Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size.

Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to newborn cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each newborn cell regardless of cell size. Disrupting both Whi5 synthesis and chromatin-based partitioning weakens G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.

PP2ACdc55 dephosphorylates Pds1 and inhibits spindle elongation in S. cerevisiae.

PP2ACdc55 (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In S. cerevisiae, Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro Pds1 hyperphosphorylation in a cdc55 deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2ACdc55 plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2ACdc55 disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

Form and function of topologically associating genomic domains in budding yeast.

The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.

Sorbitol treatment extends lifespan and induces the osmotic stress response in Caenorhabditis elegans.

The response to osmotic stress is a highly conserved process for adapting to changing environmental conditions. Prior studies have shown that hyperosmolarity by addition of sorbitol to the growth medium is sufficient to increase both chronological and replicative lifespan in the budding yeast, Saccharomyces cerevisiae. Here we report a similar phenomenon in the nematode Caenorhabditis elegans. Addition of sorbitol to the nematode growth medium induces an adaptive osmotic response and increases C. elegans lifespan by about 35%. Lifespan extension from 5% sorbitol behaves similarly to dietary restriction in a variety of genetic backgrounds, increasing lifespan additively with mutation of daf-2(e1370) and independently of daf-16(mu86), sir-2.1(ok434), aak-2(ok524), and hif-1(ia04). Dietary restriction by bacterial deprivation or mutation of eat-2(ad1113) fails to further extend lifespan in the presence of 5% sorbitol. Two mutants with constitutive activation of the osmotic response, osm-5(p813) and osm-7(n1515), were found to be long-lived, and lifespan extension from sorbitol required the glycerol biosynthetic enzymes GPDH-1 and GPDH-2. Taken together, these observations demonstrate that exposure to sorbitol at levels sufficient to induce an adaptive osmotic response extends lifespan in worms and define the osmotic stress response pathway as a longevity pathway conserved between yeast and nematodes.

DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity.

DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

UV-photoconversion of ethosuximide from a longevity-promoting compound to a potent toxin.

The anticonvulsant ethosuximide has been previously shown to increase life span and promote healthspan in the nematode Caenorhabditis elegans at millimolar concentrations. Here we report that following exposure to ultraviolet irradiation at 254 nm, ethosuximide is converted into a compound that displays toxicity toward C. elegans. This effect is specific for ethosuximide, as the structurally related compounds trimethadione and succinimide do not show similar toxicities following UV exposure. Killing by UV-irradiated ethosuximide is not attenuated in chemosensory mutants that are resistant to toxicity associated with high doses of non-irradiated ethosuximide. Non-irradiated ethosuximide extends life span at 15°C or 20°C, but not at 25°C, while irradiated ethosuximide shows similar toxicity at all three temperatures. Dietary restriction by bacterial deprivation does not protect against toxicity from irradiated ethosuximide, while non-irradiated ethosuximide further extends the long life spans of restricted animals. These data support the model that ethosuximide extends life span by a mechanism that is, at least partially, distinct from dietary restriction by bacterial deprivation and demonstrates an unexpected photochemical conversion of ethosuximide into a toxic compound by UV light.

Regulation of mRNA translation as a conserved mechanism of longevity control.

Appropriate regulation of mRNA translation is essential for growth and survival and the pathways that regulate mRNA translation have been highly conserved throughout eukaryotic evolution. Translation is controlled by a complex set of mechanisms acting at multiple levels, ranging from global protein synthesis to individual mRNAs. Recently, several mutations that perturb regulation of mRNA translation have also been found to increase longevity in three model organisms: the buddingyeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Many of these translation control factors can be mapped to a single pathway downstream of the nutrient responsive target of rapamycin (TOR) kinase. In this chapter, we will review the data suggesting that mRNA translation is an evolutionarily conserved modifier of longevity and discuss potential mechanisms by which mRNA translation could influence aging and age-associated disease in different species.

Proteasomal regulation of the hypoxic response modulates aging in C. elegans.

The Caenorhabditis elegans von Hippel-Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and beta-amyloid toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.