FIGURE 10.

The domain SgII-(333-348) functions as a transferable DCG-sorting determinant in PC12 cells. SIG-SgII-(334-348)-GFP colocalizes with secretory granule marker CgB in PC12 cells (A). Cells transfected with pCMV-SgII-(1181-1225)-GFP were processed for immunocytochemistry using a polyclonal anti-CgB primary and an Alexa Fluor 594-conjugated secondary antibodies, followed by imaging by deconvolution microscopy. Yellow regions indicate an overlap in the distribution of SIG-SgII-(334-348)-GFP (green) and endogenous secretory granules marker CgB (red), and the extent was determined using ImageMaster 5.0 (Table 1). B, accumulation of SIG-SgII-(334-348)-GFP puncta along and at the tip of neurite processes. NGF-differentiated PC12 cells were transiently transfected with pCMV-SgII-(1181-1225)-GFP and labeled with CellTracker Orange CMRA dye (red) prior to fixation to visualize the cell body and neurite processes. Deconvolution microscopy imaging reveals a substantial accumulation of SIG-SgII-(334-348)-GFP chimera at the tip of neurites, as shown in the enlarged inset. C, colocalization between SIG-SgII-(334-348)-EAP and secretory granule marker CgB. Cells expressing SIG-SgII-(334-348)-EAP were processed for immunocytochemistry using polyclonal anti-CgB and anti-human placental alkaline phosphatase primary and Alexa Fluor 594-conjugated and 488-conjugated secondary antibodies. Quantification of fluorescence overlap is reported in Table 1. D, regulated secretion of SIG-SgII-(334-348)-EAP. PC12 cells expressing SIG-EAP, SIG-SgII-EAP, or SIG-SgII-(334-348)-EAP were incubated for 15 min in secretion medium alone (mock) or 2 mm Ba²⁺. The enzymatic activity of EAP chimeras was assayed in the culture supernatant and cell lysate, and relative secretion was determined as described in the legend of Fig. 4. Values are given as the means ± S.E. of triplicate determinations. †, p > 0.05; ^***, p < 0.001 as compared with control (mock), ANOVA with Dunnett's post test. Nuclei are stained with Hoechst 33342 (blue). Scale bar, 5 μm.