Beidong Liu

The Polarisome Is Required for Segregation and Retrograde Transport of Protein Aggregates

The paradigm sirtuin, Sir2p, of budding yeast is required for establishing cellular age asymmetry, which includes the retention of damaged and aggregated proteins in mother cells. By establishing the global genetic interaction network of SIR2 we identified the polarisome, the formin Bni1p, and myosin motor protein Myo2p as essential components of the machinery segregating protein aggregates during mitotic cytokinesis. Moreover, we found that daughter cells can clear themselves of damage by a polarisome- and tropomyosin-dependent polarized flow of aggregates into the mother cell compartment. The role of Sir2p in cytoskeletal functions and polarity is linked to the CCT chaperonin in sir2Delta cells being compromised in folding actin. We discuss the findings in view of recent models hypothesizing that polarity may have evolved to avoid clonal senescence by establishing an aging (soma-like) and rejuvenated (germ-like) lineage.

Life-span extension by a metacaspase in the yeast Saccharomyces cerevisiae

Yeast metacaspase: Grim Reaper or savior? Yeast metacaspases are structural and possibly functional homologs of caspases that execute apoptosis—programmed cell death—in higher organisms. Malmgren Hill et al. tested whether yeast metacaspase Mca1 acts as an executioner or beneficial protein during replicative aging of yeast (see the Perspective by Kampinga). Boosting metacaspase levels caused a substantial and robust extension of life span. This lifespan extension was only partly dependent on the caspase activity of Mca1 but required the presence of the protein disaggregase Hsp104. Consistent with a role in proteostasis, Mca1 was recruited to chaperone-enriched aggregates during aging. Mca1 increased aggregate asymmetry during yeast cytokinesis and counteracted the age-associated accumulation of inclusions. Science, this issue p. 1389; see also p. 1341 An enzyme linked to cell death in animal cells protects yeast cells from misfolded protein aggregates. [Also see Perspective by Kampinga] Single-cell species harbor ancestral structural homologs of caspase proteases, although the evolutionary benefit of such apoptosis-related proteins in unicellular organisms is unclear. Here, we found that the yeast metacaspase Mca1 is recruited to the insoluble protein deposit (IPOD) and juxtanuclear quality-control compartment (JUNQ) during aging and proteostatic stress. Elevating MCA1 expression counteracted accumulation of unfolded proteins and aggregates and extended life span in a heat shock protein Hsp104 disaggregase– and proteasome-dependent manner. Consistent with a role in protein quality control, genetic interaction analysis revealed that MCA1 buffers against deficiencies in the Hsp40 chaperone YDJ1 in a caspase cysteine–dependent manner. Life-span extension and aggregate management by Mca1 was only partly dependent on its conserved catalytic cysteine, which suggests that Mca1 harbors both caspase-dependent and independent functions related to life-span control.

Segregation of Protein Aggregates Involves Actin and the Polarity Machinery

Document S1. Extended Experimental Procedures, One Figure, and One TablexDownload (.22 MB ) Document S1. Extended Experimental Procedures, One Figure, and One TableMovie S1. An Uncropped Full-Field Movie Showing Several Budding Events with Retrograde Movement(Yellow circle) S/early G2 phase budding events showing retrograde movement; (yellow square) budding events with a trend towards retrograde movement; (red circle) budding event showing anterograde movement; (red square) budding event showing trend towards anterograde movement; (blue circle) budding event with movements in both ways.xDownload (.69 MB ) Movie S1. An Uncropped Full-Field Movie Showing Several Budding Events with Retrograde Movement(Yellow circle) S/early G2 phase budding events showing retrograde movement; (yellow square) budding events with a trend towards retrograde movement; (red circle) budding event showing anterograde movement; (red square) budding event showing trend towards anterograde movement; (blue circle) budding event with movements in both ways.Movie S2. A Cell with Middle-Sized Bud Showing Stable Fibrillar Aggregate StructurexDownload (.03 MB ) Movie S2. A Cell with Middle-Sized Bud Showing Stable Fibrillar Aggregate StructureMovie S3. A Cell with Large-Sized Bud Showing Stable Fibrillar Aggregate Structure in Both Mother and Daughter CompartmentxDownload (.04 MB ) Movie S3. A Cell with Large-Sized Bud Showing Stable Fibrillar Aggregate Structure in Both Mother and Daughter Compartment

Absence of Mitochondrial Translation Control Proteins Extends Life Span by Activating Sirtuin-Dependent Silencing

Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.

The mystery of aging and rejuvenation — a budding topic

In the process of yeast budding, an aged and deteriorated mother cell gives rise to a youthful and pristine daughter cell. This remarkable event offers a tractable model system for identifying factors affecting life expectancy and it has been established that multiple aging factors operate in parallel. Herein, we will highlight the identity of such aging factors, how they are asymmetrically segregated, and whether the knowledge of their deteriorating effects might be utilized to approach cellular and tissue rejuvenation in metazoans, including humans.

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 Hsp104Y662A-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 Hsp104Y662A 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.

Chemogenetic fingerprinting by analysis of cellular growth dynamics

Background A fundamental goal in chemical biology is the elucidation of on- and off-target effects of drugs and biocides. To this aim chemogenetic screens that quantify drug induced changes in cellular fitness, typically taken as changes in composite growth, is commonly applied. Results Using the model organism Saccharomyces cerevisiae we here report that resolving cellular growth dynamics into its individual components, growth lag, growth rate and growth efficiency, increases the predictive power of chemogenetic screens. Both in terms of drug-drug and gene-drug interactions did the individual growth variables capture distinct and only partially overlapping aspects of cell physiology. In fact, the impact on cellular growth dynamics represented functionally distinct chemical fingerprints. Discussion Our findings suggest that the resolution and quantification of all facets of growth increases the informational and interpretational output of chemogenetic screening. Hence, by facilitating a physiologically more complete analysis of gene-drug and drug-drug interactions the here reported results may simplify the assignment of mode-of-action to orphan bioactive compounds.

Mediator Influences Telomeric Silencing and Cellular Life Span

ABSTRACT The Mediator complex is required for the regulated transcription of nearly all RNA polymerase II-dependent genes. Here we demonstrate a new role for Mediator which appears to be separate from its function as a transcriptional coactivator. Mediator associates directly with heterochromatin at telomeres and influences the exact boundary between active and inactive chromatin. Loss of the Mediator Med5 subunit or mutations in Med7 cause a depletion of the complex from regions located near subtelomeric X elements, which leads to a change in the balance between the Sir2 and Sas2 proteins. These changes in turn result in increased levels of H4K16 acetylation near telomeres and in desilencing of subtelomeric genes. Increases in H4K16 acetylation have been observed at telomeres in aging cells. In agreement with this observation, we found that the loss of MED5 leads to shortening of the Saccharomyces cerevisiae (budding yeast) replicative life span.

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

Summary Age 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.

Protein quality control in time and space – links to cellular aging

The evolutionary theory of aging regards aging as an evolved characteristic of the soma, and proponents of the theory state that selection does not allow the evolution of aging in unicellular species lacking a soma-germ demarcation. However, the life history of some microorganisms, reproducing vegetatively by either budding or binary fission, has been demonstrated to encompass an ordered, polar-dependent, segregation of damage leading to an aging cell lineage within the clonal population. In the yeast Saccharomyces cerevisiae and the bacterium Escherichia coli, such segregation is under genetic control and includes an asymmetrical inheritance of protein aggregates and inclusions. Herein, the ultimate and proximate causation for such an asymmetrical inheritance, with special emphasis on damaged/aggregated proteins in budding yeast, is reviewed.