Biology subjects

Malinsky, J.

Publications and source records attributed to Malinsky, J..

4 recordsLinked to original sources

bioRxiv2024

Live Cell Fluorescence Microscopy - From Sample Preparation to Numbers and Plots

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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=38 SRC="FIGDIR/small/587214v3_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1ffb66borg.highwire.dtl.DTLVardef@1214a99org.highwire.dtl.DTLVardef@a8b39aorg.highwire.dtl.DTLVardef@1a744d3_HPS_FORMAT_FIGEXP M_FIG C_FIG From fluorescence microscopy to numbers and plots - a generalized workflow

cell biology↗

bioRxiv2022

Two different phospholipases C, Isc1 and Pgc1, cooperate to regulate mitochondrial function

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{Delta} cells, were restored to wild-type levels in the pgc1{Delta}isc1{Delta} 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. SummaryThe 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.

cell biology↗

bioRxiv2021

Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

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{Delta} deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

cell biology↗

bioRxiv2018

The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol

The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here we present evidence that the subcellular distribution of PG is maintained by locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. We document that the Pgc1 absence leads to spreading of PG over various cellular membranes. Fluorescently labeled Pgc1 protein strongly accumulates at the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but even host no phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to membranes of the endoplasmic reticulum, mitochondria, and even of artificial phospholipid vesicles. FRAP analysis confirms continuous exchange of GFP-Pgc1 within individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate immediate phospholipase activity of Pgc1. In this model, LD serve as storage place and shelter Pgc1 preventing untimely degradation, while both phospholipase activity and degradation of the enzyme occur in membranes.

cell biology↗