The C2 domain protein Cts1 functions in the calcineurin signaling circuit during high-temperature stress responses in Cryptococcus neoformans.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Eukaryotic Cell (Impact Factor: 3.18). 12/2011; 10(12):1714-23. DOI: 10.1128/EC.05148-11
Source: PubMed


Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1Δ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurin-dependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.

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    • "Puncta-associated calcineurin may therefore affect different calcineurin substrates. In support of this hypothesis, phospholipid-binding protein, Cts1 (another calcineurin substrate), co-localized with calcineurin in cytosolic puncta and also co-immunoprecipitated with Cna1, with greater complex formation at 37°C compared to 24°C, suggesting that Cts1 is a substrate of calcineurin during high temperature stress [8]. "
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    ABSTRACT: Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, Δcna1, and a CRZ1/SP1 mutant (Δcrz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and Δcna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30-37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.
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    ABSTRACT: Cryptococcus neoformans is an environmental fungus that can cause severe disease in humans. C. neoformans encounters a multitude of stresses within the human host to which it must adapt in order to survive and proliferate. Upon stressful changes in the external milieu, C. neoformans must reprogram its gene expression to properly respond to and combat stress in order to maintain homeostasis. Several studies have investigated the changes that occur in response to these stresses to begin to unravel the mechanisms of adaptation in this organism. Here, we review studies that have explored stress-induced changes in gene expression with a focus on host temperature adaptation. We compare global messenger RNA (mRNA) expression data compiled from several studies and identify patterns that suggest that orchestrated, transient responses occur. We also utilize the available expression data to explore the possibility of a common stress response that may contribute to cellular protection against a variety of stresses in C. neoformans. In addition, we review studies that have revealed the significance of post-transcriptional mechanisms of mRNA regulation in response to stress, and discuss how these processes may contribute to adaptation and virulence. For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article.
    No preview · Article · May 2014 · WIREs RNA