JCS03475 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Interaction of the activated TC10β isoform with Exo70 in PC12 cells. (A-O) mCFP-labeled wild-type (wt) TC10β (A,C,F,H) or dominant-negative TC10β-T25N (K,M) were expressed alone (A,F,K) or co-expressed (C,H,M) with mVenus-Exo70 (D,I,N). mCFP fluorescence lifetimes (τ) (B,E,G,J,L,O) were imaged by two-photon-time domain FLIM. Lifetimes are indicated in false color ranging from 1.5 nanoseconds (blue) to 2.5 nanoseconds (red). As observed for the TC10α isoform (Fig. 2), reduced lifetimes can be detected for the mCFP-labeled wild-type form of TC10β interacting with mVenus-Exo70 upon NGF-induction (E, n=17), but not in non-treated cells (J, n=13). Lifetimes of mCFP-TC10β-T25N were not significantly reduced upon co-expression with mVenus-Exo70 (O, n=17). Bars: 5 μm (untreated cells), 10 μm (NGF-treated cells). (P) Cumulative histograms of cellular lifetime distributions for FRET between mCFP-TC10β and mVenus-Exo70. Letters at the traces refer to correspondingly indicated conditions in the above panel (E,J,O). Note the appearance of lower lifetimes (higher FRET efficiencies) for NGF-treated mCFP-TC10β-co-expressing PC12 cells only (E).

• Supplemental Figure S2 -

  Fig. S2. Transfection of control siRNA does not affect the NGF-induced activation of N-WASP. Expression levels of co-expressed proteins. (A) Representative images of N-WASP biosensor expressing PC12 cells transfected with control siRNA. Intensities of CFP (Donor), YFP (Acceptor) and the FRET ratio are shown (left) for representative cells; cumulative FRET ratios are shown for both conditions as in Fig. 4 (right). Bars: 5 μm (untreated cells), 10 μm (NGF-treated cells). (B) The expression levels of the proteins that were co-expressed with the N-WASP biosensor were estimated by Western blotting of equal protein amounts of homogenate with respective antibodies. Shown are Exo70, Cdc42-G12V and TC10α-Q67L. With an estimated transfection efficiency of 25%, these levels amount to ∼4 times the endogenous concentration. No degradation products were observed.

• Supplemental Figure S3 -

  Fig. S3. Expression of RhoGDIα reduces the activity state of N-WASP. Representative images of N-WASP biosensor expressing PC12 cells expressing HA-tagged RhoGDIα. Intensities of CFP (Donor), YFP (Acceptor) and the FRET ratio are shown (left) for representative cells; cumulative FRET ratios are shown for both conditions as in Fig. 4 (right). Bars: 5 μm (untreated cells), 10 μm (NGF-treated cells).

• Supplemental Figure S4 -

  Fig. S4. Co-expression of constitutively active TC10β-Q69L or TC10β-Q69L and Exo70 antagonize the NGF-induced activation of N-WASP. (A,B) Representative images of N-WASP biosensor expressing PC12 cells co-expressing constitutively active HA-tagged TC10β-Q69L (A) or HA-tagged TC10β-Q69L and FLAG-tagged Exo70 (B). Intensities of CFP (Donor), YFP (Acceptor) and the FRET ratio are shown (left) for representative cells; cumulative FRET ratios are shown for both conditions as in Fig. 4 (right). Bars: 5 μm (untreated cells), 10 μm (NGF-treated cells).