Figure 3.

CCDC61 Binds Microtubules

(A) Fluorescent images of RPE-1 cells, transiently overexpressing GFP-hCCDC61 or hCCDC61^F128E/D129A, showing the different CCDC61 localization patterns observed under these conditions. Bar graphs show the percentage of GFP-positive cells containing clusters-only “C” versus filament-containing cells “F” (n = 279 for GFP-hCCDC61 and n = 468 for GFP-hCCDC61^F128E/D129A counted from three biological replicates). Error bars are standard deviations. Positions of blow-up images labeled with 1 (filament-containing cell) and 2 (cluster-only cell) are indicated with white-dotted squares in the top panels. Scale bars, 20 μm.

(B) Transiently overexpressed hCCDC61 colocalizes with microtubules in cells. Immunofluorescent images of RPE-1 cells transiently overexpressing GFP-hCCDC61, GFP-hCCDC61^F128E/D129A, GFP-hCCDC61^{144−287−NES}, and GFP-hCCDC61^288−512. Anti-GFP staining is shown in green, microtubule staining in red. Magnified views of the regions indicated by the white-dotted squares in the merged images are shown either below (GFP-hCCDC61 and GFP-hCCDC61^F128E/D129A) or as insets (GFP-hCCDC61^{144−287−NES} and GFP-hCCDC61^288−512). Displayed are representative images acquired from a total of 14, 8, 10, and 11 different RPE-1 cells for GFP-hCCDC61, GFP-hCCDC61^F128E/D129A, GFP-hCCDC61^{144−287−NES}, and GFP-hCCDC61^288−512, respectively. Scale bars, 10 μm.

(C) Coiled-coil and C-terminal regions of hCCDC61 bind microtubules in vitro. Coomassie-stained SDS-PAGE gel showing a co-pelleting assay of taxol-stabilized microtubules with the head domain (1–143), PAXX-fused coiled-coil domain (144–287), or the C-terminal region (288–512) of hCCDC61. S and P indicate supernatant and pellet fraction, respectively.

(D) The coiled-coil domain of hCCDC61 directly binds microtubules. Negative-stain EM micrographs of microtubules that show their decoration with a layer of PAXX-hCCDC61^144−287 that is not observed with the corresponding 5E mutant of CCDC61. Scale bars, 200 and 50 nm in the overview panels (left) and the magnified panels (right), respectively.

(E) Quantification of the widths of microtubules decorated by PAXX-hCCDC61^144−287 or in the presence of PAXX-hCCDC61^{144−287; 5E} from (D). Widths of five different positions of ten microtubules were measured for each construct. Each point (blue for PAXX-hCCDC61^144−287 and green for PAXX-hCCDC61^{144−287; 5E}) represents a measured width at each position. Error bars (standard deviations from the mean) are shown in black lines with flat arrow ends.

See also Figure S4.