JCS007567 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 (Adobe PDF) -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Sds21.NEGFP in endocytosis. Kymograph analysis of Sds21.NEGFP foci in sds21.NEGFP dis2.Δ cells.

• Supplemental Figure S2 -

  Fig. S2. Sds21.NEGFP levels increase upon deletion of dis2⁺. (A) An image of the blot that was cropped to produce Fig. 3F to show that Dis2.NEGFP levels are much higher than those of Sds21.NEGFP. (B) The indicated strains were prepared as described for Fig. 3D and E in the text. The data clearly show that Sds21.NEGFP signals are fainter than those of Dis2.NEGFP, even when the levels are elevated (A) as a consequence of the deletion of the dis2⁺ gene. Bar, 5 μm.

• Supplemental Figure S3 -

  Fig. S3. Induction of wsh3 alleles. (A) As in Fig. 10B; images compiled by compression of three 0.3 μm slices at the indicated intervals (seconds). Bar, 5μm. (B) Western blots of the indicated strains to show the relative levels of Wsh3 protein production following derepression of nmt41 promoter of the leu1::nmt41wsh3.pkCX transgene.

• Supplemental Figure S4 -

  Fig. S4 . Recruitment of Dis2.NEGFP to Wsh3 foci at the cortex away from cell tips. Imaging of dis2.NEGFP wsh3.C2tdTom cells as described for Fig. 9B showing the transient recruitment of Dis2.NEGFP to Wsh3.C2tdTom foci that lie at cortical sites away from the cell tip. Bar, 5 μm.

• Movie 1 -

  Movie 1. Dis2.NEGFP associates with cell tips, endocytic vesicles and centromeres of interphase cells. One focal plane images were taken continuously for this movie of Dis2.NEGFP fluorescence. The time is shown in the upper left-hand corner. The blue brackets indicate Dis2.NEGFP caps at the cell tips, and the yellow arrows and brackets indicate Dis2.NEGFP foci that appear at the cell surface before moving in from the cortex and disappearing.

• Movie 2 -

  Movie 2. Dis2.NEGFP intranuclear foci migrate to the tips of separating anaphase nuclei. This is the movie from which the frames for Fig. 1C were taken.

• Movie 3 -

  Movie 3. Caps of Dis2.NEGFP foci at cell tips are not affected by the addition of DMSO. Maximal projections of nmt81atb2.NGFP (asterisk) and dis2.NEGFP cells to which 10 mM CBZ in DMSO had been added. A frame from this movie is shown in Fig. 4B. The blue brackets indicate the cap of Dis2.NEGFP that is seen at cell tips.

• Movie 4 -

  Movie 4. Caps of Dis2.NEGFP foci at cell tips are abolished following depolymerization of microtubules with CBZ. Maximal projections of nmt81atb2.NGFP (asterisk) and dis2.NEGFP cells to which 10 mM CBZ in DMSO had been added. A frame from this movie is shown in Fig. 4D.

• Movie 5 -

  Movie 5. Wsh3 is required for recruitment of Dis2.NEGFP to cell tips. Three consecutive 0.2 μm slices were compressed to give each frame of this movie showing Dis2.NEGFP fluorescence. The first few frames show a red lectin signal to identify the left-hand cell as a wild-type wsh3⁺ cell whereas the two unstained cells on the right, are wsh3.Δ. The wsh3.Δ cells lack the cap of Dis2.NEGFP fluorescence seen at the tips of the wild-type cell. A single frame from this movie is shown in the middle panel of Fig. 9A.

• Movie 6 -

  Movie 6. Dis2.NEGFP colocalises with wsh3.C2tdTomato at the cell tip. Consecutive green and red images of dis2.NEGFP wsh3.C2tdTom cells were continuously taken in three 0.2 μm slices and compressed to give each frame of this movie.