JCS037564 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Kinetics of 3′ phosphoinositide accumulation during fibroblast spreading. For the same cells used to generate the data presented in Fig. 1C,D, the normalized fluorescence intensity of EGFP-AktPH translocation, f (spatially averaged over the contact area) was quantified as a function of time, t, and normalized by each cell’s f_p,_PDGF value as in Fig. 1D (see Materials and Methods). The time axis is shifted for each cell so that t=0 corresponds to when the cell was first seen to attach by TIRF, and it is truncated at t=25 minutes because of the variable times at which PDGF and then wortmannin were subsequently added relative to t=0. The solid curve is the mean, and the dotted curves are mean ± s.d.

• Supplemental Figure S2 -

  Fig. S2. Blockade of protein synthesis by cycloheximide does not noticeably affect spreading or PI3K activation on poly-lysine. While incubated in suspension, EGFP-AktPH-expressing cells were treated with 25 µg/ml cycloheximide and then allowed to spread on poly-lysine in the continued presence of cycloheximide. A vehicle only control (DMSO) was performed in parallel. The drug did not noticeably affect the rate of spreading on poly-lysine or the spontaneous activation of PI3K signaling, ruling out the possibility that these responses were caused by deposition of cell-secreted ECM.

• Movie 1 -

  Movie 1. PI3K dynamics during spreading on fibronectin, as shown in Fig. 1A. The speed-up factor is 75×, and the scale bar is 20 µm.

• Movie 2 -

  Movie 2. PI3K dynamics during spreading on poly-lysine, as shown in Fig. 1B. The speed-up factor is 75×, and the scale bar is 20 µm.

• Movie 3 -

  Movie 3. Dynamic protrusion phenotype seen after cells spreading on fibronectin are treated with nocodazole and later with PDGF, as shown in Fig. 5A. The speed-up factor is 75×, and the scale bar is 20 µm.

• Movie 4 -

  Movie 4. Persistent movement and polarization of PI3K signaling during random migration, as shown in Fig. 6A. The speed-up factor is 600×, and the scale bar is 50 µm.

• Movie 5 -

  Movie 5. Dynamic PI3K signaling in concert with alternating protrusions and a change in the direction of migration, as shown in Fig. 6B. The speed-up factor is 600×, and the scale bar is 50 µm.