JCS011130 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Quantification of N18-HASPB-GFP expression levels. Quantitative analysis of N18-HASPB-GFP expression levels under the conditions used in Fig. 1. (A) Western blot analysis. The N18-HASPB-GFP signal (see insert) was normalized to the GAPDH loading control following quantification by the LI-COR Odyssey infrared imaging system and relative expressions levels are depicted. (B) In parallel, GFP fluorescent intensities of the cells were analyzed by flow cytometry and the relative expression levels presented are arithmetic means of three independent experiments+s.d. Values for 1000 ng/ml dox were set to 100%.

• Supplemental Figure S2 -

  Fig. S2. Characteristics of N18-HASPB-GFP-induced plasma membrane blebs. (A) Average blebbing time: the average time during expansion, stationary and retraction phase of 40 individual blebs is plotted for all analyzed blebs (light grey), stationary blebs (medium grey) and laterally motile blebs (dark gray). The s.d. is indicated by the error bars. (B) Average blebbing distances: the average distance blebs move during expansion, stationary and retraction phase is plotted for all 40 analyzed blebs (light grey), stationary blebs (medium grey) and laterally motile blebs (dark grey). The s.d. is indicated by the error bars. (C) Average speed during bleb formation and retraction: the average speed of the bleb apex relative to the PM at the base of the bleb during expansion, stationary and retraction phase is plotted for all 40 analyzed blebs (light grey), stationary blebs (medium grey) and laterally motile blebs (dark grey). The s.d. is indicated by the error bars. (D-F) Distribution of the maximum bleb speed during expansion, stationary and retraction phase: the distribution of the maximum bleb speed during expansion (D), stationary (E) and retraction (F) phase is plotted for all analyzed blebs.

• Supplemental Figure S3 -

  Fig. S3. 4D analysis of HASPB-induced PM blebbing. (A) Three representative time-points from a 4D time-lapse of N18-HASPB-GFP-expressing cells (also see Movie 2). The raw data was filtered visualized with Imaris (Bitplane) with a 3×3x3 median filter followed by an edge preserving filter (width=0.140 μm). The blebbing PM is represented by iso surface rendering. The grid size is 5 μm. (B) Three representative time-points from a 4D time-lapse of N18-Δmyr-HASPB-GFP-expressing cells (also see Movie 3). The raw data was filtered and visualized with Imaris with a 3×3×3 median filter followed by an edge preserving filter (width=0.140 μm). The blebbing PM is represented by iso surface rendering. The grid size is 5 μm.

• Supplemental Figure S4 -

  Fig. S4. Sequence alignment of the SH4 domains analyzed for plasma membrane blebbing in this study.

• Supplemental Figure S5 -

  Fig. S5. Bulk Src, MLC and filaminA phosphorylation in SH4-domain-expressing cells. Western blot analysis of the indicated cell lines without (Dox −) or following (Dox +) induction of N18-GFP expression. (A) Src and pSrc (Y⁴¹⁸). (B) MLC and pMLC (S¹⁹). (C) FLNa and pFLNa (S²¹⁵²).

• Movie 1 -

  Movie 1. 2D time-lapse of the N18-HASPB-GFP-expressing cells also used in Fig. 2. The sequence was acquired with an exposure time of 150 ms without delay between time points. Scale bar, 10 μm. Displayed is the time relative to start of the series. Video speed is approx. 14 times faster than original acquisition speed.

• Movie 2 -

  Movie 2. 4D time-lapse of N18-HASPB-GFP cells (of the data set in Fig. S3A) visualized using iso surface rendering of the PM. The grid distance is 5 μm. Video speed is approx. 7.5 times faster than the original acquisition speed.

• Movie 3 -

  Movie 3. 4D time-lapse of N18-Δmyr-HASPB-GFP cells (of the data set in Fig. S3B) visualized using iso surface rendering of the PM. The grid distance is 5 μm. Video speed is approx. 7.5 times faster than the original acquisition speed.

• Movie 4 -

  Movie 4. N18-HASPB-GFP cells show rounded and bleb-associated motility in Matrigel. Confocal sections were used to generate maximum projections from which video animations were made using Leica confocal simulator software. Shown is the GFP channel of an animation of two cells expressing N18-HASPB-GFP and mRFP-actin.

• Movie 5 -

  Movie 5. mRFP-actin fluorescence of the cells depicted in Movie 4.