Figure 1.

NtPLC3 localizes to a subapical plasma membrane domain encompassing domains occupied by PIP5K2 or PIP5K11. The localization of NtPLC3 was monitored by confocal imaging upon transient expression in tobacco pollen tubes. For these localization studies, expression levels were kept low to minimize morphological changes of the pollen tube cells. (A) Coexpression of EYFP-PIP5K11 with RedStar-PLCδ1-PH, a fluorescent biosensor for PtdIns(4,5)P₂, as indicated. B-E, Association of fluorescence-tagged NtPLC3 with an extended subapical plasma membrane domain, which spans the region occupied by coexpressed PIP5K2-mCherry or EYFP-PIP5K11. (B) Coexpression of EYFP-NtPLC3 with PIP5K2-mCherry. (C) Coexpression of NtPLC3-RFP with EYFP-PIP5K11, monitored in a time lapse experiment. The time series shown was recorded for 600 s at a frame rate of 2.14 frames min^-1. Three selected frames are shown, as indicated. (D) Kymograph analysis of median confocal LSM sections of a growing pollen tube from (C), indicating the dynamic relative movements of PIP5K2-EYFP (green) and NtPLC3-RFP (red). Right panel: merged images (overlap indicated by yellow). (E) Intensity and distribution of the EYFP-PIP5K11 (green) and NtPLC3-RFP (red) signals were further analyzed by the machine learning program Ilastik to provide nonbiased results. Note that the relative distances from the pollen tube tip are roughly constant, and that the region occupied by NtPLC3-RFP continuously spans the region occupied by PIP5K2-EYFP. Data are representative for five independent experiments. Scale bars = 10 µm.