Hsp90 and Tup1 have a stable interaction that is independent of growth temperature or developmental state.

<p><b>(A)</b> Antibody staining of whole cell protein extracts of planktonic cells grown at 30°C demonstrates that genetic depletion of <i>HSP90</i> using the <i>MAL2p</i> promoter does not affect Tup1 protein levels. <b>(B)</b> Immunoprecipitated (IP) and input samples from wild type and <i>TUP1-TAP</i> tagged strains grown at 30°C and 37°C. TAP-tagged Tup1 was pulled down using IgG agarose and blots were probed with anti-Hsp90 and anti-TAP antibodies. Enrichment of Hsp90 in the IP sample indicates that the physical interaction remains stable during standard growth (30°C) and in biofilm-inducing temperatures (37°C). <b>(C)</b> Hsp90 and Tup1 interact during biofilm and planktonic growth. Hsp90 is enriched in the immunoprecipitated <i>TUP1-TAP</i> strain relative to the input control.</p

Hsp90 is a positive regulator of transcription factor gene expression in planktonic cells.

<p><b>(A)</b><i>TEC1</i> and <i>MIG1</i> are significantly up-regulated in biofilms compared to their planktonic counterparts. <b>(B)</b> Reduction of Hsp90 levels leads to decreased expression of <i>TEC1</i>, <i>TUP1</i>, and <i>UPC2</i> during planktonic growth at 30°C with shaking at 200 rpm (*** = P < 0.001). <b>(C)</b> Reduction of Hsp90 does not affect expression of the transcription factor genes during biofilm growth (37°C, static).</p

Functional Divergence of Hsp90 Genetic Interactions in Biofilm and Planktonic Cellular States

<div><p><i>Candida albicans</i> is among the most prevalent opportunistic fungal pathogens. Its capacity to cause life-threatening bloodstream infections is associated with the ability to form biofilms, which are intrinsically drug resistant reservoirs for dispersal. A key regulator of biofilm drug resistance and dispersal is the molecular chaperone Hsp90, which stabilizes many signal transducers. We previously identified 226 <i>C</i>. <i>albicans</i> Hsp90 genetic interactors under planktonic conditions, of which 56 are involved in transcriptional regulation. Six of these transcriptional regulators have previously been implicated in biofilm formation, suggesting that Hsp90 genetic interactions identified in planktonic conditions may have functional significance in biofilms. Here, we explored the relationship between Hsp90 and five of these transcription factor genetic interactors: <i>BCR1</i>, <i>MIG1</i>, <i>TEC1</i>, <i>TUP1</i>, and <i>UPC2</i>. We deleted each transcription factor gene in an Hsp90 conditional expression strain, and assessed biofilm formation and morphogenesis. Strikingly, depletion of Hsp90 conferred no additional biofilm defect in the mutants. An interaction was observed in which deletion of <i>BCR1</i> enhanced filamentation upon reduction of Hsp90 levels. Further, although Hsp90 modulates expression of <i>TEC1</i>, <i>TUP1</i>, and <i>UPC2</i> in planktonic conditions, it has no impact in biofilms. Lastly, we probed for physical interactions between Hsp90 and Tup1, whose WD40 domain suggests that it might interact with Hsp90 directly. Hsp90 and Tup1 formed a stable complex, independent of temperature or developmental state. Our results illuminate a physical interaction between Hsp90 and a key transcriptional regulator of filamentation and biofilm formation, and suggest that Hsp90 has distinct genetic interactions in planktonic and biofilm cellular states.</p></div

Bcr1 antagonizes Hsp90’s effect on filamentation.

<p><b>(A)</b> Growing strains with wild type and reduced levels of Hsp90 under conditions that favour yeast growth (YPDM, 30°C) results in filamentation upon Hsp90 compromise. Deletion of <i>BCR1</i> enhances filamentation induced by Hsp90 depletion. <b>(B)</b> Deletion of <i>BCR1</i> alone does not affect filamentation under canonical filament-inducing conditions. Cells were imaged after 2 hours (YPD + 10% serum) and 6 hours (Lee’s and Spider media). <b>(C)</b> qRT-PCR measuring expression levels of <i>PCL1</i>, <i>PHO85</i>, and <i>HMS1</i> in wild type and <i>bcr1∆/∆</i> mutant strains grown in non-filament inducing conditions at 30°C. Bcr1 represses expression of genes in the <i>PHO85</i>-<i>PCL1</i>-<i>HMS1</i> pathway (* = P < 0.05, ** = P < 0.01, *** = P < 0.001).</p

<i>HSP90</i> genetic interactions identified in planktonic conditions do not impact biofilm formation.

<p>To genetically deplete <i>HSP90</i> the maltose-inducible promoter replaced the native promoter of the sole remaining <i>HSP90</i> allele. The wild type and <i>MAL2p-HSP90/hsp90</i> strain were then grown in RPMI 1640 (2% maltose, 0.2% glucose) planktonically and as biofilms prior to quantifying mRNA and protein levels. <b>(A)</b><i>HSP90</i> gene expression levels differ in the wild-type strain by ~50% between planktonic and biofilm cultures. In the Hsp90 depleted strain, <i>HSP90</i> is reduced in both conditions. <b>(B)</b> Western blot analysis of Hsp90 levels confirmed changes in Hsp90 protein levels. A Coomassie Blue stained 10% SDS-PAGE gel loaded with 5 μg of whole cell protein extract served as loading control. <b>(C)</b> Genetic depletion of <i>HSP90</i> using the maltose inducible promoter does not affect cell viability in strains grown planktonically or as biofilms. Biofilms were cultivated at 37°C in RPMI (2% maltose, 0.2% glucose). To ensure cells used for protein and RNA extraction were still alive following prolonged <i>HSP90</i> repression, cultures were spotted onto YPM, which promotes growth of strains with the <i>MAL2</i> promoter. Neither biofilm metabolic activity <b>(D)</b> nor dry weight <b>(E)</b> are affected by genetic depletion of Hsp90. <b>(F)</b> Homozygous deletion of <i>BCR1</i> and <i>UPC2</i> results in significantly reduced XTT conversion rates (*** = P < 0.001). <b>(G)</b> Further depleting <i>HSP90</i> in the transcription factor deletion mutants has no effect on metabolic activity as measured by XTT conversion rates.</p

Hsp90 kinase genetic interaction network.

<p>Of the 226 interactions, 34 are with kinases. Kinases are color-coded depending on their degree of connectivity, ranging from grey for one connection to orange for five connections. Kinases and test conditions (black squares) are connected with each other via edges. While the caspofungin screen shared only one of its kinase interactors (<i>CKB2</i>) with another screen, every other screen shared half or more of its interactors with another screen. For six kinases (diamonds), protein levels were measured upon Hsp90 depletion.</p

The protein kinase CK2 regulatory subunits regulate function of the Hsp90/Cdc37 protein complex.

<p>(A) Hsp90 serine and threonine phosphorylation is severely reduced in the <i>ckb1</i>Δ/<i>ckb1</i>Δ mutant, and Cdc37 serine and threonine phosphorylation is severely reduced in both the <i>ckb1</i>Δ/<i>ckb1</i>Δ and <i>ckb2</i>Δ/<i>ckb2</i>Δ mutants. Hsp90 or Cdc37 were immunoprecipitated and Western blots were hybridized with CaHsp90, TAP (to detect Cdc37-TAP), phosphothreonine, or phosphoserine antibodies. The ratio of phosphorylated to unphosphorylated Hsp90 or Cdc37 in each CK2 mutant was quantified relative to the wild type. (B) Western analysis demonstrates that Cdc37 levels are severely reduced (>50%, red box) in the mutant lacking the regulatory subunit Ckb2 (<i>ckb2</i>Δ/<i>ckb2</i>Δ) in the absence of external stress compared to the wild type (WT, BWP17); Hsp90 and Hog1 levels, however, are reduced (>25%, yellow box) in strains that lack the regulatory subunits (<i>ckb1</i>Δ/<i>ckb1</i>Δ or <i>ckb2</i>Δ/<i>ckb2</i>Δ) in response to oxidative stress in the form of a 10 minute treatment with 1 mM hydrogen peroxide. Actin served as loading control. (C) Deletion of CK2 regulatory subunits, <i>CKB1</i> or <i>CKB2</i>, phenocopies deletion of <i>HOG1</i> in terms of hypersensitivity to high osmolarity stress exerted by sorbitol. Growth is quantitatively displayed with color as indicated with the color bar. (D) Our results support a model in which the regulatory subunits of CK2 (Ckb1 and Ckb2) affect phosphorylation of Hsp90 and Cdc37, protein levels of the Hsp90-Cdc37 complex under basal or stress conditions, and levels of the target kinase Hog1.</p

Hsp90 genetic interactions identified in six screens.

<p>Hsp90 genetic interactions identified in six screens.</p

Model of high- and low-connectivity interactors identified in this screen that either modify the Hsp90/Cdc37 complex or are affected by it.

<p>The high connectivity interactors (red) modulate gene expression, protein levels, or phosphorylation of Hsp90 and Cdc37, while the chaperone complex regulates gene expression, protein levels or activation of low-connectivity interactors (blue).</p

The high-connectivity interactor Ahr1 influences <i>HSP90</i> expression and morphogenesis.

<p>(A) <i>HSP90</i> transcript levels are reduced in the <i>ahr1</i>Δ/<i>ahr1</i>Δ mutant. <i>HSP90</i> transcript levels were measured in the wild type (SN152), the <i>ahr1</i>Δ/<i>ahr1</i>Δ mutant, and the <i>AHR1</i> complemented strain by quantitative RT-PCR and normalized to <i>GPD1</i>. Data are means ± standard deviation for triplicate samples. (B) The <i>ahr1</i>Δ/<i>ahr1</i>Δ mutant filaments in rich medium at 30°C, consistent with the effects of compromised Hsp90 function. Differential Interference Contrast microscopy of strains incubated in rich medium at 30°C for 24 hours with or without 10 µM geldanamycin (GdA). Scale bar is 10 µm.</p