Biology subjects

Makarova, M.

Publications and source records attributed to Makarova, M..

6 recordsLinked to original sources

bioRxiv2022

Single-molecule analysis of receptor-beta-arrestin interactions in living cells

{beta}-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-{beta}-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in {beta}-arrestin interactions with both receptors and the lipid bilayer. In contrast to the currently widely accepted model, we show that {beta}-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, we show that following receptor interaction, the plasma membrane stabilizes {beta}-arrestin in a membrane-bound, active-like conformation, allowing it to diffuse to clathrin coated pits separately from the activating receptor. These results challenge our current understanding of {beta}-arrestin function at the plasma membrane, revealing a new critical role for {beta}-arrestin pre-association with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

cell biology↗

bioRxiv2022

Phospholipid tail asymmetry allows cellular adaptation to anoxic environments

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Here, we demonstrate the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl-tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, S. japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy whereas its sister species, the well-known model organism S. pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches. One-Sentence SummaryIn anoxic environments, saturated asymmetric acyl-tailed phospholipids can replace unsaturated ones to maintain membrane physical properties.

cell biology↗

bioRxiv2022

Topology-based fluorescence image analysis for automated cell identification and segmentation

Cell segmentation refers to the body of techniques used to identify cells in images and extract biologically relevant information from them; however, manual segmentation is laborious and subjective. We present Topological Boundary Line Estimation using Recurrence Of Neighbouring Emissions (TOBLERONE), a topological image analysis tool which identifies persistent homological image features as opposed to the geometric analysis commonly employed. We demonstrate that topological data analysis can provide accurate segmentation of arbitrarily-shaped cells, offering a means for automatic and objective data extraction. One cellular feature of particular interest in biology is the plasma membrane, which has been shown to present varying degrees of lipid packing, or membrane order, depending on the function and morphology of the cell type. With the use of environmentally-sensitive dyes, images derived from confocal microscopy can be used to quantify the degree of membrane order. We demonstrate that TOBLERONE is capable of automating this task.

bioinformatics↗

bioRxiv2022

An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus.

microbiology↗

bioRxiv2020

Asymmetric phospholipids impart novel biophysical properties to lipid bilayers allowing environmental adaptation

Phospholipids are a diverse group of biomolecules consisting of a hydrophilic head group and two hydrophobic acyl tails. The nature of the head and length and saturation of the acyl tails are important for defining the biophysical properties of lipid bilayers. It has recently been shown that the membranes of certain yeast species contain high levels of unusual asymmetric phospholipids, consisting of one long and one medium chain acyl moiety - a configuration not common in mammalian cells or other well studied model yeast species. This raises the possibility that structurally asymmetric phospholipids impart novel biophysical properties to the yeast membranes. Here, we use atomistic molecular dynamics simulations (MD) and environmentally-sensitive fluorescent membrane probes to characterize key biophysical parameters of membranes formed from asymmetric lipids for the first time. Interestingly, we show that saturated, but asymmetric phospholipids maintain membrane lipid order across a wider range of temperatures and do not require acyl tail unsaturation or sterols to maintain their properties. This may allow cells to maintain membrane fluidity even in environments which lack the oxygen required for the synthesis of unsaturated lipids and sterols.

biophysics↗

bioRxiv2019

Delineating the rules for structural adaptation of membrane-associated proteins to evolutionary changes in membrane lipidome

Membrane function is fundamental to life. Each species explores membrane lipid diversity within a genetically predefined range of possibilities. How membrane lipid composition in turn defines the functional space available for evolution of membrane-centered processes remains largely unknown. We address this fundamental question using related fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. We show that unlike S. pombe that generates membranes where both glycerophospholipid acyl tails are predominantly 16-18 carbons long, S. japonicus synthesizes unusual asymmetrical glycerophospholipids where the tails differ in length by 6-8 carbons. This results in stiffer bilayers with distinct lipid packing properties. Retroengineered S. pombe synthesizing the S. japonicus-type phospholipids exhibits unfolded protein response and downregulates secretion. Importantly, our protein sequence comparisons and domain swap experiments indicate that transmembrane helices co-evolve with membranes, suggesting that, on the evolutionary scale, changes in membrane lipid composition may necessitate extensive adaptation of the membrane-associated proteome.

cell biology↗