A polarized upstream GTPase, Cdc42, sets a permissive zone for ventral WASP puncta formation. (A) Endogenously tagged WASP forms puncta that overlap with regions of active Cdc42 (assayed via TagRFP-T–conjugated WASP-GBD; Nalbant et al., 2004). (B) Cdc42 activity exists as a gradient across the ventral surface of polarized HL-60s that extends across the front half of the cell as previously observed in Yang et al. (2016). Biosensor data are an average normalized line scan along the major axis of the cell for 49 cells collected over three experiments. Overlaid is the WASP puncta distribution presented in Fig. 1 D. (C) Trajectories of single WASP puncta rendered in the cell’s frame of reference show that puncta nucleate at the cell front and extinguish around half the cell length. Trajectories are overlaid onto the summed frames of a registered cell migrating over 40 s and are colored according to relative temporal position within a given puncta’s total duration. See also Video 6. (D) Positions of WASP puncta appearance and disappearance mapped onto a representative cell shape support this front bias across many cells. Positions were recorded for >650 puncta from 26 cells in three experiments. (E) Cumulative distribution functions for relative puncta appearance (green) and disappearance (red) positions show that the majority of events occur before one half of the cell length (vertical dashed line). (F) Endogenous WASP distributions in WT and Cdc42 KO cells reveal a loss of ventral WASP puncta in the absence of Cdc42. Images are displayed on the same intensity scale. (G) Number of WASP puncta per cell in WT and Cdc42 KO cells. nWT = 46 and nKO = 40 cells collected across three replicates. P = 0.005 by a paired two-tailed t test on replicate means. Scale bars are all 5 μm.
