Using Microscopy Tools to Visualize Autophagosomal Structures in Plant Cells
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Macroautophagy (hereafter as autophagy), is a metabolic process for sequestration of cytoplasmic cargos into a double membrane structure named as autophagosome. In plants, autophagy is required for nutrition mobilization/recycling and clearance of protein aggregates or damaged organelles during starvation or other unfavorable conditions, as well as for plant immunity during pathogen infection. Multiple experimental approaches have been developed to elucidate the autophagic activity. To facilitate further investigations on the potential involvement of autophagy in protein secretion process in plant cells, here we describe detailed protocols to measure the autophagic activity in model plant Arabidopsis. Using the autophagosome marker ATG8 and a novel autophagic regulator SH3P2 as examples, we illustrate the major cell biology tools and methods using microscopy to analyze the autophagosomal structures in plant cells, including BTH-induced autophagic response, transient expression and colocalization analysis, as well as immuno-EM labeling.
Key wordsAutophagy Autophagosome BTH Transient expression Immuno-EM labeling
This work was supported by grants from the Research Grants Council of Hong Kong (CUHK465112, 466313, 14130716, 14102417, CUHK2/CRF/11G, C4011-14R, C4012-16E, and AoE/M-05/12), Germany/Hong Kong Joint Research Scheme, CUHK Research Committee Direct Grant, NSFC (31670179, 31270226 and 31470294), CAS-Croucher Joint Lab Scheme, and Shenzhen Peacock Project (KQTD201101).
- 4.DeSelm CJ, Miller BC, Zou W, Beatty WL, van Meel E, Takahata Y, Klumperman J, Tooze SA, Teitelbaum SL, Virgin HW (2011) Autophagy proteins regulate the secretory component of osteoclastic bone resorption. Dev Cell 21(5):966–974. doi: 10.1016/j.devcel.2011.08.016 CrossRefPubMedPubMedCentralGoogle Scholar
- 5.Son SM, Cha MY, Choi H, Kang S, Choi H, Lee MS, Park SA, Mook-Jung I (2016) Insulin-degrading enzyme secretion from astrocytes is mediated by an autophagy-based unconventional secretory pathway in Alzheimer disease. Autophagy 12(5):784–800. doi: 10.1080/15548627.2016.1159375 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Kimura T, Jia J, Kumar S, Choi SW, Gu Y, Mudd M, Dupont N, Jiang S, Peters R, Farzam F, Jain A, Lidke KA, Adams CM, Johansen T, Deretic V (2017) Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy. EMBO J 36(1):42–60. doi: 10.15252/embj.201695081 CrossRefPubMedGoogle Scholar
- 7.Zhuang XH, Wang H, Lam SK, Gao CJ, Wang XF, Cai Y, Jiang LW (2013) A BAR-domain protein SH3P2, which binds to phosphatidylinositol 3-phosphate and ATG8, regulates autophagosome formation in Arabidopsis. Plant Cell 25(11):4596–4615. doi: 10.1105/tpc.113.118307 CrossRefPubMedPubMedCentralGoogle Scholar
- 9.Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, Ohsumi Y, Shirasu K (2009) Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell 21(9):2914–2927. doi: 10.1105/tpc.109.068635 CrossRefPubMedPubMedCentralGoogle Scholar
- 13.Gao CJ, Zhuang XH, Cui Y, Fu X, He YL, Zhao Q, Zeng YL, Shen JB, Luo M, Jiang LW (2015) Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation. Proc Natl Acad Sci U S A 112(6):1886–1891. doi: 10.1073/pnas.1421271112 CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Ritzenthaler C, Nebenfuhr A, Movafeghi A, Stussi-Garaud C, Behnia L, Pimpl P, Staehelin LA, Robinson DG (2002) Reevaluation of the effects of brefeldin A on plant cells using tobacco bright yellow 2 cells expressing Golgi-targeted green fluorescent protein and COPI antisera. Plant Cell 14(1):237–261. doi: 10.1105/tpc.010237 CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Bozzola JJ, Russell LD (1999) Electron microscopy: principles and techniques for biologists. Jones & Bartlett Learning, Sudbury, MAGoogle Scholar