In situ architecture of the intercellular organelle reservoir between epididymal epithelial cells by volume electron microscopy

Xia Li, Feng Qiao, Jiansheng Guo, Ting Jiang, Huifang Lou, Huixia Li, Gangcai Xie, Hangjun Wu, Weizhen Wang, Ruoyu Pei, Sha Liu, Mei Ye, Jin Li, Shiqin Huang, Mengya Zhang, Chaoye Ma, Yiwen Huang, Shushu Xu, Xiaofeng Li, Xiao Sun, Jun Yu, Kin Lam Fok, Shumin Duan & Hao Chen

Mammalian epididymal epithelial cells are crucial for sperm maturation. Historically,
vacuole-like ultrastructures in epididymal epithelial cells were
observed via transmission electron microscopy but were undefined. Here, we
utilize volume electron microscopy (vEM) to generate 3D reconstructions of
epididymal epithelial cells and identify these vacuoles as intercellular organelle
reservoirs (IORs) in the lateral intercellular space (LIS), which contains
protein aggregates, autophagosomes, lysosome-related organelles and mitochondrial
residues. Immunolabelling of organelle markers such as P62, LC3,
LAMP1 and TOMM20 confirm these findings. The IOR size or number varies
across four epididymal regions and decreases with age. Rab27a mutant mice
exhibit reduced IORs in the caput epididymis and a subfertility phenotype,
suggesting the involvement of Rab27a in the formation of IORs. Furthermore,
we observe the presence of IORs between intestinal epithelial cells besides
epididymis. Amino acid transporters at IOR edges suggest dynamic protein
recycling. Our findings reveal that the IOR is an important structure critical for
organelle turnover and recycling outside epithelial cells with limited selfdegradation
capabilities.

How Amira-Avizo Software is used

We individually segmented the cell membrane
and vacuole membrane via Amira software. […], All 3D segmentations were generated via Amira software (Version
6.5.0, ThermoFisher,USA). RawTiff images (1300 ~ 1500/sample)were
imported into Amira for alignment […],  Direct 3D reconstruction, volume rendering and
visualization of the nucleus, cellmembranes, and IOR were conducted
manually upon careful visual inspection with Amira software (Version
6.5.0, Thermo Fisher, USA). […], IORs containing autophagy-like structures were segmented with subvolume
extraction created in Amira.