Lease squares fit yielded three fitting parameters for each dataset obtained in 3 or 17 mm glucose. Na+/K+ ATPase around the cell surface, thereby providing direct evidence for a GSIS-dependent surface exposure of the ZnT8 self-antigen. Moreover, the variation in tagged-ZnT8 expression and surface labeling enabled sorting of heterogeneous beta cells to subpopulations that exhibited marked differences in GSIS with parallel changes in endogenous ZnT8 expression. The abundant surface display of endogenous ZnT8 and its coupling to GSIS exhibited the potential of ZnT8 as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations. and are zinc ions, and represent GFP tags. in the beta cell indicate lateral diffusion of ZnT8 from the insulin granule to the surface membrane, indicate zinc transport or secretion, the indicates insulin secretion, and indicate insulin molecules. indicates positions of tagged ZnT8 between the 50C75-kDa markers. indicates the position of endogenous ZnT8 below the 50-kDa marker. A small protein band shift is usually noted between ZnT8-GFP and ZnT8-FLAG/GFP due to the FLAG-tag insertion. The unique insulin biology of the beta cell requires specialized proteins like ZnT8 to support a high flux turnover of the insulin secretory pathway. Many of these proteins are potential targets of the immune system with implications for the etiology of type 1 diabetes (19,C21), an autoimmune disease characterized by specific Mouse monoclonal to EphA2 T-cell-mediated autoimmune destruction of pancreatic beta cells (22). Both CD4+ and CD8+ T-cell responses against ZnT8 (23,C27) as well as anti-ZnT8 autoantibodies (28) were found in patients or model animals that developed autoimmune diabetes. Cell- or antibody-mediated autoimmune responses may be directly targeted at ZnT8 displayed around the beta cell surface either in a form of processed ZnT8 peptides associated with MHC class I molecules or intact ZnT8 self-antigen. Tracking the surface display of intact ZnT8 is the first step in closing a knowledge gap regarding how ZnT8 self-antigen may be exposed to extracellular immune surveillance before the onset of beta cell destruction. In particular, a putative coupling between the GSIS3 and ZnT8 surface display may enforce a positive feedback loop of beta cell destruction, starting from a moderate initial beta cell loss to a compensatory increase of GSIS/ZnT8 exposure that exacerbates autoimmune insults and beta cell loss. As such, the coupling between GSIS and ZnT8 surfacing may contribute to a vicious cycle of disease progression to overt type 1 diabetes with an ultimate loss of beta cell mass and deterioration of glycemic control (29). A critical technical barrier to tracking ZnT8 on the surface of Y16 live beta cells is the lack of a specific and sensitive ZnT8 tracer. Here, we report structure-guided tagging and specific immunofluorescence staining of ZnT8 around the extracellular surface of INS-1E cells (30) that stably expressed tagged ZnT8. Flow cytometry quantification of ZnT8 surface display revealed a sigmoidal correlation with GSIS. Further analysis of endogenous ZnT8 surfacing in clonal INS-1E cells exhibited that ZnT8 was abundantly displayed around the cell surface in a GSIS-dependent fashion, thereby providing direct experimental evidence to link increased insulin secretion and ZnT8 Y16 self-antigen exposure. Moreover, specific antibody labeling of tagged-ZnT8 around the cell surface enabled flow cytometry sorting of heterogeneous INS-1E cells to subpopulations with varied GSIS phenotypes, suggesting that ZnT8 may be used as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations. Results Design of an Antibody-ZnT8 Complex The luminal face of a granular ZnT8 would be displayed around the cell surface if ZnT8 is usually translocated to the plasma membrane after insulin granule exocytosis (Fig. 1and indicate the peak positions of unbound ZnT8-FLAG/GFP and ZnT8-FLAG/GFP-antibody complex, respectively. and and or and and axis); cells in with mid-range GFP intensity showed a large spread of anti-FLAG fluorescence intensity around the axis, whereas the low or high GFP cells in or displayed limited Y16 anti-FLAG changes at a basal level (Fig. 3mRNA was detected by RT-PCR, suggesting that the large GFP buildup in high GFP cells was a result of proteolytic degradation of the ZnT8-FLAG/GFP protein as opposed to an overexpression of GFP from a truncated genomic sequence. Because the GFP signal in high GFP cells was irrelevant to the cellular ZnT8-FLAG/GFP protein level, this populace (3C4% of total cells) was excluded from analysis. Fluorescence data corresponding to each of the five sorted cell populations (group 1C5) were used to construct calibration curves that correlated the FSEC peak height to the.
