UNLABELLED: The plasma membrane is critical for the virulence of the human fungal pathogen Candida albicans. In addition to functioning as a protective barrier, the plasma membrane plays dynamic roles in a wide range of functions needed for virulence including nutrient uptake, cell wall synthesis, morphogenesis, resistance to stress, and invasive hyphal growth. Screening a collection of C. albicans mutants identified an understudied gene that is important for invasive hyphal growth, which we have termed CWR1 (Cell Wall Regulatory kinase). A mutant strain lacking CWR1 displayed defects in resisting stressful conditions that exacerbate cell wall defects. The Cwr1 protein shows strong similarity to protein kinases, suggesting it plays a regulatory role in coordinating plasma membrane and cell wall functions. A Cwr1-green fluorescent protein (GFP) fusion protein localized to punctate patches associated with the plasma membrane that partially overlapped Membrane Compartment of Can1 (MCC)/eisosome domains. In contrast to the static MCC/eisosome domains, the Cwr1-GFP patches were very dynamic. Truncation mutants lacking C-terminal sequences distal to the protein kinase domain failed to show detectable localization at the plasma membrane. Surprisingly, these mutant strains did not show the defects of a cwr1Δ mutant, suggesting that localization to punctate patches associated with the plasma membrane is not essential for Cwr1 function. Altogether, these data indicate that Cwr1 contributes to the regulation of plasma membrane functions that promote proper morphogenesis and resistance to cell wall stress, both of which are important for C. albicans virulence. IMPORTANCE: The ability of Candida albicans to grow invasively in the host and resist stress is critical for it to be an effective human pathogen. Identifying the genes that promote these processes is important for developing new strategies to block infection. Therefore, genetic methods were used in this study to identify a novel gene that is needed for invasive growth and stress resistance (Cell Wall Regulatory kinase [CWR1]). Interestingly, the Cwr1 protein localized to punctate patches in the plasma membrane, some of which co-localized with specialized subdomains of the plasma membrane known as eisosomes that are known to promote stress resistance and invasive growth in the host. Thus, these studies identified a novel regulator of traits that are critical for C. albicans pathogenesis.

• Keywords
• C2_04360W, MCC domain, ORF19.4518, Ypl150w, eisosome, eisosomes, hyphal morphogenesis, stress resistance,
• MeSH
• Cell Membrane * metabolism   MeSH
• Cell Wall * metabolism   genetics   MeSH
• Candida albicans * genetics   pathogenicity   enzymology   MeSH
• Fungal Proteins * genetics   metabolism   MeSH
• Stress, Physiological * MeSH
• Hyphae growth & development   genetics   MeSH
• Protein Kinases genetics   metabolism   MeSH
• Gene Expression Regulation, Fungal MeSH
• Virulence MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Fungal Proteins * MeSH
• Protein Kinases MeSH

The regulation of gene expression in eukaryotes relies largely on the action of exoribonucleases, evolutionarily conserved enzymes that digest decapped messenger RNAs in the 5'-3' direction. The activity of Xrn1, the major yeast exoribonuclease, is regulated by targeted changes in its cellular localisation in direct response to the cell's metabolic state. When fermentable carbon sources are available, active Xrn1 is diffusely localised in the cytosol. Upon depletion of these sources, Xrn1 is sequestered at the plasma membrane-associated protein complex, the eisosome, and becomes inactive. Although this phenomenon has been described previously, the molecular mechanisms underlying these changes remain unknown. We report that the binding of Xrn1 to the plasma membrane is subject to glycolytic flux, rather than the availability of a fermentable carbon source, is independent of TORC1 activity and requires the core eisosomal proteins Pil1 and Lsp1. We identify the SH3-like domain of the Xrn1 protein as a putative interaction domain. In addition, we show that when expressed in Saccharomyces cerevisiae, the human orthologue of Xrn1 mirrors its yeast counterpart, i.e., it segregates to the eisosome under conditions of halted glycolysis. Our results not only advance our understanding of Xrn1 regulation but also indicate that this regulatory principle is conserved from yeast to humans.

• Keywords
• Eisosome, Exoribonuclease, Glycolysis, SH3-like domain, Xrn1, Yeast,
• Publication type
• Journal Article MeSH

Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.

• Keywords
• Cellpose, Fiji, ImageJ, automated, blinded, cells, data analysis, microscopy, unbiased, yeast,
• Publication type
• Journal Article MeSH
• Review MeSH