JCS02385 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. Endogenous Pak activation after scratched-induced migration. Activation of Pak after wounding is monitored by western blot using a phospho-specific antibody directed against active, auto-phosphorylated Pak. Similar results were obtained with a phospho-Pak1/2 T423/402 antibody (data not shown). Pak3 is not expressed in these cells (data not shown).

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. Activation of Pak is not affected by inhibition of Rac. Top panel: Phospho-Pak (P-Pak) staining, and thus Pak activation, is specifically ablated in cells coinjected with pmRFP::wt Pak1 (Pak1) and pRK5-MYC::Pak1 AID (PakAID). Bottom panel: Pak activation is not affected in cells coinjected with pmRFP::wt Pak1 (Pak1) and pRK5-MYC::Rac N17 (Rac N17). Rac N17 expression induces the typical contraction of the cell and decrease in protrusion formation. Bar, 20 µm

• Movie 1
• Movie 2 -

  Movies 1 and 2. Cdc42 inhibition leads to delocalized protrusions. After scratching a confluent monolayer, leading-edge cells were co-injected with pEGFP-F together with empty plasmid (Movie 1), pRK5-MYC::WASp CRIB (Movie 2). 4.5 hours after scratching, movies were acquired with 5 min time interval. Defining ‘protrusion’ as a segment of membrane moving away from the centre of the cell between two intervals, 70% of the protrusions were found restricted to the front (Movie 1) in wound edge cells. This confirms the strong bias towards the localization of dynamic protrusions to the front. Video analysis after inhibition of Cdc42 (Movie 2) revealed unbiased protrusive activity all around the cell periphery. Bar, 20 µm.

• Movie 3 -

  Movie 3. Pak kinase is required for the localization of protrusions to the front of migrating cells. After scratching a confluent monolayer, leading-edge cells were co-injected with pEGFP-F and pRFP::Pak1 AID. The movie is representative of a minimum of five cells. Localization of the protrusions is unbiased as only 27% of the protrusions are found at the front. Bar, 20 µm.