TY - JOUR
T1 - Zn2+-Depletion Enhances Lysosome Fission in Cultured Rat Embryonic Cortical Neurons Revealed by a Modified Epifluorescence Microscopic Technique
AU - Tsao, Hung Chun
AU - Liao, Yi Feng
AU - Pratiwi, Feby Wijaya
AU - Mou, Chung Yuan
AU - Lin, Yi Jhen
AU - Pan, Chien Yuan
AU - Chen, Yit Tsong
N1 - Funding Information:
This work was supported, in part, by the Ministry of Science and Technology (MOST) of Taiwan under grant nos. 106-2113-M-002-022-MY3 and 107-2113-M-002-011-MY3 (Y.-T.C.); 107-2320-B-002-052 and 109-2311-B-002-011 (C.-Y.P.). We thank Dr. Peilin Chen of Academia Sinica for his help in the single particle tracking technique. We acknowledge the biophysical core facility at the Institute of Atomic and Molecular Sciences (IAMS) for the uses of high-resolution confocal microscopy (SP8, Leica).
Publisher Copyright:
Copyright © The Author(s), 2021. Published by Cambridge University Press on behalf of the Microscopy Society of America.
PY - 2021/4
Y1 - 2021/4
N2 - Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.
AB - Lysosomes are integration hubs for several signaling pathways, such as autophagy and endocytosis, and also crucial stores of ions, including Zn2+. Lysosomal dysfunction caused by changes in their morphology by fusion and fission processes can result in several pathological disorders. However, the role of Zn2+ in modulating the morphology of lysosomes is unclear. The resolution of conventional epifluorescence microscopy restricts accurate observation of morphological changes of subcellular fluorescence punctum. In this study, we used a modified epifluorescence microscopy to identify the center of a punctum from a series of z-stack images and calculate the morphological changes. We stained primary cultured rat embryonic cortical neurons with FluoZin3, a Zn2+-sensitive fluorescent dye, and Lysotracker, a lysosome-specific marker, to visualize the distribution of Zn2+-enriched vesicles and lysosomes, respectively. Our results revealed that treating neurons with N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine, a cell-permeable Zn2+ chelator, shrank Zn2+-enriched vesicles and lysosomes by up to 25% in an hour. Pretreating the neurons with YM201636, a blocker of lysosome fission, could suppress this shrinkage. These results demonstrate the usefulness of the modified epifluorescence microscopy for investigating the homeostasis of intracellular organelles and related disorders.
KW - autophagosome
KW - lysosome homeostasis
KW - YM201636
KW - Znhomeostasis
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U2 - 10.1017/S1431927620024940
DO - 10.1017/S1431927620024940
M3 - Article
C2 - 33487212
AN - SCOPUS:85100321376
SN - 1431-9276
VL - 27
SP - 420
EP - 424
JO - Microscopy and Microanalysis
JF - Microscopy and Microanalysis
IS - 2
ER -