Jakub Zahumensky

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.

In our approach, (cell-walled) cells in thick suspensions are placed on microscopy cover glass and overlaid by a small block of 1% agarose prepared either in buffer or cultivation medium identical to that of the respective cell culture, ensuring a close-to-natural environment. The cover glass is taped to a custom-made microscopy sample holder (Tinkercad file for 3D printing can be downloaded here). In this approach, the cells are immobilized on the cover glass just by mild mechanical forces, which does not induce a cellular stress response, as documented by normal actin structure in C. elegans (Chen and Pan, 2021) and by the absence of stress granules S. cerevisiae cells (Grousl et al., 2015; Vaškovičová et al., 2017). Furthermore, the cells spontaneously prefer a monolayer arrangement, i.e., they are mostly in the same focal plane. This makes microscopy imaging more efficient, saving both time and storage space. Since the agarose is permeable for small molecules, cells can be treated by directly adding chemicals onto the agarose block (in an inverted microscope) during time-lapse experiments (Grossmann et al., 2007; Grousl et al., 2015; Vaškovičová et al., 2017). Adding a cover glass on the other side of the sample holder (Fig. 2) prevents water evaporation and shrinking of the agarose block enabling long time-lapse experiments. The described arrangement thus represents an alternative to both surfaces coated with concanavalin A or poly-L-lysine, and expensive microfluidics systems.

Fluorescence microscopy images of biological samples contain valuable information but require rigor-ous 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 in-volves 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 cover glass. 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 addi-tion 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. Graphical overview: From fluorescence microscopy to numbers and plots – a generalized workflow

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.

Eisosomes are large hemitubular structures that underlie the invaginated microdomains in the plasma membrane of various ascomycetous fungi, lichens and unicellular algae. In fungi, they are organized by BAR-domain containing proteins of the Pil1 family. Two such proteins, Pil1 and Lsp1, participate in eisosome formation in the yeast Saccharomyces cerevisiae. Under normal laboratory conditions, deletion of the PIL1 gene results in the inability of cells to assemble wild-type-like eisosomes. We found that under certain stress conditions, Lsp1 partially substitutes for the Pil1 function and mediates assembly of eisosomes, specifically following a decrease in the activity of serine palmitoyltransferase, for example, in response to hyperosmotic stress. Besides Lsp1, the assembly of eisosomes lacking Pil1 also requires Seg1 and Nce102 proteins. Using next-generation sequencing, we found that the seg1Δnce102Δpil1Δ strain, which is unable to form eisosomes, overexpresses genes coding for proteins of oxidative phosphorylation and tricarboxylic acid cycle. By contrast, genes involved in DNA repair, ribosome biogenesis and cell cycle are downregulated. Our results identify Lsp1 as a stress-responsive eisosome organizer and indicate several novel functional connections between the eisosome and essential cellular processes.

In eukaryotic cells, mitochondria are constantly adapting to changes in the biological activity of the cell, i.e., changes in nutrient availability and environmental stresses. We propose a model in which this adaptation is mediated by lipids.

Microorganisms are challenged constantly by their rapidly changing environment. To survive, they have developed diverse mechanisms to quickly perceive stressful situations and adapt to them appropriately.

Absence of Isc1, the yeast homologue of mammalian neutral sphingomyelinase type 2, leads to severe mitochondrial dysfunction. We show that deletion of another type-C phospholipase, the phosphatidylglycerol (PG)-specific Pgc1, rescues this defect. Phosphatidylethanolamine (PE) levels and cytochrome c oxidase activity, reduced in isc1 Δ cells, were restored to wild-type levels in the pgc1 Δ isc1 Δ mutant. Pgc1 substrate, PG, inhibited in vitro activity of Isc1 and phosphatidylserine decarboxylase Psd1, an enzyme crucial for PE biosynthesis. We also identify a mechanism by which the balance between the current demand for PG and its consumption is controlled. We document that the product of PG hydrolysis, diacylglycerol, competes with the substrate of PG-phosphate synthase, Pgs1, and thereby inhibits the biosynthesis of excess PG. This feedback loop does not work in the absence of Pgc1, which catalyzes PG degradation. Finally, Pgc1 activity is partially inhibited by products of Isc1-mediated hydrolysis. The described functional interconnection of the two phospholipases contributes significantly to lipid homeostasis throughout the cellular architecture. Summary: The coordinated action of two different type-C phospholipases is documented, which provides a balance between mitochondrial phospholipid biosynthesis and sphingolipid metabolism. The regulatory role of specific lipids, phosphatidylglycerol, diacylglycerol and ceramide in this process is demonstrated.

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae , Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102 Δ deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans , suggesting a conserved principle of local sphingolipid control across species.