Isolation of Autolysosomes from Tobacco BY-2 Cells

Abstract

Autolysosomes are organelles that sequester and degrade a portion of the cytoplasm during autophagy. Although autophagosomes are short lived compared to other organelles such as mitochondria, plastids, and peroxisomes, many autolysosomes accumulate in tobacco BY-2 cells cultured under sucrose starvation conditions in the presence of a cysteine protease inhibitor. We here describe our methodology for isolating autolysosomes from BY-2 cells by conventional cell fractionation using a Percoll gradient. The autolysosome fraction separates clearly from fractions containing mitochondria and peroxisomes. It contains acid phosphatase, vacuolar H-ATPase, and protease activity. Electron micrographs show that the fraction contains partially degraded cytoplasm seen in autolysosomes before isolation although an autolysosome structure is only partially preserved.

Multiscale and Multimodal Approaches to Study Autophagy in Model Plants

“…When embarking into the field of plant autophagy, a plant cell biologist may rely on the many reviews that have flourished within the last decade, which propose many valuable tools to investigate and monitor autophagy in plant cells [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. The experimenter is also encouraged to refer to the “Guidelines for the use and interpretation of assays for monitoring autophagy” [ 12 ], which constitutes a rich source of references to embrace methods for studying autophagy in eukaryotic cells, and proposes a glossary dedicated to autophagy-related acronyms.…”

The Function of Autophagy in Lace Plant Programmed Cell Death

“…Our TEM observations of the lace plant PCD gradient confirmed the presence of double-membrane–bound autophagosome-like structures in NPCD and PCD cells (A, Figure 6 ). Additionally, numerous single-membrane vesicles (Ve, Figure 6 ) of varying shapes and sizes were found, having a similar appearance to the provacuoles formed during cellular degradation in the embryos of Norway spruce ( Picea abies ; Filonova et al, 2000; Minina et al, 2013) or autolysosomes in BY-2 cells (Takatsuka et al, 2017). The red fluorescence in LPCD cells stained with acridine orange suggests that the puncta are acidic vesicles and may serve a similar function to autolysosomes.…”