Supplementary material

S1

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization Alexander F. Palazzo, Hazel L. Joseph, Ying-Jiun Chen, Denis L. Dujardin, Arthur S. Alberts, K. Kevin Pfister, Richard B. Vallee and Gregg G. Gundersen Current Biology 2 October 2001, 11:15361541
Supplementary references
S1. Gundersen GG, Kim I, Chapin CJ: Induction of stable microtubules in 3T3 fibroblasts by TGF-beta and serum. J Cell Sci 1994, 107:645-659. S2. Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen RA, Michiels F, et al.: Matrix-dependent Tiam1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol 1998, 143:1385-1398. S3. Barr FA, Puype M, Vandekerckhove J, Warren G: GRASP65, a protein involved in the stacking of Golgi cisternae. Cell 1997, 91:253-262.

Figure S1

Table S1 Effect of serum factors on MTOC reorientation. Reoriented MTOC (%) Unstarved (10% calf serum) Control (serum-free medium) 10% calf serum LPA (1 M) PDGF (10 ng/ml) FGF (100 ng/ml) EGF (10 ng/ml) TGF1 (2 ng/ml) IGF1 (100 ng/ml) IGF2 (100 ng/ml) Insulin (10 ng/ml) IL-1 (1 g/ml) IL-6 (1 g/ml) Fibronectin (1 g/ml) 83 31 70 74 34 34 31 49 39 38 37 38 32 36 /2 /3 /4 /4 /4 /2 /2 /7 /4 /2 /3 /4 /4 /3

n 3 3 3 4 6 5 5 6 4 4 6 2 2 3

Unstarved cells were wounded after growing to confluence. All other conditions were with cells starved for two days in SFM (see Materials and methods). Serum or growth factors were added immediately after wounding, and MTOC reorientation was assesed in 100 wound-edge cells in each experiment. Data is mean standard deviation (n number of independent experiments). Treatments were for 2 hr for control, calf serum, and LPA or 7 hr for all other growth factors. Growth factors were from the following sources: human PDGF (UBI, Lake Placid, New York, USA); human IGF1, IGF2, and bovine FGF (Collaborative Research, Bedford, Massachusetts, USA); porcine TGF1 and recombinant IL-1 and IL-6 (R & D, Minneapolis, Minnesota, USA); and bovine insulin and fibronectin (Sigma). EGF was purified from mouse submaxillary gland as described previously [S1] and was the gift of F. Maxfield (Cornell University).

LPA stimulates Cdc42 GTP levels. (a) Cdc42 Western blot of Cdc42 GTP pulled down with GST-PAK-CRIB from extracts of serumstarved cells treated with serum-free media for 20 min (SFM), 1 M LPA for 20 min (LPA), or 10% CS for 20 min (CS). (b) Histogram of fold increase in Cdc42 GTP levels. Data are mean of two experiments; error bars are standard deviation. Briefly, Cdc42 GTP pull-downs were performed as follows. Serum-starved cells in 10 cm dishes were stimulated as in (a), washed twice with cold PBS, frozen at 80C for 510 min, and then extracted in lysis buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM Tris [pH 7.2], 500 mM NaCl, 10 mM MgCl2, and 10% glycerol and protease inhibitors). Cleared lysates were incubated with GSH-Sepharose beads containing GST-PAK-CRIB for 30 min and eluted as previously described [S2]. Samples were separated by SDS-PAGE and transferred to nitrocellulose. Western blotting was performed with sc87 (mouse monoclonal against Cdc42; Santa Cruz Biotechnology, California, USA) and HRP-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Pennsylvania, USA).

S2

Figure S2 (ac) Effect of G proteins on MTOC reorientation. Serum-starved wound-edge fibroblasts were microinjected with (a) GSTPAK-CRIB, which inhibits endogenous Cdc42 and Rac, (b) C3 botulinin toxin, which inhibits endogenous Rho, or (c) dominantnegative N17Rac. After 30 min, cells were treated with 1 M LPA for 2 hr to induce MTOC reorientation. (d) Serum-starved wound-edge fibroblasts were microinjected with constitutively active L63Rho and incubated for 2 hr. Cells were fixed and immunostained for pericentrin (red), Tyr MTs (green), DNA (blue), and injection marker, human IgG (shown in insets; arrows in main panels show injected cells). The bar equals 50 m. Results are quantified in Figure 1e (main text). Note that (a) inhibition of both Cdc42 and Rac by GST-PAK-CRIB microinjection interfered with LPA-triggered MTOC reorientation, but (b) dominantnegative N17Rac or inhibition of Rho by C3 microinjection, unlike (Figure 1d,e) dominantnegative Cdc42, did not inhibit LPAtriggered MTOC reorientation. (d) Microinjection of active L63Rho, active V14Rho (data not shown), or active L61Rac (data not shown) did not trigger MTOC reorientation, unlike active L61Cdc42 (see Figure 1d,e).

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Figure S3

Inhibition of dynein or dynactin blocks LPA-induced MTOC reorientation independently of its effects on the Golgi. (af) Serumstarved cells were microinjected with dynein intermediate chain mAb 74.1. After 30 min, cells were stimulated with 1 M LPA for 2 hr and then fixed and immunostained for (a,d) injected mAb 74.1, (b,e) MTs, (c,f) the Golgi marker, grasp65 [S3]. Arrows in (b) and (e) show positions of MTOC. Note that the Golgi were (c) dispersed in some injected cells but (f) not in others, even though (b,e) reorientation of the MTOC was inhibited. (gi) Serum-starved cells were microinjected with GFP-dynamitin DNA, and after allowing for expression (24 hr), cells were treated with 1 M LPA for 2 hr before fixing and immunostaining for (h) MTs and (i) grasp65. (g) Fluorescence of the expressed GFP-dynamitin is shown. Note that GFP-dynamitin overexpression (h) inhibited LPA-triggered MTOC reorientation, but (i) the Golgi in these cells remained unaffected. The bar equals 20 m.