Knockdown of mitofilin inhibits autophagy and facilitates starvation-induced apoptosis in HeLa cells

Document Type: Original Article


Zhejiang Provincial Key Laboratory of Medical Genetics, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China 325035


Objective(s): Mitofilin contributes to the maintenance of mitochondrial structure and functions. This study was undertaken to determine the mechanisms underlying its regulation of apoptosis.  
Materials and Methods: Mitofilin was knockdowned by specific short hairpin RNA (shRNA) and the stable HeLa cell clone was selected. The autophagy activity were assessed with LC3-II conversion and puncta formation by western blot and fluorescence imaging in starved and normal cultured HeLa cells. Autophagy flux was measured in the presence of NH4Cl. Wortmannin was used to inhibit autophagy. Cell viability and apoptosis were detected with cell counting kit-8 (CCK-8) and fluorescence-activated cell sorting (FACS) assay, respectively.
Results: Mitofilin expression was down-regulated in starved HeLa cells. In established mitofilin stable knockdown cell lines, LC3-II conversion and puncta formation were detected, which are both hallmarks of autophagy, under both basal and starvation conditions. Mitofilin down-regulation decreased LC3-II conversion and puncta formation, which indicates that loss of mitofilin function inhibits both basal and starvation-induced autophagy activity. CCK-8 and FACS analysis confirmed mitofilin involvement in the regulation of cell survival since mitofilin down-regulation facilitated starvation-induced apoptosis in HeLa cells.
Conclusion: Taken together, mitofilin is a potent regulator of autophagy and it may modulate cell survival through regulation of autophagy.


Main Subjects

1. Mizushima N, Levine B. Autophagy in mammalian development and differentiation. Nat Cell Biol 2010;12:823-830.
2. Thorburn A. Autophagy and disease. J Biol Chem 2018;293:5425-5430.
3. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999;402(6762):672-676.
4. Aita VM, Liang XH, Murty VV, Pincus DL, Yu W, Cayanis E, et al. Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics 1999;59:59-65.
5. Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003;112:1809-1820.
6. Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A 2003;100:15077-15082.
7. Wilde L, Tanson K, Curry J, Martinez-Outschoorn U. Autophagy in cancer: a complex relationship. Biochem J 2018;475:1939-1954.
8. Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, et al. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 2006;10:51-64.
9. White E. The role for autophagy in cancer. J Clin Invest. 2015;125:42-46.
10. Xie J, Marusich MF, Souda P, Whitelegge J, Capaldi RA. The mitochondrial inner membrane protein mitofilin exists as a complex with SAM50, metaxins 1 and 2, coiled-coil-helix coiled-coil-helix domain-containing protein 3 and 6 and DnaJC11. FEBS Lett 2007;581:3545-3549.
11. Bernert G, Fountoulakis M, Lubec G. Manifold decreased protein levels of matrin 3, reduced motor protein HMP and hlark in fetal Down’s syndrome brain. Proteomics 2002;2:1752-1757.
12. Van Laar VS, Dukes AA, Cascio M, Hastings TG. Proteomic analysis of rat brain mitochondria following exposure to dopamine quinone: implications for Parkinson disease. Neurobiol Dis 2008;29:477-489.
13. Omori A, Ichinose S, Kitajima S, Shimotohno KW, Murashima YL, Shimotohno K, et al. Gerbils of a seizure-sensitive strain have a mitochondrial inner membrane protein with different isoelectric points from those of a seizure-resistant strain. Electrophoresis 2002;23:4167-4174.
14. John GB, Shang Y, Li L, Renken C, Mannella CA, Selker JM, et al. The mitochondrial inner membrane protein mitofilin controls cristae morphology. Mol Biol Cell 2005;16:1543-1554.
15. Thapa D, Nichols CE, Lewis SE, Shepherd DL, Jagannathan R, Croston TL, et al. Transgenic overexpression of mitofilin attenuates diabetes mellitus-associated cardiac and mitochondria dysfunction. J Mol Cell Cardiol 2015;79:212-223.
16. Yang RF, Zhao GW, Liang ST, Zhang Y, Sun LH, Chen HZ, et al. Mitofilin regulates cytochrome c release during apoptosis by controlling mitochondrial cristae remodeling. Biochem Biophys Res Commun 2012;428:93-98.
17. Madungwe NB, Feng Y, Lie M, Tombo N, Liu L, Kaya F, et al. Mitochondrial inner membrane protein (mitofilin) knockdown induces cell death by apoptosis via an AIF-PARP-dependent mechanism and cell cycle arrest. Ame J Physiol Cell Physiol 2018;315:C28-C43.
18. Okamoto K, Kondo-Okamoto N. Mitochondria and autophagy: critical interplay between the two homeostats. Biochim Biophys Acta 2012;1820:595-600.
19. Tsai PI, Lin CH, Hsieh CH, Papakyrikos AM, Kim MJ, Napolioni V, et al. PINK1 Phosphorylates MIC60/Mitofilin to Control Structural Plasticity of Mitochondrial Crista Junctions. Mol Cell 2018;69:744-756 e6.
20. Akabane S, Uno M, Tani N, Shimazaki S, Ebara N, Kato H, et al. PKA Regulates PINK1 Stability and Parkin Recruitment to Damaged Mitochondria through Phosphorylation of MIC60. Mol Cell 2016;62:371-384.
21. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 2016;12:1-222.
22. Van Laar VS, Berman SB, Hastings TG. Mic60/mitofilin overexpression alters mitochondrial dynamics and attenuates vulnerability of dopaminergic cells to dopamine and rotenone. Neurobiol Dis 2016;91:247-261.
23. Jiang P, Mizushima N. Autophagy and human diseases. Cell Res 2014;24:69-79.
24. Zhi X, Feng W, Rong Y, Liu R. Anatomy of autophagy: from the beginning to the end. Cell Mol Life Sci 2018;75:815-831.
25. Razaghi A, Heimann K, Schaeffer PM, Gibson SB. Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies. Apoptosis 2018;23:93-112.
26. Betin VM, Lane JD. Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. J Cell Sci 2009;122(Pt 14):2554-2566.
27. Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I, et al. Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis 2010;1:e18.
28. Rambold AS, Lippincott-Schwartz J. Mechanisms of mitochondria and autophagy crosstalk. Cell Cycle 2011;10:4032-4038.