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.

• Klíčová slova
• C2_04360W, MCC domain, ORF19.4518, Ypl150w, eisosome, eisosomes, hyphal morphogenesis, stress resistance,
• MeSH
• buněčná membrána * metabolismus   MeSH
• buněčná stěna * metabolismus   genetika   MeSH
• Candida albicans * genetika   patogenita   enzymologie   MeSH
• fungální proteiny * genetika   metabolismus   MeSH
• fyziologický stres * MeSH
• hyfy růst a vývoj   genetika   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• regulace genové exprese u hub MeSH
• virulence MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fungální proteiny * MeSH
• proteinkinasy 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.

• Klíčová slova
• Eisosome, Exoribonuclease, Glycolysis, SH3-like domain, Xrn1, Yeast,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Cellpose, Fiji, ImageJ, automated, blinded, cells, data analysis, microscopy, unbiased, yeast,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Membrane proteins are targeted not only to specific membranes in the cell architecture, but also to distinct lateral microdomains within individual membranes to properly execute their biological functions. Yeast tetraspan protein Nce102 has been shown to migrate between such microdomains within the plasma membrane in response to an acute drop in sphingolipid levels. Combining microscopy and biochemistry methods, we show that upon gradual ageing of a yeast culture, when sphingolipid demand increases, Nce102 migrates from the plasma membrane to the vacuole. Instead of being targeted for degradation it localizes to V-ATPase-poor, i.e., ergosterol-enriched, domains of the vacuolar membrane, analogous to its plasma membrane localization. We discovered that, together with its homologue Fhn1, Nce102 modulates vacuolar morphology, dynamics, and physiology. Specifically, the fusing of vacuoles, accompanying a switch of fermenting yeast culture to respiration, is retarded in the strain missing both proteins. Furthermore, the absence of either causes an enlargement of ergosterol-rich vacuolar membrane domains, while the vacuoles themselves become smaller. Our results clearly show decreased stability of the V-ATPase in the absence of either Nce102 or Fhn1, a possible result of the disruption of normal microdomain morphology of the vacuolar membrane. Therefore, the functionality of the vacuole as a whole might be compromised in these cells.

• Klíčová slova
• eisosome, membrane microdomains, sphingolipid metabolism, vacuolar morphology, yeast,
• MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• vakuoly metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• NCE102 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH

One of the best characterized fungal membrane microdomains is the MCC/eisosome. The MCC (membrane compartment of Can1) is an evolutionarily conserved ergosterol-rich plasma membrane domain. It is stabilized on its cytosolic face by the eisosome, a hemitubular protein complex composed of Bin/Amphiphysin/Rvs (BAR) domain-containing Pil1 and Lsp1. These two proteins bind directly to phosphatidylinositol 4,5-bisphosphate and promote the typical furrow-like shape of the microdomain, with highly curved edges and bottom. While some proteins display stable localization in the MCC/eisosome, others enter or leave it under particular conditions, such as misbalance in membrane lipid composition, changes in membrane tension, or availability of specific nutrients. These findings reveal that the MCC/eisosome, a plasma membrane microdomain with distinct morphology and lipid composition, acts as a multifaceted regulator of various cellular processes including metabolic pathways, cellular morphogenesis, signalling cascades, and mRNA decay. In this minireview, we focus on the MCC/eisosome's proposed role in the regulation of lipid metabolism. While the molecular mechanisms of the MCC/eisosome function are not completely understood, the idea of intracellular processes being regulated at the plasma membrane, the foremost barrier exposed to environmental challenges, is truly exciting.

• Klíčová slova
• MCC, eisosome, ergosterol, lipids, microdomain, phosphoinositides, regulation, sphingolipids,
• MeSH
• buněčná membrána metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fungální proteiny chemie   metabolismus   MeSH
• homeostáza MeSH
• houby metabolismus   MeSH
• metabolismus lipidů MeSH
• proteinové domény MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• fosfatidylinositol-4,5-difosfát MeSH
• fungální proteiny MeSH

Phospholipase C (Plc1p) in Saccharomyces cerevisiae is required for normal degradation of repressor Mth1p and expression of the HXT genes encoding cell membrane transporters of glucose. Plc1p is also required for normal localization of glucose transporters to the cell membrane. Consequently, plc1Δ cells display histone hypoacetylation and transcriptional defects due to reduced uptake and metabolism of glucose to acetyl-CoA, a substrate for histone acetyltransferases. In the presence of glucose, Mth1p is phosphorylated by casein kinase I Yck1/2p, ubiquitinated by the SCFGrr1 complex and degraded by the proteasome. Here, we show that while Plc1p does not affect the function of the SCFGrr1 complex or the proteasome, it is required for normal protein level of Yck2p. Since stability of Yck1/2p is regulated by a glucose-dependent mechanism, PLC1 inactivation results in destabilization of Yck1/2p and defect in Mth1p degradation. Based on our results and published data, we propose a model in which plc1Δ mutation causes increased internalization of glucose transporters, decreased transport of glucose into the cells, and consequently decreased stability of Yck1/2p, increased stability of Mth1p and decreased expression of the HXT genes.

• Klíčová slova
• casein kinase I, hexose transporters, phospholipase C, transcription,
• MeSH
• fosfolipasy typu C metabolismus   MeSH
• kaseinkinasa I chemie   metabolismus   MeSH
• proteiny přenášející monosacharidy genetika   metabolismus   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   genetika   metabolismus   MeSH
• stabilita enzymů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipasy typu C MeSH
• kaseinkinasa I MeSH
• Plc1 protein, S cerevisiae MeSH Prohlížeč
• proteiny přenášející monosacharidy MeSH
• rekombinantní proteiny MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• YCK2 protein, S cerevisiae MeSH Prohlížeč