Extended Data Fig. 2: Tracking Whi3 puncta and ER co-localization.

(a) Time-lapse montages of Whi3 puncta associated with ER, including ER tubules (yellow arrowheads) and nuclear-associated ER (blue arrowheads). White dashed lines in first frame indicate cell periphery. Similar to Fig. 1d. (b) First frame from time-lapse shown in Supplementary Video 1. Yellow arrows indicate puncta that appear co-localized with the ER but moved out of the imaging plane during the movie and were not included in the tracking. (c) ER channel from the image in (b) with overlaid Whi3 tracks, colored according to the fraction of the track lifetime spent co-localized with the ER. All tracks clearly co-localize with ER structures. Not all tracks begin in the indicated frame. (d) Relative, local intensity in the ER channel as a function of time for the Whi3 tracks shown in (c), expressed as a fraction of the median intensity in the ER channel throughout the cell. Values greater than one (red region) were defined as co-localized with the ER. In this example, all tracks spend 100% of the lifetime co-localized with the ER. (e) Histogram of the tracks in (c-d), binned according to the fraction of track lifetime co-localized with the ER (similar to Fig. 1f). n = 7 tracked Whi3 puncta in this representative example from 60 hyphae. (f) Average intensity of all tracked Whi3 puncta as a function of the fraction of the track lifetime spent associated with the ER. Data points show moving average of the raw data, with lifetime fraction increments of 0.2. Data are mean ± 95% c.i. n = 83, 37, 45, 69, and 535 tracks in bins centered at 0.1, 0.3, 0.5, 0.7 and 0.9, respectively.

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