A Polybasic Motif Allows N-WASP to Act as a Sensor of PIP2 Density
Supplemental
Figure S1. PIP2 binding assays
Supplemental
Figure S2. Actin polymerization assays
Supplemental
Figure S3. PIP2 activation curve for deltaEVH1-N-WASP
Supplemental
Figure S4. Activation of N-WASP by PIP3
Supplemental
Figure S5. Co-activation by Cdc42
Timelapse Movies: Endosomal Vesicle Rocketing in Cells Transfected with Wt
and 14K N-WASP
Supplemental
Movie 1
Supplemental Movie 2
Movies were made from timelapse images taken every 5 s over a time period of 5–10 min. Live cells were visualized by fluorescence microscopy under 600× magnification 24 hr after transfection with the appropriate constructs (N-WASP variant and actin-GFP). Supplemental Movie 1 depicts a cell transfected with 14K N-WASP and actin-GFP. Actin comet tails can be seen rocketing around the cytoplasm. For comparison, Supplemental Movie 2 depicts an example of a cell transfected with 9K N-WASP and actin-GFP. No actin rocketing is observed. Only slight fluctuations of vacuoles are observed. Played with Windows Media Player, these movies run 15 s (14 K) and 8 s (9 K) each. The movies have been accelerated by a factor of approximately 35-fold above real time. See Experimental Procedures for further details.