Molecular Cell, Volume 36 Supplemental Data A Reciprocal Interdependence between Nck and PI(4,5)P 2 Promotes Localized N-WASp-Mediated Actin Polymerization in Living Cells Gonzalo M. Rivera, Dan Vasilescu, Venizelos Papayannopoulos, Wendell A. Lim, and Bruce J. Mayer Supplemental Experimental Procedures Plasmids Details of constructions in the mammalian expression vector pebb encoding fusion proteins consisting of the extracellular domain of CD16, the transmembrane domain of CD, and all three SH3domains from Nck have been previously described (Rivera et al., 2004). The wild type or catalytically inactive (D858A) phosphatase domain (residues 351-874) of human synaptojanin 1, was subcloned into pebb/cd16/7 using standard procedures. The pleckstrin homology domain of PLCδ 1 (Balla and Varnai, 2002) was subcloned N-terminal to EGFP or mrfp in pebb. N- WASp constructs were previously described (Papayannopoulos et al., 2005). The wild type murine phosphatidylinositol 5-kinase (PIP5K) type Iα or its catalytically inactive mutant (D227A) cdnas (Tolias et al., 1998) were subcloned into pebb. Wild type Nck and Nck2 constructs in pebb were previously described (Rivera et al., 2006; Tanaka et al., 1995). Several inserts were subcloned into pmscv-puro and p-migr retroviral vectors (Pear et al., 1998). Short hairpin RNAs targeting mouse Nck1 (5'-gaatcttcgccaaatgatt-3') and Nck2 (5'- ggaggagcttagtttcgagaa-3') were inserted into psuper.retro vector (Oligoengine).
Reagents and Antibodies The affinity-purified polyclonal anti-nck antibody was raised in rabbits immunized with a GST fusion of full-length human Nck1. Mouse monoclonal anti-nck1/2 (catalog no. 610100) and anti-p130cas (catalog no. 610271) antibodies were from BD Transduction Laboratories. The rabbit polyclonal anti-pip5k antibody that recognizes type I PIP5K α, β, and γ (H-300) was obtained from Santa Cruz. The mouse monoclonal anti-phosphotyrosine antibody was from Cell Signaling Technology (P-Tyr-100, catalog no. 9411) and the mouse monoclonal anti-cd16 (clone 3G8) was from Calbiochem. Texas red phalloidin and goat anti-mouse Alexa 647 were purchased from Molecular Probes. Rhodamine-conjugated goat anti-mouse IgG was from Pierce. Pull-Down Assay, Immunoprecipitation, Western Immunoblotting, and Far-Western Analysis Cells were harvested in kinase lysis buffer (25 mm Tris, ph 7.4/150 mm NaCl/5 mm EDTA/10% glycerol/1% Triton X-100/10 mm β-glycerophosphate/1 mm sodium orthovanadate/10 mm sodium pyrophosphate/10 μg/ml aprotinin/1 mm PMSF) and lysates obtained after high-speed centrifugation. For immunoprecipitation, the supernatants were precleared with 20 μl of protein A-Sepharose beads (Santa Cruz Biotechnology) for 1 h at 4 C and then incubated for 2 h at 4 C with 1.5 μg of polyclonal anti-nck per 100 μl of lysate. The immunocomplexes were collected on protein-a sepharose beads, pelleted and washed three times with cold lysis buffer. For pull-down assay, the lysates were precleared with glutathione-sepharose beads (Amersham Pharmacia Biosciences; 10 μl of 50% slurry per 100 μl of lysates) for 1 h at 4 C and then incubated for 2 h at 4 C with 10 μl of glutathione-sepharose beads precomplexed with
GST-Nck SH2 domains. The complexes were pelleted and washed three times with cold lysis buffer. The proteins were released from beads by boiling in SDS sample buffer. For Western immunoblotting, protein content from samples was determined by the Bradford Assay (Bio-Rad), and equal amounts of protein were subjected to SDS/PAGE. After transfer to nitrocellulose membranes, blots were probed with a monoclonal anti-phosphotyrosine antibody (dilution 1:5,000) or monoclonal anti-nck (dilution 1:5,000) followed, after several washes, by goat anti-mouse horseradish peroxidase-conjugated secondary antibody. Immunofluorescence Analysis Immunofluorescence of endogenous type I phosphatidylinositol 5-kinase (PIP5K ) was performed in NIH3T3 cells co-expressing actin-gfp and the fusion of CD16/7 with the three SH3 domains from Nck (CD16/7-Nck) or CD16/7 alone (CD16/7). Cells were treated with clustering antibodies before fixation. Following fixation and permeabilization, immunofluorescence staining of endogenous PIP5K was performed using a rabbit polyclonal anti-pip5k antibody that recognizes type I PIP5K α, β, and γ (H-300, Santa Cruz) followed by a goat anti-rabbit A594-conjugated IgG. Modified Algorithm A second shape recognition engine based on a new "adaptive thresholding of boundary intensity" algorithm was designed to identify and discriminate, using only one channel of information, three different formations within a cell: long actin fibers, actin comets, and circular blobs of actin. The engine samples pixel intensity on a circle surrounding the particle and uses the resulting distribution (histogram of brightness across particle's circumference) to categorize particles. Thresholding fluctuates from one particle to the other and is based on the maximum intensity of the sampled particle. "Adaptive" refers to the results validation step which is based
on the spread between the boundary intensity variation and the particle intensity. Additional information can be found at: (http://ntcnp.org/twiki/bin/view/vcell/microfilaments#4_use_the_results).
Supplemental Figures A GFP / F-actin B hnck2 F-actin R308K F-actin KSH3all F-actin Fig. S1. Nck SH2- and SH3-domain mediated interactions are required for the formation of actin comets induced by PI(4,5)P 2. A) Confocal images of NIH3T3 cells expressing wild type PIP5K type Iα, and wild type (hnck2), or mutant Nck variants (R308K or KSH3all) in conjunction with GFP from a bicistronic transcript. Scale bar represents 5 µm. B) Quantitative analysis of the phenotypic changes induced by increased cellular levels of PI(4,5)P 2 in the presence of wild type
or mutant versions of Nck. Values represent mean ± S.E.M. from images corresponding to 5-7 cells/treatment analyzed in each of three independent experiment (n=3). Bars with different letters are significantly different (p<0.05, foci: a,b; comets: x,y).
PIP5K A EBB Nck1 Nck2 Nck1+2 B Fig. S2. Requirement of Nck for the formation of actin comets induced by PI(4,5)P 2. A) Confocal images from Nck-deficient mouse embryonic fibroblasts expressing wild type PIP5K type Iα. Images from control cells (EBB) or cells rescued with Nck are shown. Scale bars
represent 5µm. G) Quantitative analysis showing percentage of cells with clearly identifiable comets. Values represent mean ± S.D. from images obtained in three independent experiments (n=3). Numbers in parenthesis indicate total number of cells analyzed/treatment.
F-actin py immunostaining Nck immunostaining merge py F-actin merge Nck F-actin Fig. S3. Subcellular distribution of tyrosine phosphorylated proteins and Nck in cells expressing catalytically inactive (D227A) PIP5K. Confocal images of NIH3T3 cells subjected to immunostaining with anti-phosphotyrosine (top panel), or anti-nck (bottom panel). Scale bars represent 5 µm.
GFP aggregation aggregation no aggregation no aggregation A PH+CD16/7 PH+CD16/7-Nck R40L+CD16/7-Nck B R40L-GFP PH-GFP F-actin A647 GFP merge F-actin A647 GFP merge F-actin A647 GFP merge F-actin A647 GFP merge C Myr-GFP R-IgG Myr-GFP / R-IgG R-IgG Myr-GFP CD16/7-Nck merge
Fig. S4. Clustering of membrane-targeted Nck SH3 domains is necessary for the localized enrichment of PI(4,5)P 2. A) Subcellular distribution of the wild type (PH) or mutant (R40L) PI(4,5)P 2 biosensor in cells overexpressing CD16/7 alone or CD16/7 fused with the SH3 domains from Nck (all constructs tagged with HA). B) Enrichment of PI(4,5)P 2 and localized actin polymerization is induced by clustering of membrane-targeted Nck SH3 domains. Cells coexpressing GFP fusions of wild type (PH-GFP) or mutant (R40L-GFP) PI(4,5)P 2 biosensor and CD16/7-Nck were left untreated (no aggregation) or treated with clustering antibodies (aggregation), and stained with Texas-red phalloidin to visualize F-actin. Clustering was performed with an unlabeled secondary antibody and the subcellular distribution of CD16/7-Nck was detected by immunofluorescence staining with an anti-ha antibody (A647). C) Membranetargeted GFP (Myr-GFP) does not colocalize to clusters of CD16/7-Nck SH3 domains. NIH3T3 cells were cotransfected with Myr-GFP and CD16/7-Nck, and treated with clustering antibodies (R-IgG).
A647 F-actin merge A647 F-actin merge A B Nck CD16 SH3 (1) SH3 (2) SH3 (3) SH2 CD7 mouse anti-cd16 Synaptojanin-1 Sac 1 SJ PR SJ Nck SH3 Y Y PI(4,5)P2 PI4P CD16/7-Nck CD16 CD7 SH3 (1) SH3 (2) SH3 (3) HA CD16/7-SJ Y Y A647-anti mouse IgG C CD16/7-SJ CD16/7-D858A CD16 CD7 SJ HA 87 - D F-actin / A647 F-actin / A647 CD16/7-Nck CD16/7-SJ Fig. S5. Strategy for the simultaneous, localized manipulation of PI(4,5)P 2 - and Nck-dependent signaling at the plasma membrane. A) Diagrams representing the domain structure of Nck, the PI 5-phosphatase synaptojanin-1, and the fusion of CD16/7 with all three Nck SH3 domains (CD16/7-Nck), or the PI 5-phosphatase domain (CD16/7-SJ). The fusion proteins were tagged with the HA epitope. B) Diagram illustrating the antibody-mediated clustering utilized to simultaneously increase the local concentration of membrane-targeted Nck SH3 domains and decrease the local concentration of PI(4,5)P 2. C) Expression of CD16/7-SJ (wild type) and CD16/7-D858A (catalytically inactive) in NIH3T3 cells demonstrated by western immunoblotting with anti-ha. D) Confocal images of NIH3T3 cells expressing CD16/7-Nck
(CD16/7-Nck, left panel), or a fusion of CD16/7 with the wild type IP 5-phosphatase domain of synaptojanin-1 (CD16/7-SJ) following treatment with clustering antibodies (A647). Scale bar represents 5 µm.
Supplemental References Balla, T., and Varnai, P. (2002). Visualizing cellular phosphoinositide pools with GFP-fused protein-modules. Sci STKE 2002, L3. Papayannopoulos, V., Co, C., Prehoda, K.E., Snapper, S., Taunton, J., and Lim, W.A. (2005). A Polybasic Motif Allows N-WASP to Act as a Sensor of PIP(2) Density. Mol Cell 17, 181-191. Pear, W.S., Miller, J.P., Xu, L., Pui, J.C., Soffer, B., Quackenbush, R.C., Pendergast, A.M., Bronson, R., Aster, J.C., Scott, M.L., and Baltimore, D. (1998). Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abltransduced bone marrow. Blood 92, 3780-3792. Rivera, G.M., Antoku, S., Gelkop, S., Shin, N.Y., Hanks, S.K., Pawson, T., and Mayer, B.J. (2006). Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B. Proc Natl Acad Sci U S A. 103, 9536-9541. Rivera, G.M., Briceno, C.A., Takeshima, F., Snapper, S.B., and Mayer, B.J. (2004). Inducible clustering of membrane-targeted SH3 domains of the adaptor protein Nck triggers localized actin polymerization. Curr Biol 14, 11-22. Tanaka, M., Gupta, R., and Mayer, B.J. (1995). Differential inhibition of signaling pathways by dominant-negative SH2/SH3 adapter proteins. Mol Cell Biol 15, 6829-6837. Tolias, K.F., Rameh, L.E., Ishihara, H., Shibasaki, Y., Chen, J., Prestwich, G.D., Cantley, L.C., and Carpenter, C.L. (1998). Type I phosphatidylinositol-4-phosphate 5-kinases synthesize the novel lipids phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 5-phosphate. J Biol Chem 273, 18040-18046.