have an autofocus system that can handle the speed of time intervals between images (i.e., every 3 s in this protocol) and can consistently focus on the cell in question before each image is taken. Additionally, without the JOBS software, which automates the entire image acquisition process, this method becomes arduous and potentially impossible depending on the number of cells being imaged as it would require manually finding and imaging each cell again at the appropriate time point. Finally, this imaging method is not immune to the issue of photobleaching. For this reason, as with any long-term acquisition method, it is important to choose fluorescent markers that are less susceptible to photobleaching and to tailor image acquisition to avoid this issue as much as possible.
This technique differs from others currently used in a crucial way. Unlike other time-lapse studies, this technique does not require imaging on the same position in the well the entire time, nor does it require manual movement of the plate to image other areas. This allows researchers the ability to image many cells in many conditions in one 24 h timeframe. Consequently, the ability to carry out this imaging and analysis on many cells in each well gives the same population information one would obtain from broadly studying a large group of cells while additionally providing specific measures from each cell imaged. While some specificities to this method may not apply to other image acquisition methods (outlined above), the benefits outweigh the complications with the type of analysis possible after acquisition. This technique allows researchers to see the exact ramifications of various treatments on the mitochondrial system, and consequently, on the cultured astrocytes.
Additionally, this method is highly customizable to many different scientific questions regarding mitochondrial behavior and roles in specific contexts. Here the outlined protocol deals specifically with cultured astrocytes. However, many other cell types can be used, and the treatments that can be tested are limited only by the questions being investigated. This type of imaging has the potential to advance the collective knowledge and understanding of mitochondrial behavior, the underlying mechanisms that lead to mitochondrial dysfunction, and the effects of many pathologies on the innate dynamics present in different types of cells.
The authors have nothing to disclose.
This study was supported by a Synapsis Foundation fellowship awarded to K.R. and the Lausanne University Hospital (CHUV). The authors thank Nikon for their help, in particular J. Gannevat.
µ-Slide 8 Well | IBIDI | 80807 | |
19 G needle | Plexus SANTE | PL001213 | |
21 G needle | Plexus SANTE | PL000142 | |
25 G needle | Plexus SANTE | PL000133 | |
Bovin Serum Albumin | LIFE TECH | 15260037 | |
Camera | HAMAMATSU | ORCA-flash4.0 V3 – C13440-20CU | |
DMEM, high glucose, GlutaMAX(TM) | THERMOFISHER | 61965059 | |
Glutamax Supplement | THERMOFISHER | 35050061 | |
Horse Serum | SIGMA | 16050122 | |
Lens | Nikon Instruments | CFI Plan Fluor 100x Oil | |
Light Engines | LUMENCOR | SPECTRA X | |
Linear-encoded motorized platine | Nikon Instruments | N/A | |
Microscope | Nikon Instruments | ECLIPSE Ti2-E MICROSCOPE INVERSE | |
Microscope Stage Incubator with 3-channel manual gas mixer and gas bubbler/ humidity module | OKOLAB | H201-NIKON-TI-S-ER | |
PBS 1x liquid | THERMOFISHER | 20012068 | |
Penicillin-Streptomycin | SIGMA | 15140122 | |
Petri dishes 100 mm | SIGMA | P5731 | |
Petri dishes 35 mm | SIGMA | CLS430165 | |
Pregnant Rats | CHARLES RIVERS | 3 | |
Software Nikon NIS-HC | Nikon Instruments | NIS-Elements HC | |
Sofware Prism | GraphPad | V8.02 | |
Stericup 500 mL | MERCK MILLIPORE | 10412701 |