Variety is the spice of life: how to explore a redox-dependent heterogeneity in genomically identical cellular populations.

Cellular heterogeneity is a widespread phenomenon, existing across organisms and serving a crucial role in evolution and cell survival. Genetically identical cells may as a result present in a variety of forms with different gene and protein expressions, as well as oxidation level. As a result, a wide range of methodologies and techniques for dissecting different types of genetic, proteomic, and phenotypic heterogeneous traits have emerged in recent years in an effort to better understand how diversity exists within a single population and its effects therein. A key area of interest seeks to establish the ways in which cellular heterogeneity and aging processes interact with each other. Here, we discuss recent developments in defining cellular heterogeneity, specifically focusing on redox-dependent heterogeneity, its characterization, quantification, and behavior. We further expand on potential applications of a cell sorting-based methodology for distinguishing between cells harboring different redox statuses. As an example, we use organelle-specific fluorescence protein-based probes to examine the crosstalk between cytosol and mitochondria in a yeast strain lacking glutathione reductase. Together, these may have wide-reaching implications for future research into redox-associated factors, as well as mechanisms of redox-dependent heterogeneity and its influence on organelles and the cell at large.

Remodeling of the focal adhesion complex by hydrogen-peroxide-induced senescence.

Cellular senescence is a phenotype characterized by cessation of cell division, which can be caused by exhaustive replication or environmental stress. It is involved in age-related pathophysiological conditions and affects both the cellular cytoskeleton and the prime cellular mechanosensors, focal adhesion complexes. While the size of focal adhesions increases during senescence, it is unknown if and how this is accompanied by a remodeling of the internal focal adhesion structure. Our study uses metal-induced energy transfer to study the axial dimension of focal adhesion proteins from oxidative-stress-induced senescent cells with nanometer precision, and compares these to unstressed cells. We influenced cytoskeletal tension and the functioning of mechanosensitive ion channels using drugs and studied the combined effect of senescence and drug intervention on the focal adhesion structure. We found that H2O2-induced restructuring of the focal adhesion complex indicates a loss of tension and altered talin complexation. Mass spectroscopy-based proteomics confirmed the differential regulation of several cytoskeletal proteins induced by H2O2 treatment.

The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation.

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.

The Cys Sense: Thiol Redox Switches Mediate Life Cycles of Cellular Proteins.

Protein homeostasis is an essential component of proper cellular function; however, sustaining protein health is a challenging task, especially during the aerobic lifestyle. Natural cellular oxidants may be involved in cell signaling and antibacterial defense; however, imbalanced levels can lead to protein misfolding, cell damage, and death. This merges together the processes of protein homeostasis and redox regulation. At the heart of this process are redox-regulated proteins or thiol-based switches, which carefully mediate various steps of protein homeostasis across folding, localization, quality control, and degradation pathways. In this review, we discuss the "redox code" of the proteostasis network, which shapes protein health during cell growth and aging. We describe the sources and types of thiol modifications and elaborate on diverse strategies of evolving antioxidant proteins in proteostasis networks during oxidative stress conditions. We also highlight the involvement of cysteines in protein degradation across varying levels, showcasing the importance of cysteine thiols in proteostasis at large. The individual examples and mechanisms raised open the door for extensive future research exploring the interplay between the redox and protein homeostasis systems. Understanding this interplay will enable us to re-write the redox code of cells and use it for biotechnological and therapeutic purposes.

Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote.

Understanding the in vivo redox biology of cells is a complex albeit important biological problem. Studying redox processes within living cells without physical disruption or chemical modifications is essential in determining the native redox states of cells. In this study, the previously characterized reduction-oxidation sensitive green fluorescent protein (roGFP2) was used to elucidate the redox changes of the genetically engineered Escherichia coli strain, SHuffle. SHuffle cells were demonstrated to be under constitutive oxidative stress and responding transcriptionally in an OxyR-dependent manner. Using roGFP2 fused to either glutathione (GSH)- or hydrogen peroxide (H2O2)- sensitive proteins (glutaredoxin 1 or Orp1), the cytosolic redox state of both wild type and SHuffle cells based on GSH/GSSG and H2O2 pools was measured. These probes open the path to in vivo studies of redox changes and genetic selections in prokaryotic hosts.

Temporal profiling of redox-dependent heterogeneity in single cells.

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.