The role of the cytoskeleton in autophagy and Crohn's disease

Biskou, Olga

Abstract

Intermediate filaments, along with microtubules, microfilaments and septins, comprise the cytoskeleton that provides cells with structure and shape. The cytoskeleton is known to participate in the organelle localization and signal transduction.
Autophagy is an intracellular degradation mechanism that allows cells to recycle damaged proteins and organelles. When cells are exposed to stress, autophagy is activated to promote cell survival. Autophagy is highly regulated, and it is reported that the cytoskeleton participates in autophagosome formation and transport. Vimentin filaments are reported to act as autophagy inhibitors, and also as a scaffold for autophagy activation during infection. However, little is known about other roles in autophagy. Autophagy is reported to be involved in autoimmune disease like Inflammatory Bowel Disease (IBD).
Crohn’s disease (CD) and Ulcerative colitis (UC), are the major forms of IBD. IBDs are multifactorial, and the contributing factors include environmental triggers, microbial dysbiosis and genetic predisposition. For CD, the genes with the strongest association include the Pattern Recognition Receptors (PRR) Nucleotide Binding Oligomerization Domain (NOD)2 and Autophagy Related Protein (ATG)16L1, that participate in autophagy induction during infection.
It was hypothesized that vimentin filaments participate in autophagy steps. Two objectives were investigated, i) the role of vimentin in NOD2 induced autophagy during infection, ii) the role of vimentin in autophagy.
The methodology used included live/fixed cell imaging, Western blot and flow cytometry.
The live cell imaging approached used for the first objective, did not provide further understanding of the underlying mechanism. For the second objective, it was shown that pharmacological vimentin inhibition through Withaferin A (WFA), results in increased autophagy as monitored through Western blot, flow cytometry and, immunostaining. Further investigation of the mechanism showed that vimentin inhibition results in re-distribution of autophagosomes and lysosomes and this results in preventing the formation of autolysosomes, as shown by the traffic light experiment, that is used to monitor the maturation of autophagosomes into autolysosomes. The main autophagy marker,Microtubule-associated protein 1-light chain 3 (LC3), is conjugated to Red Fluorescent Protein (RFP) and Green Fluorescent Protein (GFP) both expressed on the surface of autophagosome, that appear yellow. Once the autophagosome fuses with the lysosomes LC3 and GFP is degraded and autolysosomes appear red. These findings indicate that vimentin acts as a scaffold for the fusion between autophagosomes and lysosomes.

Citation

Biskou, O. The role of the cytoskeleton in autophagy and Crohn's disease. (Thesis). Edinburgh Napier University. Retrieved from http://researchrepository.napier.ac.uk/Output/2685526

• Thesis Type: Thesis
• Deposit Date: Sep 10, 2020
• Publicly Available Date: Sep 10, 2020
• DOI: https://doi.org/10.17869/enu.2020.2685526
• Keywords: autophagy; cytoskeleton; Crohn's disease; vimentin; autophagosomes; lysosomes
• Public URL: http://researchrepository.napier.ac.uk/Output/2685526
• Award Date: Jul 1, 2020