A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast
- University of Groningen, Netherlands
- Yale School of Medicine, United States
- University of Washington, United States
- DWI-Leibniz Institute for Interactive Materials, Germany
Abstract
Cellular aging is a multifactorial process that is characterized by a decline in homeostatic capacity, best described at the molecular level. Physicochemical properties such as pH and macromolecular crowding are essential to all molecular processes in cells and require maintenance. Whether a drift in physicochemical properties contributes to the overall decline of homeostasis in aging is not known. Here we show that the cytosol of yeast cells acidifies modestly in early aging and sharply after senescence. Using a macromolecular crowding sensor optimized for long-term FRET measurements, we show that crowding is rather stable and that the stability of crowding is a stronger predictor for lifespan than the absolute crowding levels. Additionally, in aged cells we observe drastic changes in organellar volume, leading to crowding on the µm scale, which we term organellar crowding. Our measurements provide an initial framework of physicochemical parameters of replicatively aged yeast cells.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1,2,3,4.
Article and author information
Author details
Liesbeth M Veenhoff
Funding
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (737.016.016)
- Liesbeth M Veenhoff
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (723.015.002)
- Arnold J Boersma
National Institutes of Health (RO1 GM105672)
- C Patrick Lusk
National Institutes of Health (P30 AG013280)
- Matt Kaeberlein
National Institutes of Health (R01 AG056359)
- Matt Kaeberlein
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Weiwei Dang, Baylor College of Medicine, United States
Version history
- Received: December 23, 2019
- Accepted: September 28, 2020
- Accepted Manuscript published: September 29, 2020 (version 1)
- Version of Record published: October 14, 2020 (version 2)
Copyright
© 2020, Mouton et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 3,951
- views
-
- 531
- downloads
-
- 25
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A physicochemical perspective of aging from single-cell analysis of pH, macromolecular and organellar crowding in yeast
eLife 9:e54707.
https://doi.org/10.7554/eLife.54707
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.
