Flo11p-independent control of "mat" formation by hsp70 molecular chaperones and nucleotide exchange factors in yeast

Abstract

The yeast Saccharomyces cerevisiae has been used as a model for fungal biofilm formation due to its ability to adhere to plastic surfaces and to form mats on low-density agar petri plates. Mats are complex multicellular structures composed of a network of cables that form a central hub from which emanate multiple radial spokes. This reproducible and elaborate pattern is indicative of a highly regulated developmental program that depends on specific transcriptional programming, environmental cues, and possibly cell-cell communication systems. While biofilm formation and sliding motility were shown to be strictly dependent on the cell-surface adhesin Flo11p, little is known about the cellular machinery that controls mat formation. Here we show that Hsp70 molecular chaperones play key roles in this process with the assistance of the nucleotide exchange factors Fes1p and Sse1p and the Hsp40 family member Ydj1p. The disruption of these cofactors completely abolished mat formation. Furthermore, complex interactions among SSA genes were observed: mat formation depended mostly on SSA1 while minor defects were observed upon loss of SSA2; additional mutations in SSA3 or SSA4 further enhanced these phenotypes. Importantly, these mutations did not compromise invasive growth or Flo11p expression, suggesting that Flo11p-independent pathways are necessary to form mats.

Figure 1.—

The S. cerevisiae Σ1278b strain forms mats on low-agar plates. A wild-type strain or a Δflo11 mutant (both MATα) was inoculated at the center of YPD plates containing 0.3% agar and grown at 23° for 7 or 14 days. Photographs of the whole plates and of a magnified portion of the plates are shown (note that in Figures 1–4 and 6 the photographs at 7 and 14 days do not necessarily show the same plate).

Figure 2.—

The Δfes1, Δsse1, and Δydj1 mutants are affected in mat formation. Mats were grown as indicated in Figure 1 (see supplemental Figure S2 at http://www.genetics.org/supplemental/ for MATa versions of these mutants and Figure 3 for quantitative measures).

Figure 3.—

Quantification of the diameter and number of spokes for each mat. For each indicated strain, eight mats were grown in parallel at 23° and the diameter (solid triangles) and number of spokes (solid circles) were measured after 7 days (the indicated numbers correspond to the average ±SE).

Figure 4.—

The overexpression of SSE1 or FES1 does not equally complement the mat defect of the Δfes1 and Δsse1 mutants. The Δfes1 and Δsse1 strains were transformed with pGPD416 (control), pGPD416-SSE1, pGPD416-FLAG-SSE2, or pGPD416-FES1 (see materials and methods) and mats were then grown as indicated in Figure 1 (see supplemental Figure S3 at http://www.genetics.org/supplemental/ for the wild-type control).

Figure 5.—

Mutations in SSA1-4 genes affect mat formation differently. Mats were grown as indicated in Figure 1 (see supplemental Figure S4 at http://www.genetics.org/supplemental/ for MATa versions of these mutants and Figure 3 for quantitative measures).

Figure 6.—

Mutations in SSA3 or SSA4 affect mat formation in a Δssa1 or Δssa2 context. Mats were formed as indicated in Figure 1 (see supplemental Figure S5 at http://www.genetics.org/supplemental/for MATa versions of these mutants and Figure 3 for quantitative measures).

Figure 7.—

Mutations in the Hsp70 system do not affect invasive growth. The indicated strains were heavily streaked on YPD plates containing 2% agar and grown for 5 days at 23°. The plates were photographed, washed with water to remove unattached cells, and photographed again. To remove the remaining cells more effectively, the plates were rubbed with a gloved finger under a gentle stream of water and photographed again.

Figure 8.—

Mutations in the Hsp70 do not affect Flo11p expression or cellular localization. (A) Cell lysates were prepared from the indicated strains (grown as mats on low-agar YPD plates at 23°) expressing HA-tagged Flo11p or from the untagged wild-type strain (control) and subjected to differential centrifugation at 13,000 × g to generate a crude membrane pellet (P13) and a supernatant (S13) containing cytosol and light vesicles. Equal amounts of proteins from each fraction were resolved by SDS–PAGE and analyzed by Western blotting with mouse anti-HA and rabbit anti-BiP antibodies. (B) The localization of Flo11p-HA was visualized by indirect immunofluorescence. The indicated strains were harvested from YPD plates, fixed, treated with mouse anti-HA antibody, and stained with Alexa Fluor 595-conjugated goat anti-mouse IgG antibody.