Figure 3. Immunoelectron microscopy analysis of PMA-induced Cx43 gap junction degradation.

To identify lysosomes, cells were incubated with 8 nm BSA–gold for 3 h and chased overnight. Cells were treated with PMA (100 ng/ml) for 30 min, fixed, prepared for electron microscopy and labelled for Cx43 (15 nm gold). (A) An annular gap junction appears to have started to be cut into smaller membrane pieces. Some parts of the gap junction membrane appear to form a small intravacuolar vesicle (arrowhead). (B) The double membrane of the annular gap junction appears to be cut and to separate. The resulting single membranes appear to form small vesicles (arrowheads), similar to vesicles inside multivesicular endosomes. (C–E) Cx43 labelling in multivesicular endosomes. Some of the intravacuolar vesicles have irregular shapes and are different from vesicles usually seen in multivesicular endosomes. Some membrane areas of the endosomes appear to be remnants of double-membrane gap junctions (arrowheads). (F) Cx43 labelling in a lysosome, identified as an electron-dense vesicle containing endocytosed 8 nm gold particles. PM, plasma membrane. Scale bars, 200 nm.