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Apoptosis Webinar

"A question of Life or Death" - differentiating between healthy and apoptotic cells

Autophagy Overview

The evolutionary conserved autophagy process is under renewed research interest with the discovery of the ATG gene products and the link between autophagy and aging as well as human diseases. A suggested role for autophagy in programmed cell death and cell cycle progression along with further elucidation of the autophagy mechanisms and their regulation are also under investigation.

We offer a selection of autophagy related antibodies and cathepsin detection kits, all performance guaranteed in many popular applications, for the study of upstream events, autophagy pathways and lysosome detection.

Cerebellar cortex stained with Goat anti human

Upstream Events

Cellular stress conditions initiate upstream events through the MAPK/Erk1/2, PI3K/AKT and mammalian target of rapamycin (mTOR) pathways with mTORC complexes leading to the autophagy mechanisms macroautophagy, microautophagy and chaperone-mediated autophagy (CMA) in eukaryotic cells.

 


Mouse peritoneal macrophage stained with MCA2293F

Macroautophagy

Macroautophagy is the most studied pathway involving the cellular degradation of cytoplasmic proteins, bulk cytoplasm, dysfunctional organelles and intracellular pathogens (innate immunity / cell defense mechanism). This process involves the formation of a phagophore which develops into a transport vesicle called the autophagosome containing the cytoplasmic constituents or organelles.

The autophagy machinery, including the phagophore assembly site / pre-autophagosomal structure (PAS), and its regulation involves more than 30 autophagy related genes (ATG Proteins) including Beclin 1, Activating molecule in beclin-1-regulated autophagy (AMBRA1), Damage-regulated autophagy modulator (DRAM), lysosome-associated membrane proteins (Lamp1 (CD107a) & Lamp2 (CD107b)) and Microtubule-associated proteins 1A/1B light chains 3A/B (MAP1LC3A/B).


Western blot of wild-type and ATG5 knockout cells

Autophagosome Marker – LC3 Antibody

Microtubule-associated proteins 1A/1B light chains 3A/LC3A and 3B/LC3B (MAP1LC3A/B) are ubiquitin-like proteins used as reliable markers for monitoring autophagy. MAP1LC3A/B is commonly further abbreviated to LC3.

A popular method for detecting autophagy uses antibodies specific to LC3 to identify LC3-positive structures such as autophagosomes. Since LC3 is the only protein identified on the inner and outer membranes of autophagosomes, MAP1LC3A/B antibodies provide a quick detection method.

Click here to learn more about our LC3 antibody, which shows superior results in western blotting.


Starvation induced autophagy

Starvation was used to induce autophagy. Untreated cells (red) and cells incubated in HBSS for 2 hr and 10 nM Bafilomycin A for 2 hr (blue). After staining with Autophagy Probe Red for 30 min, cells were washed and analyzed on the ZE5 Cell Analyzer using the 561 nm laser and 640/20 filter.

Flow Cytometry Autophagy Assay

Autophagy Probe Red is a cell-permeant aliphatic molecule that fluoresces brightly when inserted into the lipid membranes of autophagosomes and autolysosomes. Autophagy is a dynamic process typically divided into three stages. During stage one, cytoplasmic components targeted for degradation are sequestered within a double-membrane phagopore (also called the isolation membrane).

This results in the formation of a double-membrane vesicle called the autophagosome. During stage two, the autophagosome fuses with a lysosome to form the autolysosome. Degradation of the autophagosomal contents occurs during stage three (Mizushima and Komatsu (2011), Hundeshagen et al. 2011).

Autophagy Probe Red can be readily detected in cells undergoing autophagy quickly and easily by flow cytometry, with optimal excitation at 590 nm and peak emission at 620 nm (ZE5 Cell Analyzer settings: 561 nm laser and 615/24 or 640/20 filter).


Intracellular cathespin K activity detected in THP-1 cells

Lysosome Detection – Cathepsin Antibodies and Detection Kits

The autophagosome fuses to a lysosome forming an autolysosome where enzymes such as cathepsins and permeases hydrolyze and degrade the cargo for its release back into the cytoplasm for recycling.

Our Magic Red™ Cathepsin B, K and L Detection Kits are suitable for detection of lysosomes:

  • Active cathepsin analysis in whole, living cells
  • Substrate based assay
  • Fluorescence microscopy or plate reader

We also offer a range of Cathepsin Antibodies; further details about our Cathepsin Detection Kits and antibodies are available here. Our cathepsin kits are part of our apoptosis detection kits range.

Future detailed understanding of autophagy mechanisms and their role in human disease and innate and adaptive immunity is needed before potential therapeutic intervention can be developed.

Autophagy is thought to be closely linked to apoptosis; view the other sections in our quality apoptosis antibodies and kits product range:


References

Upstream Events

  • Chu CT et al. (2004). Oxidative neuronal injury. The dark side of ERK1/2.
    Eur. J. Biochem. 271: 2060–2066.
  • Flotte, TJ et al. (1983). Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets.
    Am. J. Pathol. 111:112-124.
  • Gozuacik D. and Kimchi A (2004). Autophagy as a cell death and tumor suppressor mechanism.
    Oncogene. 23: 2891-2906.
  • Ho MK and Springer TA (1983). Tissue distribution, structural characterization and biosynthesis of Mac-3, a macrophage surface glycoprotein exhibiting molecular weight heterogeneity.
    J. Biol. Chem. 258:636-642.
  • Liang XH et al. (1998). Protection against fatal Sindbis virus encephalitis by Beclin, a novel Bcl-2-interacting protein.
    J. Virol. 72: 8586-8596.
  • Liang XH et al. (1999). Induction of autophagy and inhibition of tumorigenesis by beclin 1.
    Nature. 402: 672-676.
  • Nishimoto S and Nishida E (2006). MAPK signalling: ERK5 versus ERK1/2.
    EMBO Rep. 7: 782-6.
  • Raman M. et al. (2007). Differential regulation and properties of MAPKs.
    Oncogene. 26:3100-12.
  • Reif K et al. (1993). Divergent regulation of phosphatidylinositol 3-Kinase P85 alpha and P85 beta isoforms upon T cell activation.
    J. Biol. Chem. 278: 10780-10788.
  • Semba S et al. (2002). Down-regulation of PIK3CG, a catalytic subunit of phosphatidylinositol 3-OH kinase, by CpG hypermethylation in human colorectal carcinoma.
    Clin. Cancer Res. 8:3824-3831.
  • Serajee FJ et al. (2003). Association of INPP1, PIK3CG, and TSC2 gene variants with autistic disorder: implications for phosphatidylinositol signalling in autism.
    J. Med. Genet. 40:119.
  • Shamji AF et al. (2003). Integration of growth factor and nutrient signaling: Implications for cancer biology.
    Mol. Cell 12: 271-280.
  • Springer TA (1981). Monoclonal antibody analysis of complex biological systems. Combination of cell hybridization and immunoadsorbents in a novel cascade procedure and its application to the macrophage cell surface
    J. Biol. Chem. 256:3833-3839.
  • Stern DF (2004). More than a marker… Phosphorylated Akt in prostate carcinoma.
    Clin Cancer Res. 10: 6407-10.
  • Tatin F. (2010). Sodium fluoride induces podosome formation in endothelial cells.
    Biol Cell. May 26. [Epub ahead of print]
  • Turjanski A et al. (2007). MAP kinases and the control of nuclear events.
    Oncogene. 26:3240-53.
  • Woodfield R et al. (2001). The p85 subunit of phosphoinositide 3-kinase is associated with beta-catenin in the cadherin-based adhesion complex.
    Biochem. J. 360: 335 - 344.

Autophagy Pathway

  • Espinosa,I et al. (2009). Coordinate expression of colony-stimulating factor-1 and colony-stimulating factor-1-related proteins is associated with poor prognosis in gynecological and nongynecological leiomyosarcoma. ​
    Am J Pathol. 174: 2347-2356.
  • Hundeshagen et al. (2011). Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy.​
    BMC Biol 9:38
  • Iwata A et al. (2005). HDAC6 and microtubules are required for autophagic degradation of aggregated Huntington.
    J. Biol. Chem. 280: 40282-40292
  • Kopitar-Jerala N et al. (2001). Anti-Cathepsin L monoclonal antibodies that distinguish cathepsin L from cathepsin V.
    Biol Chem. 382: 867-870.
  • Martin SL et al. (2010). Association of airway cathepsin B and S with inflammation in cystic fibrosis.
    Pediatr Pulmonol. Jul 14.[Epub ahead of print]
  • Mizushima and Komatsu (2011). Autophagy: renovation of cells and tissues. 
    Cell. 147 (4): 728-41