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. 2018 Mar 26;115(15):E3578–E3587. doi: 10.1073/pnas.1800182115

Fig. 4.

Fig. 4.

CSI1, PTL1, and the exocyst sequentially regulate the delivery of CSCs. (A) CSI1 shows a strong bias to appear before the delivery of CSCs. To visualize the delivery sequence of CSI1 and CSCs, a FRAP assay was performed in a dual-labeled line expressing RFP–CSI1 and GFP–CESA6. Kymographs were generated from a 10-min movie (5-s interval) to visualize the dynamics of particles over time. Shown is a representative kymograph displaying a CSC delivery event, in which CSC was delivered to an existing RFP–CSI1. The delivery of CSC is accompanied by pausing of the Golgi apparatus. A cartoon representation of the delivery event is shown on the Right. (B) Quantification of the time required for the delivery of CSCs and CSI1 in A. n = 16 particles for both GFP–CESA6 and RFP–CSI1. Red bar represents average time. P < 0.0001. (C) In the patrol1-2 mutants, CSI1 still appears before the delivery of CSCs. The same FRAP assay, as describe in A, was performed in the patrol1-2 mutants expressing RFP–CSI1 and YFP–CESA6. Shown is a representative delivery event and the corresponding cartoon representation. (D) Quantification of the delivery time of CSI1 and CSCs in the patrol1-2 mutants. n = 16 particles for both YFP–CESA6 and RFP–CSI1. Red bar represents average time. P < 0.001. (E) PTL1 shows a strong bias to appear at the same time as CSCs. FRAP assays were performed in dual-labeled lines of RFP–PTL1 and YFP–CESA6. Kymographs were derived from 6-min movies (1-s intervals). Arrowheads point to PTL1 particles that appeared at the same time as delivery of CSCs to the PM. n = 105 observations from eight cells. A cartoon representation of the delivery event is shown on the Right. (F) Exocyst shows a strong bias to appear at the same time as CSCs. FRAP assays were performed in dual-labeled lines of GFP–Sec5B and RFP–CESA3. Kymographs were derived from 5-min movies (2-s intervals). Shown are representative kymographs where an exocyst particle labeled by GFP–Sec5B coappeared with a CSC particle labeled by RFP–CESA3. n = 98 observations from six cells. A cartoon representation of the delivery event is shown on the Right.