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Biology

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Published: May 3rd, 2013

DOI:

10.3791/50047

1Department of Biochemistry and Molecular Medicine, University of California, Davis , 2NSF Center for Biophotonics Science & Technology, University of California, Davis , 3University of Tromsø, 4Department of Surgery (Division of Surgical Oncology), University of California, Davis , 5UC Davis Comprehensive Cancer Center, University of California, Davis , 6Department of Biological Chemistry, University of California, Davis

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

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.

1. Part 1: Cell Culture and Immuno-fluorescent Labeling

  1. Grow CWR22Rv1 human prostate tumor cells on glass coverslips (#1.5 or 170 μm thickness) placed in 6-well plates, with RPMI (Mediatech, VA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin/glutamine.
  2. Induce autophagy by treating selected samples with arginine deiminase (ADI, 0.3 μg/ml) in phosphate-buffered saline (PBS).
  3. Fix cells with 4% paraformaldehyde (PFA, Fisher Scientific, NH) diluted in PBS;.......

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The image sequence shown in Figure 1 shows the physical changes that occur in CWR22 cells during the first 80 min of autophagy induction. In this and other studies (not shown) we consistently observed: (1) displacement of the nucleus away from the cell center; (2) reduction of focal adhesion points; and (3) general translocation of autophagosomes and lysosomes towards the center of the cell. In addition, we also observed a small increase in colocalization (indicated in yellow) between autophagosomes (gre.......

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While the direct observation of cells labeled with fluorescent probes against LC3 is widely accepted as a standard method to confirm autophagic response6, quantitative 3D imaging of the same system (as we have done) provides unprecedented information and detail about the complex process of cellular autophagy. In particular, we observe that hundreds (if not thousands) of autophagosomes in live cells are formed within 80 min of autophagy induction. Similarly, we observe very interesting morphological changes in .......

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Grant support: NIH CA165263, NIH CA150197, NIH CA150197S1 (H.J. Kung), NIH CA150197S1 (C.A. Changou), NSF PHY-0120999 Center for Biophotonics Science & Technology (D.L. Wolfson, F.Y.S. Chuang), DOD PC073420 (R.J. Bold), The Research Council of Norway, Leiv Eiriksson Travel Grant 209286/F11 (B.S. Ahluwalia). H.J. Kung also acknowledges the support of the Auburn Community Cancer Endowment Fund. R.J. Bold also acknowledges the support of the J. McDonald endowment.

We thank Dr. Jenny Wei-Jen Kung and Dr. Bor-wen Wu at DesigneRx for generous supply of ADI.

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Name Company Catalog Number Comments
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  

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  2. 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).
  3. 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).
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  9. 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).
  10. 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).
  11. 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).
  12. 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).
  13. 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).
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