Asymmetric Inheritance of Aggregated Proteins and Age Reset in Yeast Are Regulated by Vac17-Dependent Vacuolar Functions

SummaryAge can be reset during mitosis in both yeast and stem cells to generate a young daughter cell from an aged and deteriorated one. This phenomenon requires asymmetry-generating genes (AGGs) that govern the asymmetrical inheritance of aggregated proteins. Using a genome-wide imaging screen to identify AGGs in Saccharomyces cerevisiae, we discovered a previously unknown role for endocytosis, vacuole fusion, and the myosin-dependent adaptor protein Vac17 in asymmetrical inheritance of misfolded proteins. Overproduction of Vac17 increases deposition of aggregates into cytoprotective vacuole-associated sites, counteracts age-related breakdown of endocytosis and vacuole integrity, and extends replicative lifespan. The link between damage asymmetry and vesicle trafficking can be explained by a direct interaction between aggregates and vesicles. We also show that the protein disaggregase Hsp104 interacts physically with endocytic vesicle-associated proteins, such as the dynamin-like protein, Vps1, which was also shown to be required for Vac17-dependent sequestration of protein aggregates. These data demonstrate that two physiognomies of aging—reduced endocytosis and protein aggregation—are interconnected and regulated by Vac17

Essential Genetic Interactors of SIR2 Required for Spatial Sequestration and Asymmetrical Inheritance of Protein Aggregates

Sir2 is a central regulator of yeast aging and its deficiency increases daughter cell inheritance of stress-and aging-induced misfolded proteins deposited in aggregates and inclusion bodies. Here, by quantifying traits predicted to affect aggregate inheritance in a passive manner, we found that a passive diffusion model cannot explain Sir2-dependent failures in mother-biased segregation of either the small aggregates formed by the misfolded Huntingtin, Htt103Q, disease protein or heat-induced Hsp104-associated aggregates. Instead, we found that the genetic interaction network of SIR2 comprises specific essential genes required for mother-biased segregation including those encoding components of the actin cytoskeleton, the actin-associated myosin V motor protein Myo2, and the actin organization protein calmodulin, Cmd1. Co-staining with Hsp104-GFP demonstrated that misfolded Htt103Q is sequestered into small aggregates, akin to stress foci formed upon heat stress, that fail to coalesce into inclusion bodies. Importantly, these Htt103Q foci, as well as the ATPase-defective Hsp104(Y662A)-associated structures previously shown to be stable stress foci, co-localized with Cmd1 and Myo2-enriched structures and super-resolution 3-D microscopy demonstrated that they are associated with actin cables. Moreover, we found that Hsp42 is required for formation of heat-induced Hsp104(Y662A) foci but not Htt103Q foci suggesting that the routes employed for foci formation are not identical. In addition to genes involved in actin-dependent processes, SIR2-interactors required for asymmetrical inheritance of Htt103Q and heat-induced aggregates encode essential sec genes involved in ER-to-Golgi trafficking/ER homeostasis

Author Correction: Mendelian neurodegenerative disease genes involved in autophagy

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Mendelian neurodegenerative disease genes involved in autophagy

Funder: Fondation Roger de Spoelberch (Roger de Spoelberch Foundation); doi: https://doi.org/10.13039/501100008236Funder: Alzheimer's Research UK (ARUK); doi: https://doi.org/10.13039/501100002283Funder: UK Dementia Research InstituteFunder: Takeda Science Foundation; doi: https://doi.org/10.13039/100007449Abstract: The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology by regulating the removal of unwanted cargoes such as protein aggregates and damaged organelles. Over the last five decades, significant progress has been made in understanding the molecular mechanisms that regulate autophagy and its roles in human physiology and diseases. These advances, together with discoveries in human genetics linking autophagy-related gene mutations to specific diseases, provide a better understanding of the mechanisms by which autophagy-dependent pathways can be potentially targeted for treating human diseases. Here, we review mutations that have been identified in genes involved in autophagy and their associations with neurodegenerative diseases

Erratum: Author Correction: Mendelian neurodegenerative disease genes involved in autophagy.

[This corrects the article DOI: 10.1038/s41421-020-0158-y.]

Damage Segregation and Cellular Rejuvenation in Saccharomyces cerevisiae

The process of aging is defined as a time-dependent decline in cellular functionality, and aging is thought to have evolved as organisms were optimized for reproduction, at the cost of an imperfect repair and maintenance system. As a consequence, different kinds of dysfunctional components and damage accumulate over time. Eventually these dysfunctional components, termed aging factors, reach critical levels at which they interfere with cellular systems, causing the age-related loss of function that ultimately leads to cell death. The investment in propagation also encompasses the retention of aging factors within the progenitor cell, so that the progeny is born rejuvenated, free from damaging aging factors. The accumulation of oxidized and aggregated proteins has been established to act as aging factors in several organisms. These damaged proteins are asymmetrically distributed during cell division, a process that in yeast relies on the actin cytoskeleton and components of the cellular protein quality control (PQC) system. In my work, I have established that this asymmetric damage segregation is an active and factor-dependent process, accomplished through the actions of two interconnected systems. Mainly, sequestration of protein aggregates into certain quality control sites within the mother cell ensures the retention of damage, but cells have also evolved a process of aggregate removal so that any damage that accidentally leaks into the daughter cell is removed. This removal is achieved either by degradation or by retrograde transport of aggregates back into the mother cell. – Text removed from public version – Additionally, we found that the process of aggregate removal includes an unexpected role for the metacaspase Mca1, acting in conjunction with the proteasome and PQC system to degrade aggregated proteins. The link between protein aggregation and aging is further reinforced by our data demonstrating that altered levels of these identified AGGs affect cellular fitness and longevity

Mendelian neurodegenerative disease genes involved in autophagy

Funder: Fondation Roger de Spoelberch (Roger de Spoelberch Foundation); doi: https://doi.org/10.13039/501100008236Funder: Alzheimer's Research UK (ARUK); doi: https://doi.org/10.13039/501100002283Funder: UK Dementia Research InstituteFunder: Takeda Science Foundation; doi: https://doi.org/10.13039/100007449Abstract: The lysosomal degradation pathway of macroautophagy (herein referred to as autophagy) plays a crucial role in cellular physiology by regulating the removal of unwanted cargoes such as protein aggregates and damaged organelles. Over the last five decades, significant progress has been made in understanding the molecular mechanisms that regulate autophagy and its roles in human physiology and diseases. These advances, together with discoveries in human genetics linking autophagy-related gene mutations to specific diseases, provide a better understanding of the mechanisms by which autophagy-dependent pathways can be potentially targeted for treating human diseases. Here, we review mutations that have been identified in genes involved in autophagy and their associations with neurodegenerative diseases

Htt103Q foci associate with Cmd1/Myo2/actin and require Cmd1, Myo2, and SEC genes for their mother cell-biased segregation.

<p>A. <i>SIR2</i> essential interactors <i>cmd1-1, myo2-14, sec53-6</i> and <i>sec18-1</i> display a reduced ability to establish mother-biased segregation of Htt-103Q aggregates. Negative values indicate a reduction in the percentage of daughter cells without aggregates compared to the wild type. Asymmetry data are presented as mean + s.d. of duplicate samples. Statistically significant differences from wild types are determined by unpaired two-tailed t-test. Asterisks denote significant differences between samples: *P,0.05. B. Relative amount of aggregated/insoluble Htt103Q proteins in the different ts-allele strains compared to that of wild type cells after induction of Htt103Q with 2% galactose. Statistically significant differences from wild types were determined by unpaired two-tailed <i>t</i>-test. C. Representative images showing co-localization (yellow arrows) of Hsp104-GFP (green) and Htt103Q-mRFP (red). Cells were heat-shocked at 42°C and allowed to recover for 30 minutes at 30°C. The co-localization of these two aggregates can be observed in about 97.1% cells displaying both type of aggregates D. Amyloid staining of cells carrying Htt103Q aggregates (right column) using Thioflavin–T. Images of cells expressing Rnq1-mRFP were used as a positive control for amyloidogenic protein aggregates and are shown in the left column. E. Co-localization of Htt103Q-mRFP aggregates with specific proteins found to be required for mother-biased segregation as indicated. The two upper panels show that Cmd1-GFP and Myo2-GFP structures overlap and co-localize with Htt103Q-mRFP aggregates in cells showing both mRFP tagged aggregates and GFP tagged structures (94.4% and 92.2% cells showed Htt103Q-mRFP co-localization with Cmd1 and Myo2 structures, respectively). The bottom two panels indicate that less cells showed co-localization between Htt103Q-mRFP and Sec18-GFP (48%) and no co-localization between Sec53-GFP and Htt103Q-mRFP aggregates. F. Heat-denatured proteins associated with Hsp104^Y662A-GFP (green) but not the Huntington protein Htt103Q-mRFP (red) required Hsp42 for foci formation. Upper panels: wild type cells, lower panels: <i>hsp42</i>Δ mutants. G. Quantification of Hsp104^Y662A and Htt103Q aggregate morphology changes in WT and <i>hsp42</i>Δ cells. More than 100 cells from Z-stack images showing aggregates were quantified. Cells were divided into 3 classes (Class 1, cells with 1 aggregate; Cclass2, cells with 2 aggregates; Class 3, cells with 3 or more aggregates). Scale bar  = 5 µm.</p

Actin/calmodulin/tubulin, phosphatidylinositol (4,5)-bisphosphate, and ER-Golgi trafficking, are required for asymmetrical inheritance of protein aggregates.

<p>A. Temperature sensitive alleles of <i>SIR2</i> essential interactors that display a reduced ability to establish aggregate asymmetry during cytokinesis. Negative values indicate a reduction in the percentage of daughter cells generated without aggregates compared to wild type. B. Alterations in aggregate inheritance and removal in the mutants displaying defects in establishing aggregate asymmetry. Data are plotted as the mutants' deviation from the wild type in aggregate inheritance (open bars; 100% increase means a two-fold increase in the aggregate inheritance values compared to wild type strains) and removal (black bars; 100% decrease means a two-fold decrease in the inheritance values compared to wild type strains). C&D. Distribution of aggregate numbers in populations of wild type and mutant cells displaying defects in aggregate inheritance. E. Hsp104 levels detected by Western blot in mutants displaying defects in aggregate inheritance. The western blot probed with anti-Hsp104 antibody is shown in the upper panel and the loading control probed with anti-pGK antibody is shown in the lower panel. F. Relative levels of aggregated/total proteins after the heat shock in different ts-allele strains. G. Analysis of Hsp104-GFP aggregate inheritance as a function of the generation time obtained during the aggregate segregation tests of the ts-mutants. Vastly different degrees of inheritance were recorded within the confidence interval, and the best linear fit shows that no statistically significant correlation trend can be observed (R² = −0.04346 and P = 0.7752). The data (blue dots), the fit line (purple), the confidence interval lines (green), and the predicted interval lines (red) for a linear regression analysis are displayed. H. Polarity defects in ts-mutants displaying aberrant aggregate segregation. Three of four tested ts-mutants, <i>cmd1-1</i>, <i>myo2-14</i> and <i>sec53-6</i>, displayed polarity defects as seen by an increase number of mother cells with more than 6 actin patches. Fold changes are calculated from 200–300 cells. The statistical significance of observed differences was determined with the two-tailed U-test. I. Pictures of ts-mutants tested for polarity defect. Upper panel shows bright field images and lower panel shows polarity phenotypes visualized by phalloidin staining. Scale bar  = 5 µm. Asymmetry, aggregate inheritance and removal and change in aggregate per cell data are presented as mean + s.d. of triplicate samples. Statistically significant differences from wild types are determined by unpaired two-tailed <i>t</i>-test. Asterisks denote significant differences between samples: *P,0.05; **P,0.01; ***P,0.001.</p