Supplementary Information for
for Kern DM, Nicholls PK, Page DC, Cheeseman IM.
A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends.
J Cell Biol 213, 315 (2016)
- Supplementary Material, Includes:
- Figure S1: Alignment and phylogeny for SKAP and its isoforms and characterization of SKAP isoforms in mouse cells and tissues.
- Figure S2: SKAP interphase interactions and validation of the SKAP depletion and replacement protocols.
- Figure S3: SKAP mitotic plus-end tracking, validation of the SKAP depletion and SKAP ?EB mutant replacement protocol, spindle positioning experiment controls, and astral microtubule phenotype.
- Figure S4: Mitotic interactions of the Astrin/SKAP complex and interphase Clasp1 localization.
- Table S1: Cell lines used in this study.
- Video 1 SKAP depletion causes multiple mitotic defects. Cells going through mitosis with SKAP depletion (related to Fig. 2). DNA, magenta; differential interference contrast microscopy, green. Total imaging time: 8h.
- Video 2 Interphase expression of long versus short SKAP-tdTomato constructs. Cells in interphase with indicated transfected constructs (related to Fig. 2). Expression of long SKAP leads to puncta in interphase and weak microtubule loation. However, short SKAP displays clear plus-end tracking. Videos are scaled identically.
- Video 3 SKAP tracks microtubule plus ends. SKAP-GFP on a spinning disc confocal microscope (see Materials and methods; related to Fig. 4). Total imaging time: 30 s.
- Video 4 SKAP delEB cells display a spindle shift in metaphase. Mitotic cells used for kymographs in Fig. 5 D. SKAP wild-type (WT; left) and ?EB cells are shown. Total time: 70 min.
- Video 5 Astral microtubule plus ends grow laterally along the cell cortex in metaphase SKAP delEB cells. Examples of EB3-tdTomato videos for control, SKAP-depleted, rescued, and SKAP ?EB cells including the two displayed in Fig. 6 D as indicated. otal imaging time 60 s; 30 s for SKAP wild type (WT). Images collected every 2 s. All videos are scaled equivalently.
- Video 6 Additional metaphase SKAP delEB cell videos. Additional videos showing EB3-marked plus ends in SKAP delEB cells (related to Fig. 6 and Video 5).