Fusión mitocondrial se midió mediante el seguimiento de la equilibración de photoconverted matriz orientada GFP través de la red mitocondrial en el tiempo. Hasta ahora, sólo una célula podría ser sometido a un análisis cinético hora larga a la vez. Se presenta un método que mide simultáneamente varias celdas, y acelera así el proceso de recopilación de datos.
Mitochondrial fusion plays an essential role in mitochondrial calcium homeostasis, bioenergetics, autophagy and quality control. Fusion is quantified in living cells by photo-conversion of matrix targeted photoactivatable GFP (mtPAGFP) in a subset of mitochondria. The rate at which the photoconverted molecules equilibrate across the entire mitochondrial population is used as a measure of fusion activity. Thus far measurements were performed using a single cell time lapse approach, quantifying the equilibration in one cell over an hour. Here, we scale up and automate a previously published live cell method based on using mtPAGFP and a low concentration of TMRE (15 nm). This method involves photoactivating a small portion of the mitochondrial network, collecting highly resolved stacks of confocal sections every 15 min for 1 hour, and quantifying the change in signal intensity. Depending on several factors such as ease of finding PAGFP expressing cells, and the signal of the photoactivated regions, it is possible to collect around 10 cells within the 15 min intervals. This provides a significant improvement in the time efficiency of this assay while maintaining the highly resolved subcellular quantification as well as the kinetic parameters necessary to capture the detail of mitochondrial behavior in its native cytoarchitectural environment.
Mitochondrial dynamics play a role in many cellular processes including respiration, calcium regulation, and apoptosis1,2,3,13. The structure of the mitochondrial network affects the function of mitochondria, and the way they interact with the rest of the cell. Undergoing constant division and fusion, mitochondrial networks attain various shapes ranging from highly fused networks, to being more fragmented. Interestingly, Alzheimer’s disease, Parkinson’s disease, Charcot Marie Tooth 2A, and dominant optic atrophy have been correlated with altered mitochondrial morphology, namely fragmented networks4,10,13. Often times, upon fragmentation, mitochondria become depolarized, and upon accumulation this leads to impaired cell function18. Mitochondrial fission has been shown to signal a cell to progress toward apoptosis. It can also provide a mechanism by which to separate depolarized and inactive mitochondria to keep the bulk of the network robust14. Fusion of mitochondria, on the other hand, leads to sharing of matrix proteins, solutes, mtDNA and the electrochemical gradient, and also seems to prevent progression to apoptosis9. How fission and fusion of mitochondria affects cell homeostasis and ultimately the functioning of the organism needs further understanding, and therefore the continuous development and optimization of how to gather information on these phenomena is necessary.
Existing mitochondrial fusion assays have revealed various insights into mitochondrial physiology, each having its own advantages. The hybrid PEG fusion assay7, mixes two populations of differently labeled cells (mtRFP and mtYFP), and analyzes the amount of mixing and colocalization of fluorophores in fused, multinucleated, cells. Although this method has yielded valuable information, not all cell types can fuse, and the conditions under which fusion is stimulated involves the use of toxic drugs that likely affect the normal fusion process. More recently, a cell free technique has been devised, using isolated mitochondria to observe fusion events based on a luciferase assay1,5. Two human cell lines are targeted with either the amino or a carboxy terminal part of Renilla luciferase along with a leucine zipper to ensure dimerization upon mixing. Mitochondria are isolated from each cell line, and fused. The fusion reaction can occur without the cytosol under physiological conditions in the presence of energy, appropriate temperature and inner mitochondrial membrane potential. Interestingly, the cytosol was found to modulate the extent of fusion, demonstrating that cell signaling regulates the fusion process 4,5. This assay will be very useful for high throughput screening to identify components of the fusion machinery and also pharmacological compounds that may affect mitochondrial dynamics. However, more detailed whole cell mitochondrial assays will be needed to complement this in vitro assay to observe these events within a cellular environment.
A technique for monitoring whole-cell mitochondrial dynamics has been in use for some time and is based on a mitochondrially-targeted photoactivatable GFP (mtPAGFP)6,11. Upon expression of the mtPAGFP, a small portion of the mitochondrial network is photoactivated (10-20%), and the spread of the signal to the rest of the mitochondrial network is recorded every 15 minutes for 1 hour using time lapse confocal imaging. Each fusion event leads to a dilution of signal intensity, enabling quantification of the fusion rate. Although fusion and fission are continuously occurring in cells, this technique only monitors fusion as fission does not lead to a dilution of the PAGFP signal6. Co-labeling with low levels of TMRE (7-15 nM in INS1 cells) allows quantification of the membrane potential of mitochondria. When mitochondria are hyperpolarized they uptake more TMRE, and when they depolarize they lose the TMRE dye. Mitochondria that depolarize no longer have a sufficient membrane potential and tend not to fuse as efficiently if at all. Therefore, active fusing mitochondria can be tracked with these low levels of TMRE9,15. Accumulation of depolarized mitochondria that lack a TMRE signal may be a sign of phototoxicity or cell death. Higher concentrations of TMRE render mitochondria very sensitive to laser light, and therefore great care must be taken to avoid overlabeling with TMRE. If the effect of depolarization of mitochondria is the topic of interest, a technique using slightly higher levels of TMRE and more intense laser light can be used to depolarize mitochondria in a controlled fashion (Mitra and Lippincott-Schwartz, 2010). To ensure that toxicity due to TMRE is not an issue, we suggest exposing loaded cells (3-15 nM TMRE) to the imaging parameters that will be used in the assay (perhaps 7 stacks of 6 optical sections in a row), and assessing cell health after 2 hours. If the mitochondria appear too fragmented and cells are dying, other mitochondrial markers, such as dsRED or Mitotracker red could be used instead of TMRE.
The mtPAGFP method has revealed details about mitochondrial network behavior that could not be visualized using other methods. For example, we now know that mitochondrial fusion can be full or transient, where matrix content can mix without changing the overall network morphology. Additionally, we know that the probability of fusion is independent of contact duration and organelle dimension, is influenced by organelle motility, membrane potential and history of previous fusion activity8,15,16,17.
In this manuscript, we describe a methodology for scaling up the previously published protocol using mtPAGFP and 15nM TMRE8 in order to examine multiple cells at a time and improve the time efficiency of data collection without sacrificing the subcellular resolution. This has been made possible by the use of an automated microscope stage, and programmable image acquisition software. Zen software from Zeiss allows the user to mark and track several designated cells expressing mtPAGFP. Each of these cells can be photoactivated in a particular region of interest, and stacks of confocal slices can be monitored for mtPAGFP signal as well as TMRE at specified intervals. Other confocal systems could be used to perform this protocol provided there is an automated stage that is programmable, an incubator with CO2, and a means by which to photoactivate the PAGFP; either a multiphoton laser, or a 405 nm diode laser.
Este método permite la formación de imágenes de alrededor de 10 células a la vez, si la adquisición se produce cada 15 minutos después de la fotoactivación. El número exacto de células dependerá de lo rápido que uno es capaz de localizar y marcar las células que expresan mtPAGFP dentro de la placa de cultivo, y la rapidez con que se puede configurar todos los parámetros del software. Para realizar la automatización sin problemas una capa uniforme de las células deben ser utilizados porque los designados z-stack márgenes se aplicará para todas las células.
El tamaño de esta zona fotoactivación inicial regirá el tiempo de equilibrado. Para ser capaz de medir la fusión mitocondrial, es importante para fotoactivar sólo 10-20% de la red, de tal manera que la propagación de la señal al resto de la red se puede controlar con el tiempo. Si es demasiado buena parte de la red se fotoactivados, es posible que la fusión completa se producen con demasiada rapidez, y el evento no será capturado.
El cuidado extremo se debe tomar paraajustar la potencia del láser del láser de dos fotones, así como la concentración TMRE para evitar fototoxicidad que conduce a la despolarización mitocondrial. Asegurarse de que la señal de mtPAGFP colocalizes con la señal de TMRE puede ayudar en la evaluación de fototoxicidad y la salud general de las células 8,15. La iluminación con luz epifluoerescent debe ser evitado. Si bien la búsqueda de células que expresan mtPAGFP, el agujero debe ser lo más abierto durante la exploración con una excitación de 488 nm de bajo poder. Ajuste de la potencia del láser de dos fotones para fotoactivar suficiente PA-GFP para medir la señal durante 1 hora, pero no a sobresaturar cualquiera de las celdas puede ser complicado 8. Sin embargo, el tiempo debe ser gastado en esta etapa debido a la optimización de programa automático se inicia, es tedioso para detenerlo, para elegir más células, y curriculum vitae.
Para el control de la calidad, la adquisición de un contraste de interferencia diferencial (DIC) de la imagen (o de luz transmitida) para controlar el foco en las células puede ser muyútil y también una buena manera de detectar las burbujas formadas en el aceite de inmersión durante la exploración, lo que ocurre a veces de los movimientos de la etapa.
Aunque el uso de este método mtPAGFP recoge datos sobre el movimiento unidireccional de proteínas de la matriz mitocondrial de las mitocondrias fotoactivados a los que no están etiquetados, es posible utilizar esta técnica para estudiar otros procesos. Por ejemplo, fluorocromos específicos se puede unir a las proteínas de membrana para observar su movimiento específico durante los eventos de fusión, como se ha demostrado para ABC-me, la fusión de la que se produce en una escala de tiempo diferente de la mezcla de proteínas de la matriz solubles 15.
The authors have nothing to disclose.
Damos las gracias al Centro Tufts para Investigación de Neurociencia, P30 NS047243 (Jackson), la afinidad de las mitocondrias de Investigación Cooperativa (mtARC) apoyado por el Centro para la Investigación Biomédica Evans Interdisciplinario de la Universidad de Boston Medical Campus, Link Corporation Medicina, y Zeiss para apoyar este trabajo.
Name of the reagent | Company | Catalogue number |
COXIII-adenoviral PA-GFP | Dr. Lippincott-Schwartz | |
TMRE | Invitrogen | T669 |
Zeiss LSM 710 confocal | Zeiss |