Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform

Important insights into aging have been generated with the genetically tractable and short-lived budding yeast. However, it is still impossible today to continuously track cells by high-resolution microscopic imaging (e.g., fluorescent imaging) throughout their entire lifespan. Instead, the field still needs to rely on a 50-y-old laborious and time-consuming method to assess the lifespan of yeast cells and to isolate differentially aged cells for microscopic snapshots via manual dissection of daughter cells from the larger mother cell. Here, we are unique in achieving continuous and high-resolution microscopic imaging of the entire replicative lifespan of single yeast cells. Our microfluidic dissection platform features an optically prealigned single focal plane and an integrated array of soft elastomer-based micropads, used together to allow for trapping of mother cells, removal of daughter cells, monitoring gradual changes in aging, and unprecedented microscopic imaging of the whole aging process. Using the platform, we found remarkable age-associated changes in phenotypes (e.g., that cells can show strikingly differential cell and vacuole morphologies at the moment of their deaths), indicating substantial heterogeneity in cell aging and death. We envision the microfluidic dissection platform to become a major tool in aging research.     

Protein kinase A and TORC1 activate genes for ribosomal biogenesis by inactivating repressors encoded by Dot6 and its homolog Tod6

Genes required for ribosome biogenesis in yeast, referred to collectively as the Ribi regulon, are tightly regulated in coordination with nutrient availability and cellular growth rate. The promoters of a significant fraction of Ribi genes contain one or more copies of the RNA polymerases A and C (PAC) and/or ribosomal RNA-processing element (RRPE) motifs. Prompted by recent studies showing that the yeast protein Dot6 and its homolog Tod6 can bind to a PAC motif sequence in vitro and are required for efficient Ribi gene repression in response to heat shock, we have examined the role of Dot6 and Tod6 in nutrient control of Ribi gene expression in vivo. Our results indicate that PAC sites function as Dot6/Tod6-dependent repressor elements in vivo. Moreover, Dot6 and Tod6 mediate different nutrient signals, with Tod6 responsible for efficient repression of Ribi genes after inhibition of the nitrogen-sensitive TORC1 pathway and Dot6 responsible for repression after inhibition of the carbon-sensitive protein kinase A signaling pathway. Consistently, Dot6 and Tod6 are required for efficient repression of Ribi gene repression immediately after nutrient deprivation and for successful adaptation to nutrient limitation. Thus, these results establish Dot6/Tod6 as a direct link between nutrient availability, Ribi gene regulation, and growth control.     

Tripartite organization of centromeric chromatin in budding yeast

The centromere is the genetic locus that organizes the proteinaceous kinetochore and is responsible for attachment of the chromosome to the spindle at mitosis and meiosis. In most eukaryotes, the centromere consists of highly repetitive DNA sequences that are occupied by nucleosomes containing the CenH3 histone variant, whereas in budding yeast, a ～120-bp centromere DNA element (CDE) that is sufficient for centromere function is occupied by a single right-handed histone variant CenH3 (Cse4) nucleosome. However, these in vivo observations are inconsistent with in vitro evidence for left-handed octameric CenH3 nucleosomes. To help resolve these inconsistencies, we characterized yeast centromeric chromatin at single base-pair resolution. Intact particles containing both Cse4 and H2A are precisely protected from micrococcal nuclease over the entire CDE of all 16 yeast centromeres in both solubilized chromatin and the insoluble kinetochore. Small DNA-binding proteins protect CDEI and CDEIII and delimit the centromeric nucleosome to the ～80-bp CDEII, only enough for a single DNA wrap. As expected for a tripartite organization of centromeric chromatin, loss of Cbf1 protein, which binds to CDEI, both reduces the size of the centromere-protected region and shifts its location toward CDEIII. Surprisingly, Cse4 overproduction caused genome-wide misincorporation of nonfunctional CenH3-containing nucleosomes that protect ～135 base pairs and are preferentially enriched at sites of high nucleosome turnover. Our detection of two forms of CenH3 nucleosomes in the yeast genome, a singly wrapped particle at the functional centromere and octamer-sized particles on chromosome arms, reconcile seemingly conflicting in vivo and in vitro observations.     

A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 → R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2^W56R can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3′-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP2^3′-UTR. Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.     

The crystal structure of yeast copper thionein: the solution of a long-lasting enigma

We report here the crystal structure of yeast copper thionein (Cu-MT), determined at 1.44-A resolution. The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biology, consisting of six trigonally and two digonally coordinated Cu(I) ions. This is at variance with the results from previous spectroscopic determinations, which were performed on MT samples containing seven rather than eight metal ions. The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues. The protein structure is somewhat different from that of Ag(7)-MT and similar, but not identical, to that of Cu(7)-MT. Besides the different structure of the metal cluster, the main differences lie in the cysteine topology and in the conformation of some portions of the backbone. The present structure suggests that Cu-MT, in addition to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones.     

De novo production of the plant-derived alkaloid strictosidine in yeast

The monoterpene indole alkaloids are a large group of plant-derived specialized metabolites, many of which have valuable pharmaceutical or biological activity. There are ∼3,000 monoterpene indole alkaloids produced by thousands of plant species in numerous families. The diverse chemical structures found in this metabolite class originate from strictosidine, which is the last common biosynthetic intermediate for all monoterpene indole alkaloid enzymatic pathways. Reconstitution of biosynthetic pathways in a heterologous host is a promising strategy for rapid and inexpensive production of complex molecules that are found in plants. Here, we demonstrate how strictosidine can be produced de novo in a Saccharomyces cerevisiae host from 14 known monoterpene indole alkaloid pathway genes, along with an additional seven genes and three gene deletions that enhance secondary metabolism. This system provides an important resource for developing the production of more complex plant-derived alkaloids, engineering of nonnatural derivatives, identification of bottlenecks in monoterpene indole alkaloid biosynthesis, and discovery of new pathway genes in a convenient yeast host.Monoterpene indole alkaloids (MIAs) are a diverse family of complex nitrogen-containing plant-derived metabolites (1, 2). This metabolite class is found in thousands of plant species from the Apocynaceae, Loganiaceae, Rubiaceae, Icacinaceae, Nyssaceae, and Alangiaceae plant families (2, 3). Many MIAs and MIA derivatives have medicinal properties; for example, vinblastine, vincristine, and vinflunine are approved anticancer therapeutics (4, 5). These structurally complex compounds can be difficult to chemically synthesize (6, 7). Consequently, industrial production relies on extraction from the plant, but these compounds are often produced in small quantities as complex mixtures, making isolation challenging, laborious, and expensive (8–10). Reconstitution of plant pathways in microbial hosts is proving to be a promising approach to access plant-derived compounds as evidenced by the successful production of terpenes, flavonoids, and benzylisoquinoline alkaloids in microorganisms (11–19). Microbial hosts can also be used to construct hybrid biosynthetic pathways to generate modified natural products with potentially enhanced bioactivities (8, 20, 21). Across numerous plant species, strictosidine is believed to be the core scaffold from which all 3,000 known MIAs are derived (1, 2). Strictosidine undergoes a variety of redox reactions and rearrangements to form the thousands of compounds that comprise the MIA natural product family (Fig. 1) (1, 2). Due to the importance of strictosidine, the last common biosynthetic intermediate for all known MIAs, we chose to focus on heterologous production of this complex molecule (1). Therefore, strictosidine reconstitution represents the necessary first step for heterologous production of high-value MIAs.Open in a separate windowFig. 1.Strictosidine, the central intermediate in monoterpene indole alkaloid (MIA) biosynthesis, undergoes a series of reactions to produce over 3,000 known MIAs such as vincristine, quinine, and strychnine.     

A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity

Protein misfolding is intimately associated with devastating human neurodegenerative diseases, including Alzheimer's, Huntington's, and Parkinson's. Although disparate in their pathophysiology, many of these disorders share a common theme, manifested in the accumulation of insoluble protein aggregates in the brain. Recently, the major disease protein found in the pathological inclusions of two of these diseases, amyotrophic lateral sclerosis (ALS) and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U), was identified as the 43-kDa TAR-DNA-binding protein (TDP-43), providing a molecular link between them. TDP-43 is a ubiquitously expressed nuclear protein that undergoes a pathological conversion to an aggregated cytoplasmic localization in affected regions of the nervous system. Whether TDP-43 itself can convey toxicity and whether its abnormal aggregation is a cause or consequence of pathogenesis remain unknown. We report a yeast model to define mechanisms governing TDP-43 subcellular localization and aggregation. Remarkably, this simple model recapitulates several salient features of human TDP-43 proteinopathies, including conversion from nuclear localization to cytoplasmic aggregation. We establish a connection between this aggregation and toxicity. The pathological features of TDP-43 are distinct from those of yeast models of other protein-misfolding diseases, such as polyglutamine. This suggests that the yeast model reveals specific aspects of the underlying biology of the disease protein rather than general cellular stresses associated with accumulating misfolded proteins. This work provides a mechanistic framework for investigating the toxicity of TDP-43 aggregation relevant to human disease and establishes a manipulable, high-throughput model for discovering potential therapeutic strategies.     

A genome-wide view of the spectrum of spontaneous mutations in yeast

The mutation process ultimately defines the genetic features of all populations and, hence, has a bearing on a wide range of issues involving evolutionary genetics, inheritance, and genetic disorders, including the predisposition to cancer. Nevertheless, formidable technical barriers have constrained our understanding of the rate at which mutations arise and the molecular spectrum of their effects. Here, we report on the use of complete-genome sequencing in the characterization of spontaneously arising mutations in the yeast Saccharomyces cerevisiae. Our results confirm some findings previously obtained by indirect methods but also yield numerous unexpected findings, in particular a very high rate of point mutation and skewed distribution of base-substitution types in the mitochondrion, a very high rate of segmental duplication and deletion in the nuclear genome, and substantial deviations in the mutational profile among various model organisms.     

High-throughput analysis of yeast replicative aging using a microfluidic system

Saccharomyces cerevisiae has been an important model for studying the molecular mechanisms of aging in eukaryotic cells. However, the laborious and low-throughput methods of current yeast replicative lifespan assays limit their usefulness as a broad genetic screening platform for research on aging. We address this limitation by developing an efficient, high-throughput microfluidic single-cell analysis chip in combination with high-resolution time-lapse microscopy. This innovative design enables, to our knowledge for the first time, the determination of the yeast replicative lifespan in a high-throughput manner. Morphological and phenotypical changes during aging can also be monitored automatically with a much higher throughput than previous microfluidic designs. We demonstrate highly efficient trapping and retention of mother cells, determination of the replicative lifespan, and tracking of yeast cells throughout their entire lifespan. Using the high-resolution and large-scale data generated from the high-throughput yeast aging analysis (HYAA) chips, we investigated particular longevity-related changes in cell morphology and characteristics, including critical cell size, terminal morphology, and protein subcellular localization. In addition, because of the significantly improved retention rate of yeast mother cell, the HYAA-Chip was capable of demonstrating replicative lifespan extension by calorie restriction.Aging and age-associated diseases are becoming the fastest-growing area of epidemiology in most developed countries (1–4). Identification of molecular mechanisms that lead to the development of interventions to delay the onset of age-associated diseases could have tremendous global impacts on public health (5). The budding yeast Saccharomyces cerevisiae was the first eukaryotic genome to be sequenced, and has been instrumental in discovering molecular pathways involved in all aspects of eukaryotic cells (6–9). S. cerevisiae is an important model for discovering evolutionarily conserved enzymes that regulate aging, such as Sir2 and Tor1 (10).Yeast replicative lifespan (RLS) is determined by manually separating the daughter cells from a mother cell on a Petri dish with a microscope-mounted glass needle, and counting the number of divisions throughout the life of the cell. Tens or hundreds of cells per strain have to be dissected and counted to determine whether the lifespans of two strains are statistically different (11–14). This method has not changed appreciably since the initial discovery of yeast replicative aging in 1959 (15). A well-trained yeast dissector can monitor and handle no more than 300 cells at once, and a typical lifespan experiment usually thus lasts ∼4 wk. Most lifespan experiments include an overnight 4 °C incubation everyday throughout the experiment for practical purposes, adding another factor that can complicate data interpretation. This tedious and low-throughput procedure has substantially hindered progress. Therefore, new strategies are required to take advantage of the power of yeast genetics and apply high-throughput unbiased genetic screen approaches to yeast aging research.Microfluidic devices have been developed to capture yeast cells for high-resolution imaging analysis during vegetative growth (16–20). Recently, such devices have been designed that enable the tracking of yeast cells throughout their lifespan, making it possible to record and study cellular phenotypic changes during aging (21–23). However, many issues prevent the use of microfluidic devices in a high-throughput manner for lifespan screens. First, although the time required to monitor the entire lifespan of the yeast cell has been dramatically reduced, the throughput is limited to 1–4 channels per device (21–23). Second, mother cells were immobilized underneath soft elastomer [polydimethylsiloxane (PDMS)] micropads (21, 22). Although several hundred trapping micropads can be assembled for each microfluidic channel, this trap design suffers from a low retention rate of ∼30% by the end of the lifespan; this seriously limits the number of usable cells in the lifespan calculation to ∼100, which restricts statistical significance of the lifespan analysis. Third, the ability for trapping micropads to retain old cells depends on the larger size of old cells compared with young cells (21, 22). However, old cells often generate large daughter cells that also become trapped by the micropads. Fourth, the micropad design often allows more than one cell to be trapped; multiple cells can be trapped underneath one micropad, whereas no cells are trapped under others. Finally, in one of the designs, cell-surface labeling and chemical modification of the device are required, which has proven to be technically challenging for fabrication and to introduce adverse effects on replicative lifespan (23).Here, we present a microfluidic platform called high-throughput yeast aging analysis chip (HYAA-Chip), which solves all of the described challenges and limitations. This innovative design can trap up to 8,000 individual yeast cells in cup-shaped PDMS structures evenly distributed to 16 discrete channels; captured cells are cultivated and aged as fresh medium continuously flows through, which removes newly budded daughter cells. The HYAA-Chip provides automated whole-lifespan tracking with fine spatiotemporal resolution and large-scale data quantification of single yeast cell aging by combining simple fabricated microfluidics with high-resolution time-lapse microscopy. The HYAA-Chip is label-free, independent of size differences between mother and daughter cells, has up to 96% single-cell trapping efficiency, and up to 92% retention rate for the initially trapped mother cells.     

F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase

The ATP synthase of yeast mitochondria is composed of 17 different subunit polypeptides. We have screened a panel of ATP synthase mutants for impaired expression of Atp6p, Atp8p, and Atp9p, the only mitochondrially encoded subunits of ATP synthase. Our results show that translation of Atp6p and Atp8p is activated by F₁ ATPase (or assembly intermediates thereof). Mutants lacking the α or β subunits of F₁, or the Atp11p and Atp12p chaperones that promote F₁ assembly, have normal levels of the bicistronic ATP8/ATP6 mRNAs but fail to synthesize Atp6p and Atp8p. F₁ mutants are also unable to express ARG8^m when this normally nuclear gene is substituted for ATP6 or ATP8 in mitochondrial DNA. Translational activation by F₁ is also supported by the ability of ATP22, an Atp6p-specific translation factor, to restore Atp6p and to a lesser degree Atp8p synthesis in the absence of F₁. These results establish a mechanism by which expression of ATP6 and ATP8 is translationally regulated by F₁ to achieve a balanced output of two compartmentally separated sets of ATP synthase genes.     

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

Transformation of both prokaryotes and eukaryotes with single-stranded oligonucleotides can transfer sequence information from the oligonucleotide to the chromosome. We have studied this process using oligonucleotides that correct a -1 frameshift mutation in the LYS2 gene of Saccharomyces cerevisiae. We demonstrate that transformation by oligonucleotides occurs preferentially on the lagging strand of replication and is strongly inhibited by the mismatch-repair system. These results are consistent with a mechanism in which oligonucleotides anneal to single-stranded regions of DNA at a replication fork and serve as primers for DNA synthesis. Because the mispairs the primers create are efficiently removed by the mismatch-repair system, single-stranded oligonucleotides can be used to probe mismatch-repair function in a chromosomal context. Removal of mispairs created by annealing of the single-stranded oligonucleotides to the chromosomal DNA is as expected, with 7-nt loops being recognized solely by MutS beta and 1-nt loops being recognized by both MutS alpha and MutS beta. We also find evidence for Mlh1-independent repair of 7-nt, but not 1-nt, loops. Unexpectedly, we find a strand asymmetry of mismatch-repair function; transformation is blocked more efficiently by MutS alpha on the lagging strand of replication, whereas MutS beta does not show a significant strand bias. These results suggest an inherent strand-related difference in how the yeast MutS alpha and MutS beta complexes access and/or repair mismatches that arise in the context of DNA replication.     

Molecular code for protein insertion in the endoplasmic reticulum membrane is similar for N(in)-C(out) and N(out)-C(in) transmembrane helices

Transmembrane α-helices in integral membrane proteins can have two orientations in the membrane: N_in–C_out or N_out–C_in. Previous studies of model N_out–C_in transmembrane segment have led to a detailed, quantitative picture of the “molecular code” that relates amino acid sequence to membrane insertion efficiency in vivo [Hessa T, et al. (2007) Molecular code for transmembrane helix recognition by the Sec61 translocon. Nature 450:1026–1030], but whether the same code applies also to N_in–C_out transmembrane helices is unknown. Here, we show that the contributions of individual amino acids to the overall efficiency of membrane insertion are similar for the two kinds of helices and that the threshold hydrophobicity for membrane insertion can be up to ≈1 kcal/mol lower for N_in–C_out compared with N_out–C_in transmembrane helices, depending on the neighboring helices.     

Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products

Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.     

Evaluating putative mechanisms of the mitotic spindle checkpoint

The mitotic spindle checkpoint halts the cell cycle until all chromosomes are attached to the mitotic spindles. Evidence suggests that the checkpoint prevents cell-cycle progression by inhibiting the activity of the APC-Cdc20 complex, but the precise mechanism underlying this inhibition is not yet known. Here, we use mathematical modeling to compare several mechanisms that could account for this inhibition. We describe the interplay between the capacities to strongly inhibit cell-cycle progression before spindle attachment on one hand and to rapidly resume cell-cycle progression once the last kinetochore is attached on the other hand. We find that inhibition that is restricted to the kinetochore region is not sufficient for supporting both requirements when realistic diffusion constants are considered. A mechanism that amplifies the checkpoint signal through autocatalyzed inhibition is also insufficient. In contrast, amplifying the signal through the release of a diffusible inhibitory complex can support reliable checkpoint function. Our results suggest that the design of the spindle checkpoint network is limited by physical constraints imposed by realistic diffusion constants and the relevant spatial and temporal dimensions where computation is performed.     

Spatiotemporal analysis of organelle and macromolecular complex inheritance

Following mitosis, daughter cells must inherit a functional set of essential proteins and organelles. We applied a genetic tool to simultaneously monitor the kinetics and distribution of old and new proteins marking all intracellular compartments in budding yeasts. Most organelles followed a general pattern whereby preexisting proteins are symmetrically partitioned followed by template-based incorporation of new proteins. Peroxisomes belong to this group, supporting a model of biogenesis by growth and division from preexisting peroxisomes. We detected two exceptions: the nuclear pore complex (NPC) and the spindle pole body (SPB). Old NPCs are stably inherited during successive generations but remained separated from new NPCs, which are incorporated de novo in mother and daughter cells. Only the SPB displayed asymmetrical distribution, with old components primarily inherited by daughter cells and new proteins equally incorporated in both cells. Our analysis resolves conflicting models (peroxisomes, NPC) and reveals unique patterns (NPC, SPB) of organelle inheritance.