Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Kal- or Trio-GEF1 overexpression induces morphological changes. AtT-20 cells were prepared as described in Fig.2. (A) Cells were visualized with TRITC-phalloidin (Sigma-Aldrich, St. Louis, MO) and Myc monoclonal antibody. Stacks of images encompassing the entire thickness of the cells were acquired and deconvolved; maximal projection images are shown, providing a two-dimensional representation of the entire volume of the cells. Kalirin and Trio GEF1 overexpression causes cell flattening, retraction of processes and dramatic remodeling of filamentous actin. Lamellipodia and stress fibers, never observed in non- and mock-transfected cells, are induced. Numbered insets are magnified to demonstrate close spatial relationship between F-actin and GEF1 in some regions of the cell. (B) Confocal images were acquired at the cell/glass substrate interface (bottom), in the middle and at the top of the cells; orthogonal views (x-z and y-z planes) are shown. Recombinant Kal- and Trio-GEF1, excluded from the nucleus, display a cytosolic localization with juxtaposition to filamentous actin apparent at the dorsal membrane and the edges of lamellipodia. Scale, 10 µm.
• Figure S2 - Kal- or Trio-GEF1 overexpression reduces cellular hormone content. For quantification, AtT-20 cells transfected with the dual promoter pCMS EGFP vector (A) encoding EGFP and Kal-GEF1, Kal-GEF1(ND/AA) or empty were stained for POMC/ACTH (JH93 antiserum, directed to the C-terminal part of ACTH(1-39), recognizes intact POMC, ACTH biosynthetic intermediate and mature ACTH; refer to Fig.4A (Sobota et al., 2006). Stacks of confocal images (with non-saturated signals) encompassing the entire volume of the cells were collapsed in the z-dimension; image analysis was carried out using SimplePCI software (Compix, Cranberry Township, PA). (B) Cell areas were quantified using EGFP. (C) POMC staining per cell was also quantified. t-test, equal variance (n=16-20); *, p <. 0.05; ***, p < 0.001.
• Figure S3 - GEF1 induced depletion of regulated cargo is not specific to AtT-20 cells. PC12 cells were transfected as described for AtT-20 cells. Kal-GEF1 overexpression caused cell flattening and depletion of a soluble cargo protein, Chromogranin B (CgB), and a granule membrane protein, Synaptotagmin-1. Transfected cells are indicated by dotted lines. Images are maximum projections. Magnified insets are shown to illustrate punctuate, granule-like staining for both antibodies. Synaptotagmin-1 monoclonal antibody (clone 41.1) was from Synaptic Systems GmbH (Gottingen, Germany) and Chromogranin B antibody (C-19 goat antiserum) was from Santa Cruz Biotechnology (Santa Cruz, CA).
• Figure S4 - Kalirin and Trio GEF1 and their activated Rho substrates induce similar morphological changes. AtT-20 cells were transfected with plasmids encoding GFP- fusion proteins of Kal-GEF1, Trio-GEF1, Rac1(Q61L) and RhoG(Q61L); based on the GFP signal, the effects of expressing similar levels of the different proteins were compared. Images of single focal planes acquired close to the substrate are shown (confocal microscopy). GFP and ACTH signals are displayed; all GFP and ACTH images were acquired under identical conditions. The lower two rows show magnified images of the insets in the upper two rows. Scale bar, 10 µm.
• Figure S5 - GEF1 overexpression does not decrease cargo synthesis or augment degradation. (A) AtT-20 cells were nucleofected with the indicated pCMS EGFP dual promoter plasmids. After 48 h, cells were subjected to depletion of mature secretory product by repetitive secretagogue challenge (Ferraro et al., 2005); a 4 h temperature blockade at 20 ºC of TGN export (Matlin and Simons, 1983) was used to accumulate intact POMC in the TGN. Lysed cells were analyzed for POMC content using antiserum JH189 (see Fig.4A). Duplicate samples from independent cultures are shown. Only unprocessed POMC is visualized, confirming depletion of mature secretory product and blockage of ISG budding and POMC processing (compare to Fig. 4B). γ-Adaptin is shown as a loading control. (B) At 24 h post-transfection, AtT-20 cells expressing Kal-GEF1 were incubated for 24 h in control medium or medium containing 2.5 mM NH4Cl, a treatment know to dissipate pH gradients across intracellular membranes (Sobota et al., 2006) and slow lysosomal hydrolase activity. POMC and LAMP1 were visualized in GEF1 expressing cells (identified by triple staining with Myc antibody; not shown). Magnified inset shows no significant colocalization of POMC and LAMP-1, suggesting that POMC and its products are not missorted to lysosomes. Maximum projections in the z-dimension are shown. POMC was visualized with JH93 antiserum; rat LAMP1 antibody (clone 1D4B) was from the Developmental Studies Hybridoma Bank, University of Iowa. Scale, 5 µm.