Fig. 7

Human KIAA0556 binds to and stabilises MTs. a In hTERT-RPE1 cells with high GFP-KIAA0556 expression levels, counterstained for acetylated alpha-tubulin (red), KIAA0556 colocalises with MTs; also, acetylated tubulin levels are enhanced, indicating a stabilised MT network. This is not observed in cells expressing low levels of GFP-KIAA0556. Scale bars, 20 μM. b Similar features are observed for SF-TAP-tagged KIAA0556 (green). Shown is a representative example of a high expressing KIAA0556 cell (low expressing cell example not shown). Images were generated by confocal laser scanning and are presented as maximum projections, to rule out differences in focal depth. *p < 0.001 (compared with low KIAA0556 expression dataset; Fisher’s exact test; n = 63). c SF-TAP-KIAA0556 transfected hTERT-RPE1 cells are resistant to nocodazole-induced destabilisation of the MT network. Cells with high SF-TAP-KIAA0556 expression (arrows) display a stabilised MT network, characterised by high levels of acetylated alpha-tubulin staining (red, normal exposure images). In non-transfected cells (arrowheads in high-exposure panel), 10 minute nocodazole (10 μM) treatment destabilised the MT network. Although some acetylated tubulin immunoreactivity remained in these cells (e.g., in the cilium and close to the nucleus), a filamentous MT network pattern was not observed. In contrast, SF-TAP-KIAA0556 transfected cells retain a stabilised MT network after nocodazole treatment. Normal and increased exposure images show that a stabilised MT network (characterised by a filamentous acetylated tubulin staining pattern) is observed in almost all DMSO treated transfected and untransfected cells (arrowheads). Data in the graph (two experiments) are derived from the analysis of >1000 untransfected cells and >110 transfected cells per condition. Error bars; standard deviation of the mean. **p < 0.01 (one-way ANOVA). Scale bar, 20 μm. d KIAA0556 binds to microtubules in vitro probed by microtubule binding protein spin-down assays either with GFP-KIAA0556 (upper panel) or GFP (lower panel, negative control) expressed in HEK293 cells. In the absence of supplementary microtubules (-MT) KIAA0556 and GFP are found in the supernatant (S). In the presence of supplementary microtubules (+MT) KIAA0556 is predominantly recovered in the pellet (P). Quantification of band densities revealed 93 % of KIAA0556 in the microtubule pellet fractions. TCL 10 % of total cell lysate (input), IB immunoblot