Supplemental Movie 1: Spk-associated Mlc1-YFP signal is maintained at septation in WT hyphae

Fluorescence time-lapse microscopic images of WT strain 7139 demonstrating that the Mlc1 tip-signal remains strongly localized to hyphal tips throughout hyphal development, including during septum formation.   Movie is run at 7 min/frame.  Images were collected with 500 ms exposures, every 2 min (range 10, step size of 5, 3 steps).

Supplemental Movie 2: Spk-associated Mlc1-YFP signal is lost at septation in rsr1Δ/Δ hyphae

Fluorescence time-lapse microscopic images of strain 9151 showing that prior to septum formation there is a strong Spk-associated Mlc1-YFP tip-signal.  During the sepation event, as marked by Mlc1-YFP at the septum, the tip-signal is briefly lost from the hyphal tip (inside the circle).  Growth continues during this time and the Mlc1-YFP tip-localized signal returns to the original site of growth. Movie is run at 7 min/frame.  Images were collected with 500 ms exposures, every 2 min (range 10, step size of 5, 3 steps).

Supplemental Movie 3: Ectopic sites of re-polarization are associated with loss of Rsr1 (1)

DIC time-lapse microscopic images of strain 9151 showing cessation of tip growth and repolarization of growth at an ectopic branch site.  Arrow indicates the cell that forms the ectopic branch.  Movie is run at 5 min/frame.  Images were collected with 500 ms exposures, every 2 min (range 10, step size of 5, 3 steps).

Supplemental Movie 4: Ectopic sites of re-polarization are associated with loss of Rsr1 (2)

Fluorescence time-lapse microscopic images of the cell shown in supplemental movie 3 that shows that loss of the Mlc1-tip associated signal and relocalization at an ectopic branch site.  Arrow indicates the cell that forms the ectopic branch.  Arrowhead indicates the site of emergence for the ectopic branch.  Movie is run at 5 min/frame.  Images were collected with 500 ms exposures, every 2 min (range 10, step size of 5, 3 steps).

Supplemental Movie 5: : Mlc1-YFP tip signal remains strongly associated with the hyphal tip in RSR1-expressing control strains

Fluorescence time-lapse microscopic images of a strain expressing RSR1 from the MET3 promoter, grown in media lacking methionine and cysteine.  The Mlc1- tip localized signal is clearly visible throughout septation event.  Movie is run at 5 min/frame.  Images were collected with 500 ms exposures, every 2 min (range 10, step size of 5, 3 steps).

Supplemental Movie 6: Loss of Mlc1-tip signal and ectopic repolarization events are recapitulated in rsr1 null control strain

Time-lapse images of Mlc1-YFP in a strain with repressed expression of RSR1 (strain in Supplemental Movie 2 grown in media containing methionine and cysteine).  Early frames of the movie show a strong Spk-associated tip-signal.  During the sepation event (marked by Mlc1-YFP at the septum), the tip-signal is lost from the hyphal tip, and then re-localizes at a tip-proximal ectopic site.  Movie is run at 5 min/frame.  Images were collected with 500 ms exposures, every 2 min (range 10, step size of 5, 3 steps).

Hausauer D, Gerami-Nejad M, Kistler-Anderson C, and Gale C. Hyphal guidance and invasive growth in Candida albicans require the Ras-like GTPase Rsr1p and its GTPase activating protein Bud2p. Eukaryotic Cell, 4:1273-1286, 2005.

Strain MG6748 (WT) expressing Spa2-YFP and grown on SDC + 10% serum-containing agar at 37°C to induce HF growth.  Spa2-YFP localizes constitutively to the hyphal tip and maintains a stable position.  In addition, Spa2-YFP localizes to the septum (arrow).

Strain DH9146 (rsr1∆/∆) expressing Spa2-YFP and grown on SDC + 10% serum-containing agar at 37°C to induce HF growth.  Spa2-YFP signal dances around the tip of the primary hypha (lower arrow) and the tip of the hyphal branch (upper arrow) prior to committing to a direction of growth

Strain MG8868 (bud2∆/∆) expressing Spa2-YFP and grown on SDC + 10% serum-containing agar at 37°C to induce HF growth.  Spa2-YFP signal dances around the hyphal tip prior to the hypha committing to a direction of growth.

Strain DH9146 (rsr1∆/∆) expressing Spa2-YFP and grown on SDC + 10% serum-containing agar at 37°C to induce HF growth.  Spa2-YFP signal disappears from the primary hyphal tips followed by signal appearance at septa.  In addition, in one of the hyphal branches, elongation pauses co-incident with the loss of Spa2-YFP signal from the branch tip.  Branch growth resumes at the time that Spa2-YFP reappears at the branch tip.