Supplemental Material

This article contains the following supporting material:

• Movie 1 - GFP-labeled microtubules in a wild-type strain, GFP-tubA. Cells were grown in glycerol-containing medium at 32°C for about 20 hours and observed at 32°C. Images were acquired with a binning factor of 1x1. The movie was speeded up 10 times.

• Movie 2 - GFP-labeled microtubules in the ΔclipA mutant strain, GFP-tubA/ΔclipA. Cells were grown in glycerol-containing medium at 32°C for about 20 hours and observed at 32°C. Images were acquired with a binning factor of 1x1. The movie was speeded up 10 times.

• Movie 3 - GFP-NUDA (dynein heavy chain) in a wild type cell. Cells were grown in glycerol-containing medium at 32°C overnight and observed at 32°C. Images were acquired with a binning factor of 1x1. The movie was speeded up 10 times.

• Movie 4 - GFP-NUDA (dynein heavy chain) in a ΔclipA mutant cell. Cells were grown in glycerol-containing medium at 32°C overnight and observed at 32°C. Images were acquired with a binning factor of 1x1. The movie was speeded up 10 times.

• Movie 5 - GFP-NUDM (p150^Glued dynactin) in a wild type cell. Cells were grown in glycerol-containing medium at 32°C overnight and observed at 32°C. Images were acquired with a binning factor of 2x2. The movie was speeded up 10 times.

• Movie 6 - GFP-NUDM (p150^Glued dynactin) in a ΔclipA cell. Cells were grown in glycerol-containing medium at 32°C overnight and observed at 32°C. Images were acquired with a binning factor of 2x2. The movie was speeded up 10 times.

• Movie 7 - GFP-CLIPA in an elongating hyphal tip. Cells were grown and observed on agarose pads at 32°C after one day of growth from spores. The medium was minimal medium with 100 mM threonine and 4 mM glucose as carbon sources plus supplements. This medium is a moderate inducer of the alcA(p) promoter that controls GFP fusion expression: threonine is a strong inducer of alcA(p), while a small amount of glucose suppresses alcA(p) and improves hyphal growth. 0.6 second exposure was taken every 2 seconds for a total of 30 images. Bar, 5 μm.

• Movie 8 - GFP-NUDF in an elongating hyphal tip. Cells were grown under the same conditions as that in Movie 7.

• Movie 9 - GFP-CLIPA in an internal region. Nuclei (n) and septum (S) are marked. Asterisks track two comets that originate from the same spot on the leftmost nucleus. Cells were grown under the same conditions as that in Movie 7.

• Movie 10 - GFP-CLIPA/ΔnudA strain. Several comets are seen pausing their movement. Asterisks on the first frame mark two such comets. The top one eventually resumes movement, while the bottom one either disappears or moves out of focus. Cells were grown under the same conditions as that in Movie 7.

• Movie 11 - GFP-CLIPA in wild-type cells grown at 42°C. Cells were grown in glycerol-containing medium at 42°C for about 16 hours and observed at 42°C. Images were acquired with a binning factor of 2x2. The movie was speeded up 10 times.

• Movie 12 - GFP-CLIPA in the ΔkipA mutant cells grown at 42°C. Cells were grown in glycerol-containing medium at 42°C for about 16 hours and observed at 42°C. Images were acquired with a binning factor of 2x2. The movie was speeded up 10 times.

• Movie 13 - GFP-CLIPA in the Δkina mutant cells grown at 42°C. Cells were grown in glycerol-containing medium at 42°C for about 16 hours and observed at 42°C. Images were acquired with a binning factor of 2x2. The movie was speeded up 10 times.

• Movie 14 - GFP-NUDE in an elongating hyphal tip of a wild-type strain. Cells were grown under the same conditions as that in Movie 7. Images were captured with 1-second exposure.

• Movie 15 - GFP-NUDE in the ΔnudA strain. Two internal hyphal segments are shown. Comets are seen in the horizontal hypha and specks in the vertical hypha. Cells were grown under the same conditions as that in Movie 7.

• Movie 16 - Overexpression of GFP-NUDF in the ΔnudE/ΔclipA strain did not force GFP-NUDF to accumulate in comets. (A) GFP-NUDF/ΔnudE/ΔclipA; (B) GFP-NUDF/ΔnudE; (C) GFP- NUDA/ΔnudE/ΔclipA. Strains were grown on minimal medium with 100 mM threonine as the only carbon source to maximize GFP fusion expression. The fluorescence signals determined from original 12-bit data (first frame only, maximum possible is 4096) are 1200-1300 inside hyphae in A, 1600-1700 inside hypha in B. For C, the inside signal is 266±8 (mean±standard deviation), while the signal outside hyphae is 208±4. The GFP-NUDF comets are visible in B despite high background.