The protein IA-2, which is a member of the transmembrane protein tyrosine phosphatase family and a major autoantigen in type 1 diabetes, is found in dense-core vesicles (DCVs) of many neuroendocrine tissues. The luminal domain of IA-2 has a significant homology with the protein RESP18, which is also found in the lumen of the endoplasmic reticulum of neuroendocrine cells. Very little is known about the function of RESP18, but it has been hypothesized that the protein is implicated in the regulation of dense-core vesicle exocytosis in pancreatic islet cells. We have tested this hypothesis by localizing RESP18 using immunoelectron microscopy with anti-RESP18 polyclonal antibody, detected by colloidal gold conjugated to secondary antibody. We found that RESP18 is mainly expressed in the lumen of DCVs, and only to a lesser extent in the ER and Golgi network. Furthermore, double labeling experiments with 5-nm and 10-nm gold nanoparticles showed that RESP18 is co-localized with insulin in pancreatic beta cell vesicles, as well as with glucagon in pancreatic alpha cell vesicles. One of the limitations of immunolabeling of plastic sections is difficulty in penetration of the labels after embedding. Experiments are therefore being performed to immunolabel cells that have been frozen and cryosectioned after cryoprotection with gelatin and sucrose (Tokuyasu method). Frozen Islets are cryosectioned using a Leica Ultracut UCT ultramicrotome with an FCS cryosectioning accessory. After thawing, immuno-gold labeling and embedding the sections, it is planned to perform electron tomography to determine the penetration of gold nanoparticles of different diameters, in multiple labeling experiments.
| Cai, Tao; Hirai, Hiroki; Fukushige, Tetsunari et al. (2009) Loss of the transcriptional repressor PAG-3/Gfi-1 results in enhanced neurosecretion that is dependent on the dense-core vesicle membrane protein IDA-1/IA-2. PLoS Genet 5:e1000447 |
