JCS054460 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

• Supplemental Table S2 -

• Supplemental Figure S1 -

  Fig. S1. Yeast two-hybrid assays reveal that Dph1 interacts with the tail of Myo52 (left plate) and Tip1 (right plate). Tip1 specifically associated with Dph1, and not other ubiquitin receptor proteins such as Rhp23. Cells containing the specified plasmids were cultured on YNB plates lacking indicated amino acids at 30°C for 3 days.

• Supplemental Figure S2 -

  Fig. S2. (A) Graph showing changes in relative Tip1-HA levels in wild-type (filled diamonds), myo52Δ (empty diamonds), dph1Δ (empty triangles), mts3-1 (crosses) cells and Tip1-HA levels in myo52Δ dph1Δ (filled diamonds) cells upon addition of cycloheximide. Relative Tip1/Tip-HA levels were determined by comparing ratio of band intensities from anti-HA, anti-Tip1 and anti-Cdc8 (loading control) alkaline phosphatase western blots. (B) The distributions of microtubule number within wild type (white), myo52Δ (grey) and myo52Δ dph1Δ (black) cells, averaged from over 300 cells / genotype, fitted to Gaussian distributions with resultant mean values of 3.05, 3.97 and 4.54 respectively.

• Movie 1 -

  Movie 1. Timelapse movie showing maximum projections of 21 z-sections through a mal3-gfp nmt81mCherry−atb2 myo52-1 cell. Mal3 localises normally upon microtubules in these cells, even in cells with a bent cell morphology (arrowhead). Z-sections separated by 0.2 µm. Frames of two wavelength z-stacks were acquired every 5 seconds. Total duration of movie was 35 seconds. Scale bar: 5 µm

• Movie 2 -

  Movie 2. Simultaneous timelapse movie of maximum projections from 21 z-sections through tip1-GFP myo52Δ cells mixed with TRITC-lectin coated tip1-GFP myo52⁺ cells. GFP signal is shown in the left, and the TRITC signal (highlighting the myo52⁺ cells) is shown in the right. Cytoplasmic GFP signal is increased and cell tip associated GFP signal. Z-sections separated by 0.2 µm. Frames of z-stacks were acquired every 2 seconds. Total duration of movie was 40 seconds. Scale bar: 5 µm

• Movie 3 -

  Movie 3. Timelapse movie of maximum projections from 10 z-sections through a tip1-tdTomato gfp-atb2 cells. Tip1-labelled microtubules polymerise until contacting the cell tip, when Tip1 is deposited at the cortex and the microtubules undergo catastrophe. Z-sections separated by 0.2 µm. Frames of two wavelength z-stacks were acquired every 5 seconds. Total duration of movie was 300 seconds. Scale bar: 5 µm

• Movie 4 -

  Movie 4. Timelapse movie of maximum projections from 10 z-sections through a tip1-tdTomato gfp-atb2 myo52Δ cell. Microtubules continue to polymerise upon contact with the cell tip, curl around the cell end, and grow back into the cytoplasm. Z-sections separated by 0.2 µm. Frames of two wavelength z-stacks were acquired every 5 seconds. Total duration of movie was 60 seconds. Scale bar: 5 µm.

• Movie 5 -

  Movie 5. Timelapse movie of serial section (7) maximum projections through a tip1-tdTomato gfp-atb2 myo52-1 cell. Tip1-loaded microtubules continue to polymerise upon contact with the cell tip, curl around the cell end, and grow back into the cytoplasm before undergoing catastrophe. Z-sections separated by 0.2 µm. Frames of two wavelength z-stacks were acquired every 5 seconds. Total duration of movie was 60 seconds. Scale bar: 5 µm