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. 2009 Jun 15;20(12):2932–2942. doi: 10.1091/mbc.E08-12-1193

Figure 3.

Figure 3.

Outer membrane fusion precedes fusion of the inner membrane and transfer of NPCs. (A) Transfer of HMG1-GFP compared with Sik1p-mRFP (and Spc42p-mRFP). Note that the HMG1-GFP is detected in the trans-nucleus long before Sik1p-mRFP (asterisk, arrow; ATY1917 × ATY1528). (B) Transfer of the tagged inner membrane protein, GFP-Prm3p, compared with Sik1p-mRFP (and Spc42p-mRFP). GFP-Prm3p and Sik1p-mRFP are detected in the trans-nucleus at essentially the same time (asterisk, arrow). The cell that expresses the mRFP-tagged proteins also expresses Mid2p-GFP (ATY1919 × ATY3149). Note: GFP-Prm3p was not preinduced. (C) Transfer of the tagged protein of the periphery of the nucleoplasm, GFP-Esc1p, compared with Sik1p-mRFP (and Spc42p-mRFP). GFP-Esc1p and Sik1p-mRFP are detected in the trans-nucleus at approximately the same time (asterisk, arrow; ATY1917 × ATY1550). (D) A cell expressing Sik1p-mRFP and Spc42p-mRFP (ATY1917) was crossed with a cell expressing Nup49p-GFP (ATY2226). Sik1p-mRFP arrives in the trans-nucleus well before the NPCs (asterisk), and the tagged NPCs invade the trans-NE (arrow) when dilation of the nexus is seen. Dilation of the nexus is accompanied by separation of the tagged SPBs from one face of the NE. Note: Experiments that follow a mRFP-tagged inner membrane protein along with HMG1-GFP also indicate initial fusion of the outer membrane, but the mRFP signal is not sufficiently strong to allow definitive imaging. (E) Summary diagram of the sequential events of NE fusion. The steps are (I) SPB contact, (II) appearance of the apical discontinuity of the NE, (III) initial transfer of outer membrane proteins, (III′) continued transfer of outer membrane and spreading of the trans-NE signal to the cortical ER, (IV) transfer of inner membrane proteins and the lamina, as well as nucleoplasmic continuity, and (V) visible dilation of the nexus, transfer of NPCs and disengagement of the SPB from one face of the NE. There are obvious delays between steps III and IV and between IV and V, suggesting that, at the molecular level, these intervening periods allow appropriate preparations for the succeeding events. HMG1-GFP, green; NPCs, yellow.