1. Volume 10, Issue 22, Pages 1403-1412 (November 2000)
DAPP1 undergoes a PI 3-kinase-dependent cycle of plasma-membrane recruitment and endocytosis upon cell stimulation 
K.E Anderson, P Lipp, M Bootman, S.H Ridley, J Coadwell, L Rönnstrand, J Lennartsson, A.B Holmes, G.F Painter, J Thuring, Z.-Y Lim, H Erdjument-Bromage, A Grewal, P Tempst, L.R Stephens, P.T Hawkins 
Current Biology 
Volume 10, Issue 22, Pages (November 2000)
DOI: /S (00)

2. Fig. 1
Time-lapse confocal imaging of the effects of PDGF stimulation in PAE cells, and antigen receptor ligation in DT40 chicken B cells, on the subcellular localisation of GFP–DAPP1. (a) PAE cells were transiently transfected with 20μg DNA encoding GFP–DAPP1. Transfected cells were plated onto glass coverslips in complete medium for 14h, then serum-starved for 11h before being mounted into a 37°C cell chamber on the confocal microscope. Cells were stimulated with PDGF (10ng/ml) and images captured at the indicated times. (b) Quantitation of membrane and cytosolic staining of GFP–DAPP1 over time, at the positions indicated by the circles in (a). (c) DT40 cells were transiently transfected with 30μg/ml DNA encoding GFP–DAPP1. The cells were seeded onto poly-l-lysine-coated coverslips and incubated in full media for 12h, then serum-starved for 8h. Cells were stimulated with mouse anti-chicken IgM (10μg/ml) and imaged by confocal microscopy. The time-lapse movie sequences of GFP–DAPP1 and GFP–PKB, with images collected every 3sec, are available as Movies 1–4 in the Supplementary material.
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3. Fig. 2
GFP–DAPP1 vesicle formation in response to PDGF is dependent on PI 3-kinase activity and an intact DAPP1 PH domain, and prevented by inhibition of endocytosis by GTPase-deficient dynamin. PAE cells expressing (a,b,d–f) wild-type PDGF-β receptor or (c) the Y740/751F mutant PDGF-β receptor, which is unable to signal via PI 3-kinase, were transiently transfected with 20μg DNA encoding GFP fusion proteins of either (a–c) wild-type DAPP1 or (d) DAPP1 containing a point mutation (R184A) in the PH domain, or (e,f) cotransfected with 10μg DNAencoding GFP–DAPP1 and (e) 10μg DNA encoding either a wild-type bovine dynamin 1a or (f) the GTPase-deficient dynamin DynS45N. Cells were plated onto coverslips in complete medium for 14h, then serum-starved for 11h. Cells were stimulated with PDGF-β (10ng/ml) for 5min. In (b), the cells were treated with 50μM LY for 30min before stimulation. The cells were fixed in paraformaldehyde and processed for immunocytochemistry as described in the Materials and methods.
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4. Fig. 3
Colocalisation of GFP–DAPP1-containing vesicles and internalised PDGF-β receptor and EEA1 following 5min of stimulation. PAE cells were transiently transfected with 20μg DNA encoding GFP–DAPP1 and treated as described in Fig. 2. Cells were stimulated with PDGF-β (10ng/ml) for 5min, and then fixed and processed for immunocytochemistry as described in the Materials and methods. (a) The cells were processed using PDGF-β receptor monoclonal antibody, and goat antibody to mouse IgG coupled to rhodamine isothiocyanate (RITC). Cells were viewed under fluorescent microscopy for GFP and RITC and the two images overlaid (merged). The final panel is an enlarged view of the merged image of the cell, with arrows pointing to a number of vesicles positive for both GFP–DAPP1 and PDGF-β receptor. (b) The cells were processed using EEA1 monoclonal antibody, and goat antibody to mouse IgG coupled to RITC. The cells were viewed as described in (a), with arrows indicating vesicles positive for both GFP–DAPP1 and EEA1.
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5. Fig. 4
The PI 3-kinase-dependent tyrosine phosphorylation of DAPP1 in PAE cells stimulated by PDGF requires an intact PH and SH2 domain, and occurs on Y139. PAE cells expressing either the wild-type (WT) or Y740/751F mutant PDGF-β receptor were transiently transfected with 20μg DNA encoding full-length EE–DAPP1; or an SH2 domain (residues 1–157); or containing a point mutation in the PH domain (R184A), SH2 domain (R61K), or central tyrosine residue in a consensus sequence for Src tyrosine kinase phosphorylation (Y139F). Cells were plated onto 9cm dishes and serum-starved and treated as described in the Materials and methods. (a) Cells were stimulated with PDGF (10ng/ml) for 5min, with or without 30min pretreatment with wortmannin (100nM) as indicated. The cells were lysed and anti-EE antibody immunoprecipitation performed as described in the Materials and methods. Membranes containing transferred anti-EE antibody immunoprecipitated proteins were probed for phosphotyrosine using 4G10 monoclonal antibody (upper panel), stripped with 5% acetic acid, then reprobed for DAPP1 expression using polyclonal antibody raised in sheep (lower panel). (b) Time course of EE–DAPP1 phosphorylation by PDGF. PAE cells were transfected and treated as described above. Cells were stimulated with PDGF (10ng/ml) for the indicated times. Cells were lysed, and samples immunoprecipitated as described in (a). Phosphorylation was quantitated on a Biorad Gel Doc system. A representative result is shown. (c) Cells were stimulated with PDGF (10ng/ml) for 5min, and samples immunoprecipitated and treated as described in Materials and methods. Membranes containing transferred proteins were probed for phosphotyrosine (upper panel) before being stripped and reprobed for DAPP1 expression (lower panel).
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6. Fig. 5
Time-lapse confocal imaging of the subcellular localisation of wild-type and mutant GFP–DAAP1 in PAE cells in response to PDGF. PAE cells expressing wild-type PDGF receptor were transiently transfected with DNA encoding GFP fusion proteins with wild-type DAPP1, tyrosine-phosphorylation mutant (Y139F) or the PH domain (residues 153–280), and treated as described in Fig. 1. The bottom panels show subcellular localisation of wild-type GFP–DAPP1 in PAE cells expressing the Y579/581F mutant PDGF receptor, which is unable to support signalling through Src family tyrosine kinases. The cells were either unstimulated (control) or stimulated with PDGF for 10 min, as described in Fig. 1. Time-lapse movie sequences of the localisation of these proteins are available as Supplementary material.
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