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First published online August 16, 2005
doi: 10.1242/10.1242/jcs.02503

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Ubiquitously expressed secretory carrier membrane proteins (SCAMPs) 1-4 mark different pathways and exhibit limited constitutive trafficking to and from the cell surface

Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, VA 22908, USA

View larger version (83K): [in a new window]   Fig. 1. Distribution of the four ubiquitously expressed mammalian SCAMPs in NRK cells. (A-D) low magnification wide-field images of SCAMP1-4 (SC1-SC4), emphasizing their concentration in perinuclear compartments. (E-G) Digitally deconvolved sections comparing the distribution of SCAMP1 to each of the other SCAMPs individually. SCAMP1 was detected using a biotinylated antibody 1 and SCAMP2 using 2, SCAMP3 using 3 and SCAMP4 using 4. Insets at lower left illustrate the extent of overlap (yellow) at perinuclear sites while the insets at lower right are representative views showing limited overlap of isoforms in the peripheral cytoplasm. Bar, 10 µm.

View larger version (165K): [in a new window]   Fig. 2. Distribution of SCAMP1-3 on transferrin-loaded endosomes as viewed by immunoelectron microscopy on A431 cell whole mounts. (A-C) Examples of specimens stained for all three SCAMPs using primary antibody-gold conjugates: SCAMP1 (SC1), 5 nm gold; SCAMP2 (SC2), 10 nm gold; and SCAMP3 (SC3), 15 nm gold. The electron-dense areas represent HRP stain within endosomes. Clusters of SCAMP1, 2 and 3 are circled and numbered accordingly. The arrow in C identifies the plasma membrane and enables visualization of the absence of extracellular background staining. (D) Control specimen stained with affinity-purified rabbit IgG-10 nm gold at the same concentration used in the other samples. Bar, 100 nm.

View larger version (134K): [in a new window]   Fig. 3. Comparison of the distributions of each of the SCAMPs (SC1-SC4) to markers that localize to perinuclear and peripheral compartments in differing patterns. (A-D) Transferrin receptor (TfR), (E-H) EEA-1 (EEA1), (I-L) TGN-38 (TGN38) and (M-P) syntaxin 6 (syn6). SC1 and SC3 staining is also compared to mannose-6-phosphate receptor (M6PR) (Q,R) and to CD63 staining (CD63) (S,T). All images are digitally deconvolved sections, and the insets highlight differing degrees of overlap in the perinuclear region. Bar, 10 µm.

View larger version (116K): [in a new window]   Fig. 4. SCAMPs on the plasma membrane. Plasma membrane sheets were labeled for SCAMP1 (A), SCAMP2 (B) or transferrin receptor (TfR) (C). (D) EM image of a portion of plasma membrane sheet ripped off the upper surface of an NRK cell and double labeled with antibodies against SCAMP1 and 2 conjugated directly to 5nm and 10 nm colloidal gold. Asterisk indicates clathrin-coated patch. Staining of SCAMP1 (E-G) and SCAMP2 (H-J) on plasma membrane sheets is also compared to clathrin (clat, E,H), adaptin AP2 (F,I) and dynamin 2 (dyn2, G,J). Insets illustrate limited colocalization. Bar, 10 µm (C); 100 nM (D).

View larger version (100K): [in a new window]   Fig. 5. SCAMPs are not detected in post-Golgi carriers for VSV-G. NRK cells transfected with GFP-VSVG were incubated at 39°C for 4 hours, then at 20°C for 2 hours to accumulate VSV-G in the TGN (A-C) and were warmed to 32°C for 30 minutes in the presence of 0.5% tannic acid to trap VSV-G in secretory carriers prevented from fusing with the plasma membrane (D-F). Deconvolved sections of cells immunostained for SCAMP1 (B,E) and SCAMP4 (C,F) compare the distributions of the SCAMPs to GFP-VSV-G. Bar, 10 µm.

View larger version (99K): [in a new window]   Fig. 6. Time course of fluorescent transferrin uptake and recycling and its association with SCAMP-containing compartments. (A-P) Alexa 594-transferrin (100 µg/ml) was bound to NRK cells at 0°C and uptake was initiated by warming to 37°C for the specified time. Whole cell images of uptake after 1 minute (A) and 3 minutes (B). (C,D) Deconvolved images 10 seconds (C) or 20 seconds (D) after uptake in cells stained for clathrin. (E-P) Deconvolved images comparing internalized transferrin at indicated times to the staining of SCAMP1-4. (E-I) portions of peripheral areas are shown. (M-P) portions of perinuclear areas are shown. (Q-T) To follow recycling carriers, cells were labeled with Alexa 594-transferrin for 10 minutes and chased for 60 minutes at 37°C. Deconvolved sections of perinuclear areas comparing transferrin and the staining of the SCAMPs are shown. Bar, 10 µm (B); 2 µm (D,T).

View larger version (105K): [in a new window]   Fig. 7. Trafficking of transferrin in compartments marked by GFP-SCAMP1 at early and late times after uptake. (A) Overall image of a cell expressing GFP-SCAMP1. (B) Portion of a plasma membrane sheet from a GFP-SCAMP1 expressing cell labeled with anti-clathrin antibody (clat). (C,D) transferrin uptake in NRK cells stably expressing GFP-SCAMP1 was performed as in Fig. 6E-L and the internalization stopped after 20 seconds (C) and 1 minute (D). Cells were fixed and observed by fluorescence, and deconvolved sections are shown. (E,F) Cells expressing GFP-SCAMP1 were labeled for 10 minutes with Alexa 594-transferrin and chased for 90 seconds. (E) Image of a live cell. (F) Selected frames from supplementary material Movie 1 of the cell shown in (E). The open arrowheads track the appearance (upper left panel) and movement of both transferrin and GFP-SCAMP1. The apparent separation of transferrin and GFP-SCAMP1 is due to sequential capture of images from the two different channels. (G-J) Live imaging of transferrin and GFP-SCAMP1 in carriers recycling transferrin to the surface. (G) Live cell expressing GFP-SCAMP1 imaged at 40 minutes of chase following a 15-minute labeling with Alexa 594-transferrin. (H) Selected images from Movie 2 in supplementary material showing transferrin movement (arrowhead) while GFP-SCAMP1 remains stationary. (I) Three panels from supplementary material Movie 3 showing movement of GFP-SCAMP1 (arrowhead) and not transferrin. (J) Selected images of supplementary material Movie 4 showing that distinct puncta of transferrin and GFP-SCAMP1 are tethered to one another and appear to be trying to separate during relocation (arrowhead). Bars, 10 µm (A,E,G); 2 µm (D,J).

View larger version (108K): [in a new window]   Fig. 8. Distribution of GFP-tagged Rabs 5, 4, and 11 in relation to endogenous SCAMPs illustrated using SC1 and SC4 as examples. (A-F) deconvolved images of cells expressing wild-type rabs: rab5 (A,B), rab4 (C,D) and rab11 (E,F) and stained for SCAMP1 (A,C,E) and SCAMP4 (B,D,F). Insets show extent of rab-SCAMP overlap at higher magnification from marked peripheral (A-D) and perinuclear (E,F) cytoplasmic regions. Left insets in A,B illustrate enlarged circular endosomal profiles typically observed in cells expressing rab5-Q67L with focal peripheral SCAMP staining. (G-R) Whole cell images of cells expressing GFP-tagged dominant-negative rab5-S34N (G-J), rab4-I121N (K-N) and rab11-S25N (O-R) and comparing GFP staining (G,I,K,M,O,Q) to SCAMP1 (H,L,P) and SCAMP4 (J,N,R). Cells expressing GFP-rab are indicated by arrows. Bar, 10 µm.

View larger version (86K): [in a new window]   Fig. 9. The effect of Eps15-DPF expression and of methyl-ß-cyclodextrin on the distribution of constitutively recycling cargo and SCAMP1. COS-7 cells were transiently transfected with GFP-tagged Eps15-DPF, fixed, immunostained for SCAMP1 or TfR and visualized by fluorescence (A-D) or TIRF-M (G-H) to compare SCAMP or TfR staining to GFP in paired images. (E,F) COS-7 cells were treated with 10 mM methyl-ß-cyclodextrin (CD, 30 minutes, 37°C), fixed, double-labeled for SCAMP1 and TfR, and examined by fluorescence. Bar, 10 µm.

View larger version (116K): [in a new window]   Fig. 10. Distribution of AP-1 and AP-3 adaptor complexes in relation to SCAMPs as viewed in deconvolved images of NRK cells. (A,B) Cells immunostained for AP-1 (-adaptin) and SCAMP2 (A) or SCAMP3 (B). (C,D) Cells immunostained for AP-3 (3) and SCAMP2 (C) or SCAMP3 (D). Insets in each panel highlight staining in the perinuclear region. Bar, 10 µm.