Fig. 6

MT asters are anchored by FSD1 at mother centrioles. a RPE-1 cells transfected with control, FSD1, or Kif3a siRNAs were stained with anti-Ninein (green), anti-C-Nap1 (red), and anti-Ac-tubulin (purple) antibodies. Scale bar, 1 μm. b RPE-1 cells stained with indicated antibody were visualized using the stimulated emission depletion microscopy (STED). Schematic representation of the precise FSD1 localization on mother centrioles. Scale bar, 500 nm. c RPE-1 cells subjected to a microtubule regrowth assay were fixed and permeabilized with cold methanol and stained with antibodies against FSD1 and α-tubulin. The sample was then imaged through super-resolution 3D-SIM. Scale bars, 5 μm (main image) and 500 nm (magnified region). d Ectopically expressed full-length FSD1 co-localized with microtubule asters regrown after nocodazole release 10 min. Magnified centrioles are shown in the insets. Scale bars, 5 μm (main image) and 1 μm (magnified region). e Schematic diagram illustrates the different domains of FSD1 for its microtubule (MT) binding and centriolar (CEN) localization. f Endogenous FSD1 binds to microtubules. RPE-1 cell extracts were incubated with increasing amounts of Taxol-stabilized microtubules and then centrifuged (100,000 × g for 40 min) to form supernatant and pellet fractions. Samples from both fractions were probed with the indicated antibodies. The p38 and γ-tubulin were used as negative and positive controls for microtubule binding, respectively. g The FSD1-SPRY domain could directly bind to microtubules. The different purified MBP-tagged FSD1 truncations were incubated in the presence (+) or absence (−) of Taxol-stabilized microtubules (MTs) and were separated into supernatant (S) and pellet (P) fractions after high-speed centrifugation. Coomassie blue staining of both tubulins and FSD1 is indicated. h FSD1 could bind to microtubules (MTs), as shown by TIRF microscopy. GMPCPP seed MTs (with Alexa-647 and biotin labeled tubulin) were attached to a neutravidin-coated coverslip. Unpolymerized tubulin labeled with Alexa 561 was added and allowed to polymerize at room temperature for 5 min, then, 10 nM Flag-GFP-Vector or Flag-GFP-FSD1 (green) was added to the solution. The sample was imaged after reaction setup for 5 min through TIRF microscopy. Scale bars, 5 μm (main image) and 2 μm (magnified region)