JCB. Supplemental material THE JOURNAL OF CELL BIOLOGY. Hong et al.,

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1 Supplemental material JCB Hong et al., THE JOURNAL OF CELL BIOLOGY Figure S1. Analysis of purified proteins by SDS-PAGE and pull-down assays. (A) Coomassie-stained gels of purified GST-tagged proteins. (B) Coomassiestained gels of purified proteins used in vitro actin branching and debranching assays. (C) Pull-down of GST-tagged cortactin by liposomes bearing the indicated PIPs and blotted with anticortactin antibody. The relative affinity of GST cortactin for liposomes containing different phosphoinositides was quantified by densitometric analysis of the anticortactin immunoblots from three independent experiments. Note the similarity in result with Fig. 1, in which the immunoblots were probed with anti-gst antibody. Mean ± SE. **, P < JCB 2015 PI(3,5)P 2 regulates cortactin actin interactions Hong et al. S18

2 Figure S2. Effect of inhibition of PI(3,5)P 2 synthesis on endosomal actin, endosomal compartments, and cofilin phosphorylation. (A) Immunolocalization of Rab7 (green), actin (red), and cortactin (blue) in DMSO or YM treated SCC61 cells. Bars, 20 µm (3 µm for magnifications). (B) Quantitation of percentage of colocalization of cortactin or actin with Rab7. Note decrease in actin localization to late endosomes in cortactin KD (KD) cells treated with YM independent experiments, n 45 in each condition. (C) Immunolocalization of cortactin (green), actin (red), and Rab7 (blue) in DMSO or apilimod-treated MDA-MB-231 cells. Bars, 20 µm. (D) Quantitation of percentage of colocalization of cortactin or actin with Rab7. ***, P < 0.001; ****, P < (E) Representative images from MDA-MB-231 cells showing localization of early (EEA1, green) and late (Rab7, red) endosome markers upon 2 h treatment with 800 nm YM Bars, 20 µm (5 µm for magnifications). (F) Representative Western blot of whole-cell lysates from cells treated with YM201636, DMSO, or no diluent, as indicated, and probed for cofilin phosphorylated on serine 3 (p-cofilin), total cofilin (t-cofilin), or β-actin as a loading control. Ratio of phospho-cofilin to total cofilin in whole-cell lysates was quantified by densitometric analysis of Western blot data from three independent experiments. Mean ± SE plotted. S19

3 Figure S3. Analysis of the contribution of lysines in the cortactin fourth repeat domain to F-actin and PI(3,5)P 2 binding. (A) Schematic cartoon of mutant cortactin containing K to Q mutations within the fourth repeat. (B) Coomassie stained of gels of purified GST-tagged cortactin mutants. (C) GST-tagged WT and mutant cortactin proteins were incubated with F-actin, and co-sedimented. Supernatants (S) and pellets (P) were subjected to Western blotting with an anti-gst antibody. The proportion of bound (detected in P) and unbound (detected in S) cortactin to F-actin was plotted. (D) Pull-down of GST-tagged WT and mutant cortactin by PI(3,5)P 2 -liposomes and blotted with anti-gst antibody. The relative affinity of GST cortactin for liposomes containing PI(3,5)P 2 was quantified by densitometric analysis of the anti-cortactin immunoblots from 4 independent experiments. Mean ± SE. **, P < JCB 2015 PI(3,5)P 2 regulates cortactin actin interactions Hong et al. S20

4 Figure S4. Measurement and calculation of phosphoinositide and cortactin levels in MDA-MB-231 cells. Deacylated PIP samples for yeast (used as a positive control) and MDA-MB-231 cells were analyzed by HPLC, as described in the Materials and methods. (A) Osmotic shock of the yeasts with 1 M KCl for 10 min increased the PI(3,5)P 2 level (red trace) compared with control cells (in black). This experiment was completed once. (B) From the yeast experiment shown in A, the total counts of PI(3,5)P 2 were calculated by adding counts up from fractions 30, 31, and 32, and subtracting the basal level, whereas the total counts of PI(4,5)P 2 were calculated by adding up counts from fractions 33, 34, and 35 and subtracting the basal level. (C) Phospholipid profiles in MDA-MB-231 cells were analyzed from three independent experiments. (D) In MDA cells, fractions 30, 31, and 32 include both PI(3,5)P 2 and PI(3,4)P 2 as they are eluted in same fractions. Thus, the total counts of PI(3,5)P 2 and PI(3,4)P 2 were calculated by adding up counts from fractions 30, 31, and 32 and subtracting the basal level, and the total counts of PI(4,5)P 2 were calculated by adding up counts from fractions 33, 34, and 35 and subtracting the basal level. (E) Representative immunoblot (from n = 3 experiments) of purified cortactin that we used for a standard curve and total cell lysate from 500,000 MDA-MB-231 cells. (F) Table contents indicate all values and sources that we used for calculating cellular concentrations of PI(3,5)P 2 and cortactin. S21

5 Figure S5. PI(3,5)P 2 regulates actin assembly in cells with varying cortactin expression levels. (A) Immunoblotof cortactin expression and β-actin loading control in MDA-MB-231 cells. Relative expression level of cortactin to actin in cells was calculated based on the band intensities. This experiment was completed once. (B) Representative images showing cortactin (green) and actin (red) localization on Rab7 + endosomes (blue) after 2-h treatment with 800 nm YM or DMSO diluent control in cortactin OE MDA-MB-231 cells. Bars, 20 µm (3 µm for magnifications). (C) Images were analyzed for percentage of colocalization of cortactin or actin with Rab7. 2 independent experiments, n 50 cells in each condition. The DMSO and YM data for parental cells are the same data as that shown in Fig. 3 (C and D) and Fig. 4 (B and C). ##, P < 0.01; ****, P < Asterisks compare DMSO versus YM condition of cortactin OEs and # symbols compare parental versus OE in YM treated condition JCB 2015 PI(3,5)P 2 regulates cortactin actin interactions Hong et al. S22

6 Video 1. Rab7 + endosome enlargement by YM treatment. Time-lapse confocal microscopy of live MDA-MB-231 cells stably expressing mrfp-rab7. 20 min after YM treatment, images were acquired using a laser-scanning confocal microscope (LSM710; Carl Zeiss). Frames were taken every 10 s for 75 min. Video 2. Actin branching reaction, +buffer condition. TIRF microscopy movie showing polymerization reaction containing 0.75 µm 33% Oregon Green actin, 10 nm Arp2/3 complex, 50 nm GST-VCA. Single-wavelength (488 nm) images were acquired using Nikon TiE inverted microscope every 15 s with an exposure time of 30 ms and played back at 10 frames per second (fps). Bar,5 µm. This video corresponds to Fig. 6 A. Video 3. Actin branching reaction, +cortactin condition. TIRF microscopy movie showing polymerization reaction containing 0.75 µm 33% Oregon Green actin, 10 nm Arp2/3 complex, 50 nm GST-VCA, 50 nm cortactin. Single-wavelength (488 nm) images were acquired using Nikon TiE inverted microscope every 15 s with an exposure time of 30 ms and played back at 10 fps. Bar, 5 µm. This video corresponds to Fig. 6 A. Video 4. Actin branching reaction, +PI(3,5)P 2 -liposomes condition. TIRF microscopy movie showing polymerization reaction containing 0.75 µm 33% Oregon Green actin, 10 nm Arp2/3 complex, 50 nm GST-VCA, 200 nm PI(3,5)P 2 -liposomes. Single-wavelength (488 nm) images were acquired using a Nikon TiE inverted microscope every 10 s with an exposure time of 30 ms and played back at 15 fps. Bar, 5 µm. This video corresponds to Fig. 6 A. Video 5. Actin branching reaction, +cortactin+pi(3,5)p 2 -liposomes condition. TIRF microscopy movie showing polymerization reaction containing 0.75 µm 33% Oregon Green actin, 10 nm Arp2/3 complex, 50 nm GST-VCA, 50 nm cortactin, and 200 nm PI(3,5)P 2 -liposomes. Single-wavelength (488 nm) images were acquired using a Nikon TiE inverted microscope every 10 s with an exposure time of 30 ms and played back at 15 fps. Bar, 5 µm. This video corresponds to Fig. 6 A. Video 6. Actin branching reaction, +cortactin+pi(3,4)p 2 -liposomes condition. TIRF microscopy movie showing polymerization reaction containing 0.75 µm 33% Oregon Green actin, 10 nm Arp2/3 complex, 50 nm GST-VCA, 50 nm cortactin, and 200 nm PI(3,4)P 2 -liposomes. Single-wavelength (488 nm) images were acquired using a Nikon TiE inverted microscope every 10 s with an exposure time of 50 ms and played back at 15 fps. Bar, 5 µm. This video corresponds to Fig. 6 A. S23

7 Video 7. Actin stability on Rab7 + endosomes. MDA-MB-231 control and cortactin KD (KD) cells expressing megfp-f-tractin were treated with DMSO or YM for 2 h, and then cells were analyzed by live imaging on a DeltaVison deconvolution microscope (Applied Precision). Frames were taken every 2 s for 52 s, immediately after Latrunculin A treatment. Bars, 5 µm. This video corresponds to Fig. 7. S24 JCB 2015 PI(3,5)P 2 regulates cortactin actin interactions Hong et al. S24