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Fig. 6. SDF-1 secreted from transfected COS cells acts as an extracellular signal regulating axonal patterning of CXCR4-GFP-expressing neurons. (A,B) COS cells transfected with CXCR4-GFP were incubated with or without 100 nM SDF-1 for 2 hours. In unstimulated cells, CXCR4-GFP was diffusely distributed throughout the cells (A) whereas SDF-1 stimulation induced the formation of CXCR4-GFP-containing intracellular clusters (B). (C) COS cells were separately transfected with either CXCR4-GFP (green) or SDF-1 (red, identified with an anti-SDF-1 antibody), and were subsequently co-cultured. CXCR4-GFP distribution exhibits a typical internalization pattern only if an SDF-1-expressing cell was located in the proximity of the former. By contrast, in cells distant from any SDF-1-transfected cells, CXCR4-GFP was distributed diffusely over the cell. (D,E) Hippocampal neurons were transfected at the time of plating with CXCR4-GFP. COS cells previously transfected with SDF-1 were added to the neuron culture at day 1 and the co-culture was fixed at day 2. CXCR4-GFP-expressing neurons in contact with SDF-1-expressing COS cells showed a reduced axonal length (E1,E2) compared to neurons distant from any SDF-1-transfected cells (D1,D2). Bar, 20 µm.
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