The induction of autophagic vacuoles and the unique endocytic compartments, C-shaped multivesicular bodies, in GH4C1 cells after treatment with 17beta-estradiol, insulin and EGF.
The mechanisms for the formation of autophagic vacuoles were investigated using GH4C1 cells, a rat pituitary tumor cell line, whose induction increases intracellular levels of lysosomal proteinases and their mRNA by treatment with a combination of hormones (17beta-estradiol, insulin and EGF). By ordinary electron microscopy, autophagic vacuoles containing various undigested structures with or without limiting membranes were abundant in the hormone-induced cells. These vacuoles, also containing numerous small vesicles, appeared to be derived from multivesicular bodies. In fact, there were also numerous C-shaped multivesicular bodies which enclosed cytoplasmic portions, suggesting that these unique structures are involved in the production of the autophagic vacuoles. Moreover, the cytoplasmic portions enlapped by the C-shaped multivesicular bodies were high in electron density and contained filamentous structures. By the cryothin-section immunogold method, the C-shaped multivesicular bodies in some cases contained lysosomal marker proteins such as cathepsins B and H, and Igp 120. Using an anti-actin monoclonal antibody, immunogold particles clearly labeled the cytoplasmic portions enclosed by the C-shaped multivesicular bodies. Pulse-chase experiments with horse radish peroxidase, a fluid-phase endocytic marker, revealed that the incidence of the C-shaped multivesicular bodies labeled with horse radish peroxidase peaked at 30 min after the beginning of chase incubation, whereas no C-shaped multivesicular body with horse radish peroxidase was detected in the cells by cytochalasin D treatment. These results suggest that the C-shaped multivesicular bodies occur in a transitional process from endosomes to lysosomes by the action of actin filaments, and that this morphological change may be essential for the production of autophagic vacuoles in the hormone-induced GH4C1 cells.
Waguri
S
S
Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Suita, Japan. waguri@anat1.med.osaka-u.ac.jp
Kohmura
M
M
Gotow
T
T
Watanabe
T
T
Ohsawa
Y
Y
Kominami
E
E
Uchiyama
Y
Y
D000199:Actins / Q000502:physiology; D000818:Animals; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D011992:Endosomes / Q000502:physiology* / Q000648:ultrastructure; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology*; D006735:Horseradish Peroxidase; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D008247:Lysosomes / Q000502:physiology / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D010587:Phagocytosis / Q000502:physiology*; D010902:Pituitary Gland / Q000166:cytology; D051381:Rats; D014617:Vacuoles / Q000502:physiology* / Q000648:ultrastructure
D000199:Actins; D007004:Hypoglycemic Agents; D007328:Insulin; D004958:Estradiol; D004815:Epidermal Growth Factor; D006735:Horseradish Peroxidase