एक तेज़, उच्च संकल्प, एमटीपीए GFP-आधारित Mitochondrial फ्यूजन में समानांतर Confocal माइक्रोस्कोपी का उपयोग एकाधिक कक्षों की काइनेटिक डेटा हासिल परख
Summary
Mitochondrial संलयन मैट्रिक्स – लक्षित समय के साथ mitochondrial नेटवर्क के भर photoconverted GFP के संतुलन पर नज़र रखने के द्वारा मापा गया था. इस प्रकार अब तक, केवल एक कक्ष एक समय में एक घंटे तक गतिज विश्लेषण किया जा सकता है. हम एक तरीका है कि एक साथ एकाधिक कक्षों के उपाय है, जिससे डेटा संग्रह प्रक्रिया तेजी से प्रस्तुत करते हैं.
Abstract
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.
Protocol
Discussion
यह विधि एक समय में 10 के आसपास कोशिकाओं की इमेजिंग के लिए अनुमति देता है, अगर अधिग्रहण photoactivation के बाद हर 15 मिनट होता है. कक्षों की सही संख्या पर निर्भर करता है कि कैसे जल्दी से एक को खोजने और संस्कृति पकवान के भीतर mtPAGFP व्यक्त कोशिकाओं, और कैसे जल्दी से एक सॉफ्टवेयर के सभी मानकों सेट कर सकते हैं को चिह्नित करने में सक्षम है. बनाने स्वचालन सुचारू रूप से चलाने कोशिकाओं की एक परत भी क्योंकि नामित Z-ढेर हाशिये सभी कक्षों के लिए लागू होगी किया जाना चाहिए.
आकार इस प्रारंभिक photoactivation के क्षेत्र के संतुलन को नियंत्रित करेंगे. Mitochondrial संलयन मापने के लिए सक्षम होना करने के लिए, यह महत्वपूर्ण है करने के लिए नेटवर्क का केवल 10-20% photoactivate, ऐसी है कि नेटवर्क के आराम करने के लिए संकेत के प्रसार को समय पर नजर रखी जा सकती है. यदि नेटवर्क की भी बहुत photoactivated है, यह संभव है कि पूरा संलयन भी जल्दी हो जाएगा, और घटना पर कब्जा कर लिया नहीं किया जाएगा.
चरम देखभाल के लिए लिया जाना चाहिएदो photon लेजर लेजर शक्ति के रूप में के रूप में अच्छी तरह से TMRE phototoxicity जो mitochondrial विध्रुवण की ओर जाता है से बचने की एकाग्रता समायोजित. यह सुनिश्चित करना कि mtPAGFP संकेत TMRE संकेत के साथ colocalizes की phototoxicity और सामान्य सेल 8,15 स्वास्थ्य का आकलन करने में मदद कर सकते हैं. Epifluoerescent प्रकाश के साथ रोशनी से बचा जाना चाहिए. जबकि mtPAGFP व्यक्त कोशिकाओं के लिए खोज, pinhole ज़्यादा से ज़्यादा खुला होना चाहिए, जबकि एक कम बिजली की 488nm उत्तेजना के साथ स्कैनिंग. दो photon लेजर शक्ति का समायोजन करने के लिए फिलीस्तीनी अथॉरिटी GFP 1 घंटे से अधिक समय संकेत को मापने के लिए, लेकिन नहीं oversaturate कक्षों की किसी भी मुश्किल हो आठ कर सकते हैं पर्याप्त photoactivate. बहरहाल, समय के इस अनुकूलन कदम में खर्च किया जाना चाहिए क्योंकि एक बार स्वचालित कार्यक्रम शुरू कर दिया है, यह इसे रोकने के लिए, अधिक कक्षों का चयन करें, और फिर से शुरू करने के लिए कठिन है.
गुणवत्ता नियंत्रण के लिए एक अंतर हस्तक्षेप विपरीत (डीआईसी) के अधिग्रहण छवि (या प्रेषित प्रकाश) कोशिकाओं पर ध्यान केंद्रित करने की निगरानी बहुत हो सकता हैऔर भी स्कैनिंग के दौरान विसर्जन के तेल में गठन बुलबुले का पता लगाने के लिए एक अच्छा तरीका उपयोगी, इस मंच के आंदोलनों से कभी कभी होता है.
हालांकि इस mtPAGFP पद्धति का उपयोग करके photoactivated उन है कि लेबल नहीं हैं को mitochondria से mitochondrial मैट्रिक्स प्रोटीन की यूनिडायरेक्शनल आंदोलन पर डेटा इकट्ठा, यह अन्य प्रक्रियाओं का अध्ययन करने के लिए इस तकनीक का उपयोग करने के लिए बोधगम्य है. उदाहरण के लिए, विशिष्ट fluorochromes झिल्ली प्रोटीन करने के लिए संलग्न किया जा सकता है संलयन की घटनाओं के दौरान अपने विशिष्ट आंदोलन का पालन करने के लिए, के रूप में एबीसी मुझे, जो घुलनशील मैट्रिक्स प्रोटीन का मिश्रण 15 से एक अलग समय के पैमाने पर होता है संलयन के लिए दिखाया गया है.
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम तंत्रिका विज्ञान अनुसंधान के लिए Tufts केंद्र NS047243 p30 (जैक्सन), Mitochondria आत्मीयता रिसर्च सहयोगात्मक (mtARC) के अंतःविषय जैव चिकित्सा अनुसंधान के लिए बोस्टन यूनिवर्सिटी मेडिकल कैम्पस, लिंक चिकित्सा निगम में इवांस केंद्र द्वारा समर्थित है, और इस काम का समर्थन करने के लिए Zeiss धन्यवाद.
Materials
| Name of the reagent | Company | Catalogue number |
| COXIII-adenoviral PA-GFP | Dr. Lippincott-Schwartz | |
| TMRE | Invitrogen | T669 |
| Zeiss LSM 710 confocal | Zeiss |
References
- Braschi, E., McBride, H. M. Mitochondria and the culture of the Borg: understanding the integration of mitochondrial function within the reticulum, the cell, and the organism. Bioessays. 32, 958-966 (2010).
- Chan, D. C. Mitochondria: dynamic organelles in disease, aging, and development. Cell. 125, 1241-1252 (2006).
- Chan, D. C., Detmer, S. A. Functions and dysfunctions of mitochondrial dynamics. Nature Reviews Molecular Cell Biology. 8, 870-879 (2007).
- Han, X. J., Tomizawa, K., Fujimura, A., Ohmori, I., Nishiki, T., Matsushita, M., Matsui, H. Regulation of mitochondrial dynamics and neurodegenerative diseases. Acta Med Okayama. 65, 1-10 (2011).
- Huang, H., Choi, S. Y., Frohman, M. A. A quantitative assay for mitochondrial fusion using Renilla luciferase complementation. Mitochondrion. 10, 559-566 (2010).
- Karbowski, M., Arnoult, D., Chen, H., Chan, D. C., Smith, C. L., Youle, R. J. Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J. Cell. Biol. 164, 493-499 (2004).
- Legros, F., Lombes, A., Frachon, P., Rojo, M. Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol. Biol. Cell. 13, 4343-4354 (2002).
- Molina, A. J., Shirihai, O. S. Monitoring mitochondrial dynamics with photoactivatable [corrected] green fluorescent protein. Methods Enzymol. 457, 289-304 (2009).
- Molina, A. J., Wikstrom, J. D., Stiles, L., Las, G., Mohamed, H., Elorza, A., Walzer, G., Twig, G., Katz, S., Corkey, B. E., Shirihai, O. S. Mitochondrial networking protects beta-cells from nutrient-induced apoptosis. Diabetes. 58, 2303-2315 (2009).
- Otera, H., Mihara, K. Molecular mechanisms and physiologic functions of mitochondrial dynamics. J. Biochem. 149, 241-251 (2011).
- Patterson, G. H., Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 297, 1873-1877 (2002).
- Schauss, A. C., Huang, H., Choi, S. Y., Xu, L., Soubeyrand, S., Bilodeau, P., Zunino, R., Rippstein, P., Frohman, M. A., McBride, H. M. A novel cell-free mitochondrial fusion assay amenable for high-throughput screenings of fusion modulators. BMC Biol. 8, 100 (2011).
- Sheridan, C., Martin, S. J. Mitochondrial fission/fusion dynamics and apoptosis. Mitochondrion. 10, 640-648 (2010).
- Twig, G., Elorza, A., Molina, A. J., Mohamed, H., Wikstrom, J. D., Walzer, G., Stiles, L., Haigh, S. E., Katz, S., Las, G., Alroy, J., Wu, M., Py, B. F., Yuan, J., Deeney, J. T., Corkey, B. E., Shirihai, O. S. Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. EMBO J. 27, 433-446 (2008).
- Twig, G., Graf, S. A., Wikstrom, J. D., Mohamed, H., Haigh, S. E., Elorza, A., Deutsch, M., Zurgil, N., Reynolds, N., Shirihai, O. S. Tagging and tracking individual networks within a complex mitochondrial web with photoactivatable GFP. Am. J. Physiol. Cell Physiol. 291, C176-C184 (2006).
- Twig, G., Hyde, B., Shirihai, O. S. Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochim. Biophys. Acta. 1777, 1092-1097 (2008).
- Twig, G., Liu, X., Liesa, M., Wikstrom, J. D., Molina, A. J., Las, G., Yaniv, G., Hajnoczky, G., Shirihai, O. S. Biophysical properties of mitochondrial fusion events in pancreatic beta-cells and cardiac cells unravel potential control mechanisms of its selectivity. Am. J. Physiol. Cell. Physiol. 299, C477-C487 (2010).
- Twig, G., Shirihai, O. S. The interplay between mitochondrial dynamics and mitophagy. Antioxid. Redox Signal. 14, 1939-1951 (2011).
