3D उन्नत प्रतिदीप्ति माइक्रोस्कोपी का उपयोग कर भोजी के मात्रात्मक विश्लेषण
Summary
भोजी कोशिकाओं को प्रोटीन और organelles नीचा और पुनरावृत्ति करने के लिए सक्षम बनाता है एक सर्वव्यापी प्रक्रिया है. हम कल्पना और छोटे यों को उन्नत प्रतिदीप्ति माइक्रोस्कोपी लागू होते हैं, लेकिन autolysosomes में गठन और autophagosomes और लाइसोसोम के वितरण, और उनके संलयन सहित भोजी की प्रेरण के साथ जुड़े आवश्यक, शारीरिक परिवर्तन,.
Abstract
Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear – in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells – and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.
Protocol
Representative Results
Discussion
LC3 के खिलाफ जांच फ्लोरोसेंट के साथ लेबल कोशिकाओं का प्रत्यक्ष अवलोकन व्यापक रूप autophagic प्रतिक्रिया 6, मात्रात्मक 3 डी में एक ही प्रणाली (हम कुछ किया है के रूप में) की इमेजिंग सेलुलर भोजी की जटिल प्रक्रिय…
Disclosures
The authors have nothing to disclose.
Acknowledgements
अनुदान का समर्थन: एनआईएच CA165263, एनआईएच CA150197, एनआईएच CA150197S1 (एच.जे. कुंग), एनआईएच CA150197S1 (सीए Changou), Biophotonics विज्ञान और प्रौद्योगिकी (डीएल वोल्फसन, FYS चुआंग), डीओडी PC073420 (आरजे बोल्ड) के लिए NSF बनावट-0120999 सेंटर, रिसर्च नॉर्वे की परिषद, Leiv Eiriksson यात्रा अनुदान 209286/F11 (बी एस अहलूवालिया). एच.जे. कुंग भी कपिश समुदाय कैंसर बंदोबस्ती कोष का समर्थन मानता है. आरजे बोल्ड भी जे मैकडॉनल्ड्स बंदोबस्ती का समर्थन मानता है.
हम आदि की उदार आपूर्ति के लिए डॉ. जेनी वी जेन कुंग और DesigneRx में डॉ. बोर-वेन वू धन्यवाद.
Materials
| Name of Reagent/Material | Company | Catalogue Number | Comments |
| Arginine Deiminase (ADI) | DesigneRx | ||
| HEPES | Sigma | H4034 | |
| Casein | Sigma | C5890 | |
| Paraformaldehyde | Fisher | 4042 | |
| Saponin | Sigma | S4521 | |
| Alexa anti-mouse 555 | Invitrogen | A21422 | |
| Alexa anti-rabbit 647 | Invitrogen | A21244 | |
| LysoTracker Red DND-99 | Invitrogen | L7528 | |
| anti-Lamp1 | DSHB | H4A3 | |
| anti-Cadherin | Cell Signaling | #3195 | |
| SlowFade Gold | Invitrogen | S36936 | |
| 35 mm poly-d-lysine coated glass bottom plate | MatTek | P35GC-1.5-1.4-C | |
| No.1, 22 mm coverslip | Corning | #2865-22 | |
| Microscope slides | Globe Scientific | 1324G |
References
- Kim, R. H., Coates, J. M., Bowles, T. L., McNerney, G. P., Sutcliffe, J., Jung, J. U., Gandour-Edwards, R., Chuang, F. Y., Bold, R. J., Kung, H. J. Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Res. 69 (2), 700-708 (2009).
- Miyazaki, K., Takaku, H., Umeda, M., et al. Potent growth inhibition of human tumor cells in culture by arginine deiminase purified from a culture medium of a Mycoplasma-infected cell line. Cancer Res. 50, 4522-4527 (1990).
- Takaku, H., Matsumoto, M., Misawa, S., Miyazaki, K. Anti-tumor activity of arginine deiminase from Mycoplasma argini and its growth-inhibitory mechanism. Jpn. J. Cancer Res. 86, 840-846 (1995).
- Levine, B., Klionsky, D. J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell. 6, 463-477 (2004).
- Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature. 451, 1069-1075 (2008).
- Kabeya, Y., Mizushima, N., Ueno, T. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720-5728 (2000).
- Pattingre, S., Espert, L., Biard-Piechaczyk, M., Codogno, P. Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie. 90, 313-323 (2008).
- Maiuri, M. C., Criollo, A., Tasdemir, E., et al. BH3-only proteins and BH3mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L). Autophagy. 3, 374-376 (2007).
- Crighton, D., Wilkinson, S., O’Prey, J., et al. DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell. 126, 121-134 (2008).
- Dillon, B. J., Prieto, V. G., Curley, S. A., et al. Incidence and distribution of argininosuccinate synthetase deficiency in human cancers: a method for identifying cancers sensitive to arginine deprivation. Cancer. 100, 826-833 (2004).
- Dale, B. M., McNerney, G. P., Thompson, D. L., et al. Cell-to-cell transfer of HIV-1 via virological synapses leads to endosomal virion maturation that activates viral membrane fusion. Cell Host & Microbe. 10 (6), 551-562 (2011).
- Cogger, V. C., McNerney, G. P., Nyunt, T., et al. Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations. J. Struct. Biol. 171 (3), 382-388 (2010).
- York, A. G., Parekh, S. H., Nogare, D. D., et al. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy. Nat. Methods. 9 (7), 749-754 (2012).
- Gustafsson, M. G. L. Surpassing the lateral resolution limit by factor of two using structured illumination microscopy. Journal of Microscopy. 198, 82-87 (2000).
- Shao, L., Kner, P., Rego, E. H., Gustaffson, M. G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Method. 8 (12), 1044-1046 (2011).
