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Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane

Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5435, USA

View larger version (46K): [in a new window]   Fig. 2. The APC armadillo repeat domain localizes to cortical APC clusters. Basal sections of cells expressing different APC domains (shown schematically on left) fused to GFP (GFP: green) co-stained for endogenous full-length APC (APC: red) or for ß-tubulin (Tub: red). Note that the APC antiserum (anti-APC) does not recognize the deleted GFP-APC fusion proteins because they lack the epitope. (a-c) Full-length GFP-APC co-aligns along MTs (white arrowheads in a-c) and accumulates in cortical clusters at the tip of cell extensions distal to the MT ends (black arrowheads in a-c). (d-f) GFP-APCwoE1 co-aligns with MTs but does not colocalize with endogenous APC in clusters at the tip of cell extensions (black arrowheads in d-f). (g-n) GFP-APCE1X1 localizes to the cortical APC clusters (black arrowheads in g-i), but does not co-align with MTs (black arrowheads in k-n). (o-q) APCARM-GFP does not co-align with MTs but colocalizes with endogenous APC in the cortical clusters (black arrowheads in o-q). (r-t) APCARM-Rep.-GFP shows weak colocalization in some APC clusters (black arrowheads in r-t) but not others (white arrowheads in r-t).

View larger version (66K): [in a new window]   Fig. 1. Subcellular distribution of endogenous APC and EB1 in MDCK cells. Basal sections of MDCK cells co-stained for EB1 (green in a-e and a''-e'') and APC (red in a'-e' and a''-e''). Arrowheads, cortical APC clusters at the tip of cell extensions; black arrowheads, APC clusters without EB1; white arrowheads, APC clusters partially overlapping with EB1. Bars, 10 µm. Insets, higher magnification of the upper left cell extension in a-a" co-stained for EB1 (green) and APC (red). Bar, 2.5 µm.

View larger version (121K): [in a new window]   Fig. 4. Subcellular localization of DsRed-EB1, DsRed-EB1CT and DsRedEB1NT in MDCK cells. (a-f) Basal sections of cells expressing full-length DsRed-EB1 (a-c) or DsRed-EB1CT (d-f) and co-stained with a mouse monoclonal antibody to ß-tubulin (green). (a-c) DsRed-EB1 predominantly localizes to the distal (plus) ends of MTs (arrows in a-c). (d-f) DsRed-EB1CT is enriched in cortical clusters at the tip of the cell extension (arrowheads in d-f) and occasionally localizes to the plus end of MTs (arrows in d-f). (g-i) Basal sections of cell extensions of a MDCK cell expressing DsRed-EB1NT (red, marked with `+') and an untransfected MDCK cell marked as `-'. Cells were costained with a mouse monoclonal antibody to EB1 (green) that does not recognize DsRed-EB1NT (see Material and Methods). DsRed-EB1NT co-aligns along MTs including their distal (plus) ends, which are marked by endogenous EB1 (green) but is not as well restricted to the distal (plus) ends of MTs as endogenous EB1 (arrows in g-i). Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 3. Binding of EB1 and the C-terminal EB1 domain to MTs or APC in vitro. (a) MT pelleting assay. Lanes 1-3 show 1/20 of the respective MBP fusion proteins after preclearance by centrifugation and before incubation with polymerized tubulin. Lanes 4-6 show 2/3 of the MT pellets after incubation with the respective MBP fusion proteins and centrifugation through a glycerol cushion. Lane 7, 2.5 µg purified bovine brain tubulin. (b) Binding of MBP-EB1 fusion proteins to the EB1-binding domain in GST-APCCT. Precipitation of the respective MBP fusion proteins by GST-APCCT bound to APC AB/Protein A (APC AB+GST-APCCT) (lane 7-9). GST-APCCT appears as a double band of which the faster migrating one is probably caused by partial degradation. In control assays, MBP fusion proteins were incubated with anti-GST antibody (GST AB) bound to Protein A (lane 1-3); with GST bound to GST AB/Protein A (GST AB+GST) (lane 4-6) and with anti-APC antibody (APC AB) bound to Protein A (lane 10-12). Approximately 1/15 of the total MBP fusion proteins used for each binding assay are shown in lane 13-15. M, Molecular weight standards.

View larger version (63K): [in a new window]   Fig. 5. Colocalization of DsRed-EB1 and DsRed-EB1CT but not of DsRed-EB1NT in cortical APC clusters. (a-i) Basal sections of cell extensions of cells expressing full-length DsRed-EB1 (a-c), DsRed-EB1NT (d-f) or DsRed-EB1CT (g-i). Cells were co-stained with APC antiserum (green). DsRed-EB1 predominantly localizes to the distal (plus) ends of MTs (arrows in b, c). (Fig. 4a-c) and colocalizes with APC in cortical clusters at the tip of the extension (white arrowheads in a-c). DsRed-EB1NT shows filamentous staining at MT distal (plus) ends and along MTs (arrows in e, f) (Fig. 4g-i) but does not colocalize with APC in cortical clusters (black arrowheads in d-f). DsRed-EB1CT colocalizes with APC at the tip of the extension (white arrowheads in g-i). Bar, 10 µm.

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