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Files in this Data Supplement:
Fig. S1. Anti-DNC-1 immunofluorescence staining. Anti-DNC-1 staining shows that (A) DNC-1 accumulation at the P2-EMS border in wild types (WT) is cell-cycle dependent, whereas DNC-1 accumulation is reduced in (B) mes-1(bn7), (C) mom-5 (or57) and (D) mom-5;src-1(RNAi) embryos. Note that anti-DNC-1 antibody staining has a higher background than live GFP-labeled embryos, in that cell borders between other cells are also stained. The P2 cell is to the right and EMS is at the bottom. White arrows mark P2-EMS dynactin accumulation. (E) Anti-DNC-1 staining in the WT one-cell embryo showing DNC-1 localization to centrosomes, spindle, MT plus-ends (shown by *), kinetochore (shown by arrowheads) and posterior cortex. This localization is gone in dnc-1(RNAi) embryos (F). (G) Western blot blotted with anti-DNC-1 antibody (1:3000) shows a single band about 150 kD in size.
Fig. S2. Defective spindle rotations in P0 and P1 blastomeres in dnc-1(or404ts) embryos. Multi-photon time series of embryos expressing tubulin::GFP in (A) wild-type (WT) one-cell embryo and (B) dnc-1(or404ts) shifted to restricted temperature at pronuclear centration showed defected P0 spindle rotation; (C) dnc-1(or404ts) shifted to restricted temperature at two-cell interphase showed normal P1 spindle rotation; (D) dnc-1(or404ts) shifted to restricted temperature at one-cell anaphase showed defective P1 spindle rotation. 0:00 represents the beginning of temperature shift. Scale bar: 10 µm.
Movie 1. Multi-photon movie of a DNC-1::GFP embryo. P2 cell is to the right and EMS is at the bottom. Frames are 4.5 seconds apart and played back at eight frames per second. Arrow points the dynactin accumulation at the P2-EMS border. See Fig. 1A.
Movie 2. Multiphoton movie of an DNC-1::GFP; mom-5 RNAi embryo. P2 cell is to the right and EMS is at the bottom. Frames are 4.5 seconds apart and played back at eight frames per second. Arrow points the dynactin accumulation at the P2-EMS border. Double-headed arrows show the misaligned spindle. See Fig. 2B.
Movie 3. Multi-photon movie of an DNC-1::GFP; src-1 RNAi embryo. P2 cell is to the right and EMS is at the bottom. Frames are 4.5 seconds apart and played back at eight frames per second. Arrow points the dynactin accumulation at the P2-EMS border. Double-headed arrows show the misaligned spindles. See Fig. 2C.
Movie 4. Multi-photon movie of DNC-1::GFP; mom-5 &src-1 RNAi embryo. P2 cell is to the right and EMS is at the bottom. Frames are 4.5 seconds apart and played back at eight frames per second. Double-headed arrows show the misaligned spindles. See Fig. 2G′.
Movie 5. Multi-photon movie of DNC-1::GFP; gpa-16(it143ts); goa-1 RNAi embryo. P2 cell is to the right and EMS is at the bottom. Frames are 4.5 seconds apart and played back at eight frames per second. Arrow points to the dynactin accumulation at the P2-EMS border. Double-headed arrows show the misaligned spindles. Correspondence to Fig. 2H.
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