Figure 6: GBP effect on a reporter assay for Metchnikowin expression in Drosophila plasmatocytes and S2 cells, and a model of the GBP signalling pathway.

(a) Low magnification view of Drosophila plasmatocytes. Larvae expressing GFP under the control of the Mtk promoter were injected with ~20 nmol per larva of GBP, 1 h before plasmatocytes were prepared. Non-spread (blue arrowhead) and spread (red arrowhead) plasmatocytes were observed by laser scanning confocal microscopy (EZ-Ti, Nikon). Scale bar, 20 μm. (b) High magnification view of Drosophila plasmatocytes. Other explanations are as in a. (c) Quantitative analysis of GFP expression in non-spread and spread cells. Cell morphology was scored by counting ~200 cells from a randomly selected view. Average fluorescent signal intensities of each cell were quantified by the confocal microscope software EZ-C1 (Nikon). Each value represents the mean±s.d. for four independent determinations (**P<0.01): Significant differences are indicated by Tukey’s HSD. (d) Effect of GBP on GFP expression in S2 cells expressing GFP under the control of the Mtk promoter. One hour after addition of 50 nM GBP, S2 cells were analysed by confocal microscopy as described in a. Scale bar, 10 μm. (e) Overview of the new positive and negative regulatory features of the GBP signalling pathway described in the current study: Stimulation of PLC by activated GBPR produces IP₃, which promotes extracellular Ca²⁺ influx. We have previously demonstrated the presence of GBPR together with its adaptor protein (P77) ref. 38,42. Elevated Ca²⁺ triggers secretion of Pvf which stimulates Pvr. Activation of Pvr in turn promotes ERK phosphorylation, which activates cellular immune responses (cellular immunity) such as encapsulation while simultaneously repressing the expression of immune effector molecules such as Mtk (humoral immunity). See the text for discussion of the possibility that Pvf2 may be activated by proteolysis.