Figure 5.

Addition of PDBu identifies the nuclear membrane as a selective target for Cys1–GFP translocation. (A) Series of three confocal fluorescence images of RBL cells expressing Cys1–GFP and stimulated by addition of PDBu (1 μM). The image on the left was recorded before PDBu addition, the middle image 1 min after PDBu addition, and the image on the right 10 min after PDBu addition. PDBu induced an initial localization of the Cys1–GFP to the nuclear membrane (middle). This nuclear localization became weaker in time, possibly due to the translocation of Cys1–GFP to the plasma membrane (right). Images were corrected for photobleaching. (B) PDBu concentration dependence of the plasma and nuclear membrane translocation of Cys1–GFP. Maximum translocation is reached at ∼40 nM PDBu for the plasma membrane and at 300 nM for the nuclear membrane. (C) Time course of translocation of Cys1–GFP to plasma and nuclear membrane in response to PDBu (1 μM). The relative increase in plasma membrane fluorescence reached a plateau after ∼60–120 s, whereas the relative increase in nuclear membrane fluorescence reached a maximum during a similar time period but then slowly decreased over time. (D–F) Analysis of plasma and nuclear membrane photobleaching recovery experiments. In D, the raw fluorescence recovery traces are shown. Interestingly, the recovery was faster for the nuclear membrane than for the plasma membrane. E shows a graph of the square radius of the bleach profile as a function of time for nuclear and plasma membrane. The apparent lateral diffusion coefficient of the Cys1–GFP in the two membranes was similar in both membranes. F shows the calculated time course of dissociation of Cys1–GFP away from the nuclear versus plasma membrane. The significantly faster apparent dissociation time from the nuclear membrane suggests that Cys1-domains have a lower affinity for the nuclear membrane compared with the plasma membrane.