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First published online 17 February 2004
doi: 10.1242/jcs.00939

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Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes

Ryan K. Louie^1,*, Shirin Bahmanyar^1,*, Kathleen A. Siemers^1,*, Violet Votin¹, Paul Chang², Tim Stearns², W. James Nelson^1,2 and Angela I. M. Barth^1,

¹ Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA
² Deptartment of Biological Sciences, Stanford University, Stanford, CA 94305, USA

View larger version (75K): [in a new window]   Fig. 1. Preferential localization of APC and EB1 to a subset of centrioles. (A) Schematic representation of centrosome duplication during the cell cycle and localization of centrosome marker proteins. Pericentrin and -tubulin localize to the pericentriolar material (lilac area); centrin localizes to the centrioles (light blue tubes). MT-anchoring appendages on the mother centriole are marked in black. (B) Basal section of a MDCK cell co-stained for APC (red) and -tubulin (green). Arrowheads mark cortical APC clusters, arrow marks localization of APC with -tubulin. Bar, 10 µm. Insets show another example of the centrosome region of an MDCK cell at higher magnification. Bar, 2 µm. (C) Sections of U-2 OS cells showing the centrosome regions of cells immunostained for -tubulin (green in a-c), pericentrin (red in g-j) and centrin (green in d-f,k-m) and co-stained for APC (red in a-f) and EB1 (green in g-m). Cells with two centrosomes marked by pericentriolar pericentrin (red in g-j) or -tubulin (green in a-c) or with four centrioles marked by centrin (green in d-f,k-m) are also shown. APC and EB1 localize preferentially to one of two centrosomes (a-c for APC, g-j for EB1) and to two of four centrioles (d-f for APC) or to one of four centrioles (k-m for EB1). See also Movie 1 (http://jcs.biologists.org/supplemental). Bar, 2 µm.

View larger version (47K): [in a new window]   Fig. 6. APC localizes to the mother centriole. (A) APC co-localizes with DsRed-EB1 to the mother centriole. Sections of MDCK cells expressing DsRed-EB1 (red in a-n) showing the centrosome regions immunostained for centrosome markers pericentrin (blue in a-d), -tubulin (blue in e-h) or centrin (blue in j-n) and co-stained for the mother centriole marker p150Glued/dynactin (green in b-d) or APC (green in f-h,k-n). DsRed-EB1 co-localizes with p150Glued/dynactin at only one of the two centrosomes stained by pericentrin (a-d). DsRed-EB1 and APC preferentially localize to the same of two centrosomes marked by -tubulin (e-h) and to the same two centrioles of four centrioles marked by centrin (j-n). APC precedes EB1 at the second centrosome (e-h). Only two out of 25 analysed U-2 OS and MDCK cells in asynchronous cultures had no APC at the second centrosome, whereas seven out of 28 analysed cells had no EB1 at the second centrosome, indicating that APC localizes earlier to the second mother centriole than EB1. Bar, 1 µm. (B) U-2 OS centrosome region expressing GFP-centrin as a marker for centrioles (blue) and co-immunostained for APC (green) and for the mother centriole with p150Glued/dynactin (red). Sections of this region have been deconvolved and recombined in a 3D rendering. The first column shows four centrioles in multiple angles of counterclockwise (ccw) rotation, with the two central centrioles too close to each other to be resolved as separate spots. The second and third columns show that APC tightly surrounds the mother centriole as marked by p150Glued/dynactin and extends to a second centriole. (B') Schematic representation of 3D localization of proteins shown in B at a 205° angle. See Movie 4 (http://jcs.biologists.org/supplemental) for a 360° rotation and Materials and Methods for the resolution limits of the optical system. (C) U-2 OS centrosome region expressing GFP-centrin as a marker for centrioles (blue) and co-immunostained for EB1 (green) and for the mother centriole with p150Glued/dynactin (red). Sections of this region have been deconvolved and recombined in a 3D rendering. The first column shows two centrioles in multiple angles of ccw rotation. The second and third columns show that EB1 and p150Glued/dynactin cap one end of the mother centriole and continue in a MT-like filamentous extension from this end of the centriole. (C') Schematic representation of 3D localization of proteins shown in C at a 0° angle. See Movie 5 (http://jcs.biologists.org/supplemental) for a 360° rotation. (D) APC-EB1 interaction is not essential for localization of APC and EB1 to centrosomes. SW480 centrosome regions were co-immunostained for the centrosomal markers -tubulin (green in a,c) or pericentrin (green in d,f,h,j) and APC (red in b,c), EB1 (red in e,f) or p150Glued (red in i,j). N-APC, EB1 and p150Glued localize to centrosomes in SW480 cells that express a truncated form of APC without the EB1 binding site.

View larger version (39K): [in a new window]   Fig. 2. Localization of EB1 and its C-terminal binding partner APC to centrosomes is independent of intact cytoplasmic MTs. (A) MDCK cells (a-c) and MDCK cells treated with nocodazole to disrupt the MT cytoskeleton (a'-c') were immunolabelled for -tubulin (a,a'), APC (b,b') or EB1 (c,c'). Filamentous MTs (a), MT-dependent cortical APC clusters (arrowheads in b) and MT plus-end localization of EB1 (c) are disrupted by nocodazole (a', black arrowheads in b',c'). Localization of APC to cell-cell contact sites is independent of intact MTs and seems to be enhanced in nocodazole-treated MDCK cells (arrows in b,b'). Bar, 50 µm. (B) Sections of nocodazole-treated MDCK cultures shown in (A) were stained for centrosome markers -tubulin (green in a-c) or pericentrin (red in d-f) and co-stained for APC (red in a-c) or EB1 (green in d-f). APC and EB1 remain localized around the centrosome in nocodazole-treated cells, whereas their MT-dependent localizations are disrupted (see A). Images in d-f show an example in which EB1 localizes to both centrosomes of a cell. Bar, 2 µm. (C) Section of a nocodazole-treated U-2 OS cell expressing GFP-APC (green in a-c) stained with the centriolar marker centrin (red in a-c). GFP-APC preferentially localizes to one of the two centrioles in this cell [Movie 2 (http://jcs.biologists.org/supplemental)]. Bar, 2 µm.

View larger version (60K): [in a new window]   Fig. 5. EB1 localizes to the mother centriole. (A) Centrosome region of a nocodazole-treated U-2 OS cell expressing GFP-centrin as a marker for centrioles (blue) and co-immunostained for EB1 (red) and for the mother centriole with -tubulin (green). Sections of this region were deconvolved and recombined in a 3D rendering. Multiple angles of a counterclockwise (ccw) rotation show that EB1 localizes with -tubulin to one end of the mother centriole. EB1 caps this end of the mother centriole like a wizard's hat, whereas -tubulin forms a more ring-like structure around the same end. (A') Schematic representation of 3D localization of proteins shown in A at the 0° angle. See Movie 3 (http://jcs.biologists.org/supplemental) for a 360° rotation and Materials and Methods for the resolution limits of the optical system. (B) Schematic representation of -tubulin distribution (green) during centrosome maturation as shown in the three panels in (C). (C) U-2 OS cell sections showing different centrosome maturation stages as marked by immunostaining for -tubulin (green in a,c,d,f,g,j). Sections are also stained for EB1 (red in b,c,e,f,h,j) to define EB1 localization during centrosome maturation. The first column shows increasing -tubulin accumulation at the second mother centriole (a,d,g). The second and third columns show that EB1 has a similar distribution pattern to -tubulin at different maturation stages (b-j) but that -tubulin precedes EB1 at the second mother centriole (c,f,j). In three out of 12 analysed cells, -tubulin was localized to the second centrosome, with EB1 being only at one centrosome. Bar, 2 µm.

View larger version (30K): [in a new window]   Fig. 3. EB1 and its C-terminal binding partner APC co-purify with centrosomes. Centrosomes were purified from nocodazole-treated MDCK cells (a-o) or U-2 OS cells (a'-o'), stained for the centrosome markers -tubulin (green in a-c,a'-c'), centrin (red in d-f,d'-f') or pericentrin (red in g-n,g'-n') and co-stained for APC (red in a-c,a'-c' and green in d-f,d'-f'), EB1 (green in g-j,g'-j') or p150Glued/dynactin (green in k-n,k'-n'). Most of the purified centrosomes contain APC and EB1 (white arrowheads in a-j,a'-j'). An example of a centrosome without APC is shown (black arrowhead in d-f). By contrast, most of the centrosomes have little p150Glued/dynactin (arrows in k-n,k'-n') or no p150Glued/dynactin (black arrowheads in k-n). Examples of centrosomes that contain p150Glued/dynactin are shown (white arrowheads in k'-n'). Purified MDCK (o) or U-2 OS (o') centrosomes are functional as measured by induction of MT aster formation in vitro (o,o'). Bar, 10 µm.

View larger version (95K): [in a new window]   Fig. 4. EB1 localization to the centrosome is mediated by its C-terminal domain. Sections of MDCK cells expressing full-length EB1 fused to DsRed (DsRed-EB1, red in a-c), the N-terminal domain of EB1 fused to DsRed (DsRed-EB1NT, red in d-f) or the C-terminal domain of EB1 fused to DsRed (DsRed-EB1CT, red in g-j). Cells were co-stained for -tubulin (green in b,c,e,f,h,j). DsRed-EB1 localizes along MTs (arrows in a,c) and forms a ring around the centrosome (white arrowheads in a-c). DsRed-EB1NT localizes along MTs (arrows in d,f) but does not localize around the centrosome (black arrowheads in d-f). DsRed-EB1CT does not localize along MTs but forms a ring around the centrosome (white arrowheads in g-j). DsRed-EB1 and DsRed-EB1CT show preferential localization to one of the two centrosomes in a cell (insets in a-c for DsRed-EB1 and insets in g-j for DsRed-EB1CT). Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 7. Depletion of EB1 with small interfering RNA inhibits MT regrowth from centrosomes after nocodazole washout. (A) SDS lysates from untreated HeLa cultures (1) and cultures incubated with control (2) or EB1 (3) siRNA were immunoblotted for EB1 (red), tubulin (green) and actin (green). EB1 levels were reduced in cultures treated with siRNA against EB1. Polyclonal antiserum to EB1 showed weak cross-reaction with a second slower migrating protein (red signal above actin), which was not reduced in response to EB1 siRNA. (B,C) HeLa cells incubated with siRNA against GFP as a control (B) or with siRNA against EB1 (C) were treated with nocodazole to depolymerize MTs, incubated at room temperature for the indicated times after nocodazole removal to allow regrowth of MTs and immunostained for -tubulin (green) and EB1 (red). Some cultures were incubated with siRNA but not treated with nocodazole (–Noc) to control for preservation of MTs during fixation and, in some cultures, the nocodazole was not washed out (–wash) to control for MT regrowth during the time that was needed for the washes (compare `–wash' to `0 minutes'). Immunofluorescence images of EB1 were taken with identical exposure times to allow comparison of fluorescence intensity between images. (D,E) Analysis of MT aster areas after 20 minutes of regrowth at room temperature. HeLa cells incubated with siRNA to GFP (a,b) or siRNA to EB1 (c,d) were immunostained for EB1 (a,c) and -tubulin (b,d). Arrows mark cells depleted of EB1; an asterisk marks an unaffected cell with regular EB1 level in the same culture. (E) The areas covered by MT asters in EB1-depleted cells were compared with the areas covered by MT asters in cells of control cultures incubated with siRNA to GFP. Control, n=37; EB1 knock down, n=15.

View larger version (47K): [in a new window]   Fig. 8. Depletion of EB1 with small interfering RNA reduces MT minus-end anchoring at the centrosome. (A) Cos-7 cells incubated with siRNA against GFP as a control or with siRNA against EB1 were immunostained for EB1 (red), and -tubulin (green) and co-stained with DAPI for DNA (blue). Immunofluorescence images of EB1 were taken with identical exposure times to allow comparison of fluorescence intensity between images. Examples of cells with MT minus ends focused at the centrosome are marked as `F' and examples of cells with reduced MT minus-end focus are marked as `D' for diffuse. (B) The number of `F' and `D' type cells with EB1 knock down was quantified and compared with the number of these cell types in control cultures (`GFP RNAi control'). Knock down was defined by reduced immunostain for EB1 (`EB1 RNAi knock down') and cells in the EB1 siRNA-treated cultures that did not show EB1 depletion were quantified as an internal control (`EB1 RNAi unaffected'). Cells depleted for EB1 show reduced focus of MT minus ends at the centrosome. GFP RNAi control, n=165; EB1 RNAi unaffected, n=101; EB1 RNAi knock down, n=108.

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