Model depicting hypothetical effect of CaM on the structure of the C termini of EEA1 and consequent binding to PI(3)P on membranes. Solid bars represent the coiled-coil region of each monomer, leading into the splayed FYVE domains.

Effect of Ca²⁺/CaM inhibition on full-length EEA1, the C termini of EEA1, and the p40Phox and CISK PX domains in live cells. COS-7 cells were transfected with the construct indicated on the left and exposed to 100 μM W7. Stacks of 21 optical sections were obtained every 20 s, restored, and projected into single 2D images. Projections for the times indicated on top of each panel are shown. Arrow in bottom right panel indicates the formation of tubules in response to W7. The complete set of images for the p40Phox construct can be seen as a QuickTime video (Video 1).

(A) Effects of CaM inhibitors on the localization of GFP-EEA1 and Alexa 594-transferrin in COS-7 cells. Cells expressing GFP-EEA1 were incubated with Alexa 594-transferrin for 10 min and washed once before addition of 100 μM W7. Stacks of 21 optical sections were collected every 30 s imaging alternatively with lasers tuned to 488- and 594-nm wavelengths. After image restoration, stacks were projected into a single 2D image. Projections for the times indicated are shown. The arrow indicates the concentration of transferrin in the recycling endosome after 15 min of treatment with W7. (B) Effects of diverse CaM inhibitors on endogenous EEA1 localization. COS-7 cells were treated with the indicated inhibitors at the concentrations indicated above each panel. After 10 min, cells were fixed and stained with human antibody to EEA1 and imaged by epifluorescence.

Effects of Ca²⁺/CaM inhibition on EEA1 association with activated Rab5 or PI(3)P. (A) Cytosolic extracts of 3T3 cells were incubated with nothing or with 100 μM W7 for 60 min and then incubated with GST alone or recombinant GST-tagged Rab5 immobilized on glutathione-Sepharose. GST-Rab5 was loaded with GTPγS or GDPβS as indicated. Bound proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to EEA1. The position of EEA1 on the gel is indicated. Duplicates of each condition were done in each experiment, and three experiments with similar results were obtained. (B) Cytosolic extracts of 3T3 cells were incubated with nothing or with 100 μM W7 for 60 min and incubated with PS/PI liposomes containing increasing concentrations of PI(3)P as indicated above each lane. Liposomes were pelleted and analyzed by immunoblotting with antibodies to EEA1. The exposed films were scanned and the intensity of each band quantified. The inset represents the immunoblot from one experiment, and the graph represents the results from a different experiment. Similar results were obtained in three independent experiments.

(A) Effect of CaM inhibitors on PI 3-kinase activity. COS-7 cells were transfected with myc-tagged Vps34, lysed, and immunoadsorbed with anti-myc antibody immobilized on Sepharose beads. Beads were washed and incubated with [γ-³²P]ATP and liposomes in the presence of the concentrations of inhibitor indicated under each lane. Lipids were separated by TLC and analyzed by autoradiography. The position of PI(3)P on the chromatogram is indicated by the arrow. (B) Postnuclear extracts from 3T3 cells were prepared as described in MATERIALS AND METHODS and incubated with wortmannin (Wort) or W7 at 100 nM or 100 μM final concentration, respectively, for 1 h. Cytosol was separated by high-speed centrifugation and incubated with PS/PI liposomes for 30 min. Liposomes were pelleted and spotted on nitrocellulose, which was probed with GST-SARA-FYVE and antibodies to GST. The specificity of the probe is depicted in the bottom row, in which 1 μg of the indicated lipids was spotted.

Association of GFP-tagged wild-type and mutant EEA1 with immobilized CaM. (A) The cytoplasmic extract from COS-7 cells expressing GFP-EEA1wt was incubated in the absence (C) or presence of 10 mM BAPTA, 10 mM EGTA or with an additional 2 μM CaCl₂ for 30 min at room temperature. The extract was then incubated with CaM-Sepharose or Sepharose alone as described in MATERIALS AND METHODS. Bound proteins were analyzed by immunoblotting with antibody to GFP. (B) Cytoplasmic extracts from COS-7 cells expressing either eGFP-EEA1 or eGFP-EEA1 containing mutations in the IQ-like motif (eGFPEEA1Q1289L/R1293G) were incubated in the presence or absence of EGTA. Binding to immobilized CaM was then determined as described in A. The concentration of the eGFP constructs in 20% of the starting cell extract (input) is shown. (C) Cytosol from cells expressing either GFP alone, GFP-EEA1wt, or EEA1 deletion constructs comprised of the amino acids indicated were incubated with or without EGTA and assessed for binding to CaM-Sepharose as described in A.

(A) Effects of W7 on eGFP-EEA1 and endogenous EEA1 association to enlarged endosomes containing persistently activated Rab5. COS-7 cells were cotransfected with dsRed-Rab5Q79L and eGFP-EEA1. Cells expressing both constructs were localized and imaged as described in Figure 1. Stacks of 21 optical sections were obtained every 30 s, restored, and projected into single 2D images. Two time points are illustrated. The overlap of the red and green signal is displayed as pure white in regions of equal pixel intensity. (B) COS-7 cells were transfected with dsRed-Rab5Q79L, and after 36 h treated for 10 min with 100 nM wortmannin (Wort) or 100 μM W7. Cells were fixed and endogenous EEA1 was detected using a human antiserum, and Alexa 488 secondary antibodies. Cells shown express similar levels of Rab5Q79L based on fluorescence intensity. A nontransfected cell (NT) is shown for comparison. (C) The fluorescence intensity of EEA1 on the endosomes containing Rab5Q79L and on the cytoplasmic region surrounding the endosomes was quantified and the ratio plotted.

Effects of the Ca²⁺/CaM inhibitor W7 on the association of EEA1 with cell membranes and on the membrane levels of PI(3)P. (A) Dose response of EEA1 to Ca²⁺/CaM inhibitors: A postnuclear supernatant from 3T3 cells was obtained as described in MATERIALS AND METHODS, dispensed into aliquots, and incubated for 15 min with CaM inhibitors at the concentrations indicated. Cytosol was separated from membranes by high-speed centrifugation. The membrane pellet was resuspended in cytosol buffer to the same volume as the cytosol and equal volume aliquots analyzed by SDS-PAGE and immunoblotting with antibodies to EEA1 and chemiluminescence. The exposed films were scanned and the intensity of each band quantified. The ratio of the cytosol and membrane intensities was plotted. Bars represent the mean, and lines the SE of three independent experiments. (B) The experiment was performed as described in A with no additions, 100 nM wortmannin (Wort), or 100 μM W7 for 60 min. The insets illustrate the immunoblots of the membrane and cytosolic extracts. Plotted are the intensities of each band. This experiment was repeated five times with similar results (C) The resuspended membrane pellets were extracted with chlorophorm/methanol as described in MATERIALS AND METHODS. Extracted lipids were spotted on nitrocellulose and probed with GST-SARA-FYVE as described in MATERIALS AND METHODS and in Figure 2. Two experiments are illustrated, with their corresponding standards. This experiment was repeated four times with similar results.

Anti-CaM antibody causes a release of EEA1 from endosomal membranes. (A) Postnuclear supernatant from 3T3 cells was obtained as described in MATERIALS AND METHODS, and aliquots were incubated without or with 100 nM wortmannin (Wort), 100 μM W7, or the indicated concentration of nonimmune mouse IgG (IgG) or anti-CaM IgG (anti-CaM) for 60 min. Membranes and cytosol were separated by high-speed centrifugation and analyzed by SDS-PAGE and immunoblotting with antibodies to EEA1 and chemiluminescence. The exposed films were scanned and the intensity of each band quantified and plotted. Inset illustrates the immunoblot from the membrane fractions. Bars represent the mean and lines the SE of the mean from three independent experiments. (B) The membrane fraction from 3T3 cells was isolated by high-speed centrifugation, resuspended in cytosol buffer, and incubated without or with 100 nM wortmannin (Wort), 100 μM W7, or 5 μM nonimmune mouse IgG (IgG) or anti-CaM IgG (Anti-CaM) for 60 min at room temperature. The membrane pellet was separated from supernatant and equal quantities of the resulting fractions immunoblotted for EEA1. Immunoblots were scanned, and the intensities of the bands quantified. Plotted are the ratios of supernatant and pellet. Bars represent the mean and lines the SE of the mean from four independent experiments. Inset illustrates a Western blot of the supernatant from one experiment. (C) Anti-CaM antibody was microinjected into COS-7 cells, which were fixed within 5 min of injection (top) or after 45 min postinjection. Injected antibody was detected with anti-mouse secondary antibodies and endogenous EEA1 with human anti-EEA1 antiserum and antihuman secondary antibodies. Arrows in top panels point to sites of colocalization of the two probes and in lower panels point to the injected cell.