Role of VAMP8/endobrevin in constitutive exocytotic pathway in HeLa cells.

To evaluate the role of VAMP8/endobrevin in constitutive exocytosis, we have examined the exocytotic pathways of VAMP8 and human growth hormone, both GFP-tagged, by total internal reflection fluorescence microscopy (TIRF-M). Human GH-GFP and VAMP8-GFP were similarly expressed in small round vesicles and elongated tubular vesicles in HeLa cells, and were mostly exocytosed at the peripheral area of the cells. VAMP8-GFP gave 2 types of exocytotic images: a burst type and a non-burst type. The burst type showed a sharp transient increase in the peak fluorescence intensity and a much slower decrease in the average intensity in the active windows, where exocytosis took place, as observed in the "full-fusion" type of exocytosis. The non-burst type showed a relatively long-lasting fusion to the plasma membrane with little transfer of VAMP8-GFP to the plasma membrane, as observed in the so-called "kiss-and-run" type of exocytosis. Endogenous VAMP8 and hGH-GFP were colocalized on the same vesicles at least in part. However, the constitutive exocytosis of hGH-GFP and CLuc, a secreted luciferase from Cypridina noctiluca, was normal, even when siRNAs for VAMP8 and VAMP3 robustly decreased their proteins. These results suggest that VAMP8 is not essential for constitutive exocytosis, although it can be involved in the exocytosis.