Figure 2.

Kal- or Trio-GEF1 overexpression causes depletion of cell hormone content. AtT-20 cells were mock-transfected (i.e., treated with Lipofectamine 2000 in the absence of plasmid) or transfected with myc-tagged Kal- or Trio-GEF1 constructs. Stacks of images encompassing the entire thickness of the cells were acquired (by confocal or deconvolution microscopy); maximum projections in the z-dimension are shown in B–D and the right-side panels in A; the middle and left panels in A display single focal planes acquired close to the substrate. (A) Recombinant proteins (Myc) and mature ACTH were visualized. (B) Secretory granule markers were visualized: Carboxypeptidase E (CPE), Chromogranin A (CgA), PAM-1 (in PAM-1 AtT-20 cells), and VAMP4. (C) Golgi/TGN markers were visualized: TGN38, GM130, Carboxypeptidase D (CPD), and β-COP. (D) Cells were fixed and stained for Rab11 or incubated with AlexaFluor 546-transferrin (Molecular Probes) for 15 min and then chased in nonfluorescently labeled transferrin for 15 min before fixation. Dotted and solid lines outline GEF1- and nontransfected cells, respectively. Scale, 10 μm.