Autophagy: The unsolved murder

Autophagy, a process through which cells perform auto-digestion on targeted cellular components, is essential for maintenance of cellular homeostasis during various stress conditions. Excessive levels of autophagy have also been observed in association with various forms of cell death, however autophagy’s role in this cell death is a controversial topic.  It’s uncertain whether autophagy has a causative role, or whether it is merely a side effect as the cell attempts to prolong its life.

There are several pathways through which autophagy can be performed: macroautophagy, microautophagy and chaperone-mediated autophagy (CMA).  The most common pathway, macroautophagy, consists of the formation of a double membrane vesicle around targeted cellular components.  This vesicle, known as an autophagosome, then migrates through the cytosol to merge with a lysosome filled with hydrolytic enzymes, resulting in the breakdown of the autophagosomes contents.  The direct engulfment of cellular components by the lysosome is known as microautophagy.  Lastly, CMA uses a binding complex that attaches to a specific protein recognition site, allowing transport and entry into the lysosome for digestion.

We decided to observe the mysteries of autophagy using the 3D Cell Explorer, allowing us to observe the cells without the use of chemical stains, but instead by using the unique refractive indices found in individual cellular compartments. 

Experiment:

Using glioblastoma cells, autophagy was induced by the addition of a drug.  The cells were incubated at 37 degrees with 5% CO2 and imaged every 60s over a period of 9 hours.  Check out the video to see the whole process!

 

  • Special thanks to Prof. Douglas Hanahan and Laboratory of Translational Oncology for the samples they gave us.
  • The cells used are LN18, and the drugs used are a combination of imipramine and ticlopidine, from the following publication:
    Dual Targeting of the Autophagic Regulatory Circuitry in Gliomas with Repurposed Drugs Elicits Cell-Lethal Autophagy and Therapeutic Benefit.
    PMID: 26412325 DOI:10.1016/j.ccell.2015.08.012