scholarly journals Quantitative characterisation of low abundant yeast mitochondrial proteins reveals compensation for haplo-insufficiency in different environments

2021 ◽  
Author(s):  
Alkisti Manousaki ◽  
James Bagnall ◽  
David Spiller ◽  
Michael White ◽  
Daniela Delneri
Keyword(s):  
Bound State ◽  
Correlation Spectroscopy ◽  
Yeast Cells ◽  
Compensatory Mechanism ◽  
Protein Abundance ◽  
Fluorescence Correlation ◽  
Non Invasive ◽  
Quantitative Characterisation ◽  
Membrane Bound ◽  
The Impact

Quantification of low abundant membrane-bound proteins such as transcriptional factors and chaperones has been proved difficult even with the most sophisticated analytical technologies. Here we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for quantitation of low abundance protein and as proof of principle we choose two interacting membrane-bound proteins involved in fission of mitochondria in yeast. In Saccharomyces cerevisiae the recruitment of Fis1p and Mdv1p fission proteins to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We used FCS in single, GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells, and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism which controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.

scholarly journals Intramolecular dynamics of single molecules in free diffusion

2017 ◽  
Author(s):  
Charles Limouse ◽  
Jason C. Bell ◽  
Colin J. Fuller ◽  
Aaron F. Straight ◽  
Hideo Mabuchi
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Correlation Spectroscopy ◽  
Biomolecular Systems ◽  
Optimal Position ◽  
Biochemical Processes ◽  
Fluorescence Correlation ◽  
Dna Molecule ◽  
Feedback Scheme ◽  
The Impact

AbstractBiomolecular systems such as multiprotein complexes or biopolymers can span several tens to several hundreds of nanometers, but the dynamics of such “mesocale” molecules remain challenging to probe. We have developed a single-molecule technique that uses Tracking Fluorescence Correlation Spectroscopy (tFCS) to measure the conformation and dynamics of molecular assemblies specifically at the mesoscale level (~100-1000 nm). tFCS is non-perturbative, as molecules, which are tracked in real-time, are untethered and freely diffusing. To achieve sub-diffraction spatial resolution, we use a feedback scheme which allows us to maintain the molecule at an optimal position within the laser intensity gradient. We find that tFCS is sufficiently sensitive to measure the distance fluctuations between two sites within a DNA molecule separated by distances as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted chromatin, and can assay transient protein mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the impact that tFCS can have in the study of a wide variety of biochemical processes involving mesoscale conformational dynamics.

scholarly journals Understanding the Impact of Industrial Stress Conditions on Replicative Aging in Saccharomyces cerevisiae

2021 ◽  
Vol 2 ◽  
Author(s):  
Marco Eigenfeld ◽  
Roland Kerpes ◽  
Thomas Becker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Real Time ◽  
High Throughput ◽  
Stress Conditions ◽  
Yeast Cells ◽  
Industrial Processes ◽  
Non Invasive ◽  
Age Related ◽  
Yeast Aging ◽  
The Impact

In yeast, aging is widely understood as the decline of physiological function and the decreasing ability to adapt to environmental changes. Saccharomyces cerevisiae has become an important model organism for the investigation of these processes. Yeast is used in industrial processes (beer and wine production), and several stress conditions can influence its intracellular aging processes. The aim of this review is to summarize the current knowledge on applied stress conditions, such as osmotic pressure, primary metabolites (e.g., ethanol), low pH, oxidative stress, heat on aging indicators, age-related physiological changes, and yeast longevity. There is clear evidence that yeast cells are exposed to many stressors influencing viability and vitality, leading to an age-related shift in age distribution. Currently, there is a lack of rapid, non-invasive methods allowing the investigation of aspects of yeast aging in real time on a single-cell basis using the high-throughput approach. Methods such as micromanipulation, centrifugal elutriator, or biotinylation do not provide real-time information on age distributions in industrial processes. In contrast, innovative approaches, such as non-invasive fluorescence coupled flow cytometry intended for high-throughput measurements, could be promising for determining the replicative age of yeast cells in fermentation and its impact on industrial stress conditions.

scholarly journals The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors

2016 ◽  
Vol 44 (2) ◽  
pp. 624-629 ◽  
Author(s):  
Laura E. Kilpatrick ◽  
Stephen J. Hill
Keyword(s):  
G Protein ◽  
Fluorescence Correlation Spectroscopy ◽  
Photon Counting ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
G Protein Coupled Receptors ◽  
Fluorescence Correlation ◽  
Membrane Bound ◽  
Molecular Brightness ◽  
G Protein Coupled

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

scholarly journals Bacterial pore-forming proteins induce non-monotonic dynamics due to lipid ejection and crowding

2020 ◽  
Author(s):  
Ilanila Ilangumaran Ponmalar ◽  
K. G. Ayappa ◽  
J. K. Basu
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Bacterial Virulence ◽  
Correlation Spectroscopy ◽  
Listeriolysin O ◽  
Oligomeric State ◽  
Fluorescence Correlation ◽  
Confocal Fluorescence ◽  
Membrane Bound ◽  
Lipid Diffusion ◽  
Free Area

ABSTRACTDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors require an understanding of the target cellular response to combat rising antimicrobial resistance. Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane disruption and related lipid mediated active repair processes during attack by PFTs remain largely unexplored. We report counter intuitive and non-monotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane bound oligomers of a prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO). The observed protein concentration dependent dynamical cross-over is correlated with transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events due to a preponderance of ring-like pore states. At higher protein concentrations where membrane inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodelling and mechanical property variations, providing unique insights into developing strategies to combat virulent bacterial pathogens responsible for several infectious diseases.SIGNIFICANCEDeveloping alternate strategies against pore forming toxin (PFT) mediated bacterial virulence factors requires understanding target cellular responses and cellular defence strategies to combat rising antimicrobial resistant strains. While it is well understood that PFTs exist in a wide variety of oligomeric states, the underlying membrane response to these states is unexplored. Using confocal fluorescence correlation spectroscopy and a membrane free area based model we relate non-monotonic variations in the lipid diffusivity arising from an interplay of lipid ejection events and membrane crowding due to variations in concentration of membrane bound listeriolysin O. Our observations have a direct bearing on understanding cellular defense and repair mechanisms effective during initial stages of bacterial infection and intrinsically connected to the underlying membrane fluidity.

scholarly journals Transbilayer Coupling of Lipids in Cells Investigated by Imaging Fluorescence Correlation Spectroscopy

2022 ◽  
Author(s):  
Nirmalya Bag ◽  
Erwin London ◽  
David A Holowka ◽  
Barbara Baird
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Fluorescence Correlation Spectroscopy ◽  
Immunoglobulin E ◽  
Correlation Spectroscopy ◽  
Model Systems ◽  
Live Cells ◽  
Membrane Diffusion ◽  
Fluorescence Correlation ◽  
The Impact

Plasma membrane hosts numerous receptors, sensors, and ion channels involved in cellular signaling. Phase separation of the plasma membrane is emerging as a key biophysical regulator of signaling reactions in multiple physiological and pathological contexts. There is much evidence that plasma membrane composition supports the co-existence liquid-ordered (Lo) and liquid-disordered (Ld) phases or domains at physiological conditions. However, this phase/domain separation is nanoscopic and transient in live cells. It is recently proposed that transbilayer coupling between the inner and outer leaflets of the plasma membrane is driven by their asymmetric lipid distribution and by dynamic cytoskeleton-lipid composites that contribute to the formation and transience of Lo/Ld phase separation in live cells. In this Perspective, we highlight new approaches to investigate how transbilayer coupling may influence phase separation. For quantitative evaluation of the impact of these interactions, we introduce an experimental strategy centered around Imaging Fluorescence Correlation Spectroscopy (ImFCS), which measures membrane diffusion with very high precision. To demonstrate this strategy we choose two well-established model systems for transbilayer interactions: crosslinking by multivalent antigen of immunoglobulin E bound to receptor FcεRI, and crosslinking by cholera toxin B of GM1 gangliosides. We discuss emerging methods to systematically perturb membrane lipid composition, particularly exchange of outer leaflet lipids with exogenous lipids using methyl alpha cyclodextrin. These selective perturbations may be quantitatively evaluated with ImFCS and other high-resolution biophysical tools to discover novel principles of lipid-mediated phase separation in live cells in the context of their pathophysiological relevance.

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