Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie01 - Supplemental Movie 1. Bimolecular Fluorescence Complementation Assay. S2 cells transfected with EB1-VN and EB1-VC (left), NH2-(VN) and COOH-terminal (VC) halves of Venus alone (middle) and VN-Shot-VC in the cis orientation (right). EB1 is an obligate dimer and is a positive control for the reconstitution of fluorescence, and untagged VN and VC should not fluoresce. We observed fluorescence at the tips of microtubules in cells expressing VN-Shot-VC indicating that Shot's NH2- and COOHtermini are close enough for reconstitution of the Venus fluorescent protein. Images were acquired by Hawk-VT multi-point array scanning confocal microscope (Visitech International, Sunderland, UK) for 2 min.
- Movie02 - Supplemental Movie 2. The central rod domain is required for the interaction between Shot's ABD and EF-hand-GAS2 domain. S2 cells transfected with VN-Shot-VC (left), VN-ShotΔRod-VC (middle), and ShotΔRod- EGFP (right). Without the central rod domain Shot's NH2- and COOH are unable to interact. Images were acquired by Hawk-VT multi-point array scanning confocal microscope (Visitech International, Sunderland, UK) for 2 min.
- Movie03 - Supplemental Movie 3. EF-hand motifs contribute to Shot's regulation. S2 cells transfected with VC-ShotΔEF-hand-VN (left), VC-ShotEF-handMut-VN (middle), and VC-Shot-VN (right). Without the EF-hand motifs, Shot is trapped in an “open” conformation and is unable to reconstitute fluorescence, however, a mutant version of Shot where key calcium coordinating residues in the EF-hand motif where mutated (D5080A, D5082A, N5084A) reconstituted fluorescence at the tips of microtubules similar to VC-Shot-VN. Images were acquired by Hawk-VT multi-point array scanning confocal microscope (Visitech International, Sunderland, UK) for 45 sec.
- Movie04 - Supplemental Movie 4. Rapamycin induced folding targets Shot to the microtubule plus end. S2 cells co-transfected with FKBP-Shot-FRB-EGFP and EB1-mRFP following perfusion with 500 nM rapamycin (left) and DMSO (right). Perfusion of rapamycin induces dimerization and targets Shot to the tips of microtubules while DMSO treated cells appeared wild-type. Images were acquired on laser TIRF system (Nikon,Tokyo, Japan) for 3 min.
- Movie05 - Supplemental Movie 5. Live-Cell imaging of rapamycin perfusion. S2 cells expressing FKBP-Shot-FRB-EGFP perfused with either DMSO (left) or 500 nM rapamycin (right). Images were acquired on laser TIRF system (Nikon,Tokyo, Japan) for 3 min.