Temporal profiling of redox-dependent heterogeneity in single cells

Abstract

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe Grx1-roGFP2. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach, we defined a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

Proteasome lid bridges mitochondrial stress with Cdc53/Cullin1 NEDDylation status

“…In order to directly link the intracellular redox state the yCul1 rubylation status, we applied a flow cytometry methodology to cells expressing a redox-sensitive Grx1-roGFP2 probe, which allows measurement of the redox state of individual cells within a heterogeneous population and sorting them according to their oxidative status, resulting in “oxidized” and “reduced” subpopulations [62] . The sorted populations (oxidized, reduced or “mixed” populations) were tested by immunoblotting for yCul1 R /yCul1 ratio ( Fig.…”

“…Cells were then grown at 30 °C on 2–5 ml minimal medium supplemented with casein amino acid and -URA selection under aerobic conditions, diluted to approximately 0.25 OD 600 , and harvested at relevant time points following dilution. Sorting was conducted using predefined gates as described in [62] , with Grx1-roGFP2 expression under oxidized and reduced conditions determining subpopulations. These gates were adjusted according to probe expression in each of the strains.…”

A molecular switch for Cdc48 activity and localization during oxidative stress and aging

“…S6F). This accumulation of ribosomes, which most probably are not direct interactors of Cdc48 itself, might indicate potential proteotoxic stress conditions, including the accumulation of misfolded proteins or oxidative stress 30 .…”

Large‐Scale Analysis of Redox‐Sensitive Conditionally Disordered Protein Regions Reveals Their Widespread Nature and Key Roles in High‐Level Eukaryotic Processes

“…It may also contribute to the pathogenesis of many different diseases, as well as to degenerative processes associated with aging . However, ROS can also serve as cellular signaling molecules and regulate various biological processes such as the immune response, cell proliferation, development, and more . The specific effects of ROS are captured in large part through the covalent and reversible modifications of specific cysteine residues, which can in turn modify the structural or functional properties of the redox‐sensitive proteins.…”

“…[10,11] However, ROS can also serve as cellular signaling molecules and regulate various biological processes such as the immune response, cell proliferation, development, and more. [12,14] The specific effects of ROS are captured in large part through the covalent and reversible modifications of specific cysteine residues, which can in turn modify the structural or functional properties of the redox-sensitive proteins. Such posttranslational modifications are tightly regulated by changes in levels of diverse physiological oxidants, and are generally faster than expression of regulatory proteins.…”