A histone deacetylase 3 and mitochondrial complex I axis regulates toxic formaldehyde production.

Cells produce considerable genotoxic formaldehyde from an unknown source. We carry out a genome-wide CRISPR-Cas9 genetic screen in metabolically engineered HAP1 cells that are auxotrophic for formaldehyde to find this cellular source. We identify histone deacetylase 3 (HDAC3) as a regulator of cellular formaldehyde production. HDAC3 regulation requires deacetylase activity, and a secondary genetic screen identifies several components of mitochondrial complex I as mediators of this regulation. Metabolic profiling indicates that this unexpected mitochondrial requirement for formaldehyde detoxification is separate from energy generation. HDAC3 and complex I therefore control the abundance of a ubiquitous genotoxic metabolite.

Differential scaling between G1 protein production and cell size dynamics promotes commitment to the cell division cycle in budding yeast.

In the unicellular eukaryote Saccharomyces cerevisiae, Cln3-cyclin-dependent kinase activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in their protein production rate. Rather than scaling with cell size dynamics, these pulses follow the intrinsic metabolic dynamics, peaking around Start. Using a viral-based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population levels, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells across growth conditions. Thus, uncoupling between two fundamental physiological parameters drives cell cycle commitment.

Calorie restriction does not elicit a robust extension of replicative lifespan in Saccharomyces cerevisiae.

Calorie restriction (CR) is often described as the most robust manner to extend lifespan in a large variety of organisms. Hence, considerable research effort is directed toward understanding the mechanisms underlying CR, especially in the yeast Saccharomyces cerevisiae. However, the effect of CR on lifespan has never been systematically reviewed in this organism. Here, we performed a meta-analysis of replicative lifespan (RLS) data published in more than 40 different papers. Our analysis revealed that there is significant variation in the reported RLS data, which appears to be mainly due to the low number of cells analyzed per experiment. Furthermore, we found that the RLS measured at 2% (wt/vol) glucose in CR experiments is partly biased toward shorter lifespans compared with identical lifespan measurements from other studies. Excluding the 2% (wt/vol) glucose experiments from CR experiments, we determined that the average RLS of the yeast strains BY4741 and BY4742 is 25.9 buds at 2% (wt/vol) glucose and 30.2 buds under CR conditions. RLS measurements with a microfluidic dissection platform produced identical RLS data at 2% (wt/vol) glucose. However, CR conditions did not induce lifespan extension. As we excluded obvious methodological differences, such as temperature and medium, as causes, we conclude that subtle method-specific factors are crucial to induce lifespan extension under CR conditions in S. cerevisiae.

Does autophagy mediate age-dependent effect of dietary restriction responses in the filamentous fungus Podospora anserina?

Autophagy is a well-conserved catabolic process, involving the degradation of a cell's own components through the lysosomal/vacuolar machinery. Autophagy is typically induced by nutrient starvation and has a role in nutrient recycling, cellular differentiation, degradation and programmed cell death. Another common response in eukaryotes is the extension of lifespan through dietary restriction (DR). We studied a link between DR and autophagy in the filamentous fungus Podospora anserina, a multicellular model organism for ageing studies and mitochondrial deterioration. While both carbon and nitrogen restriction extends lifespan in P. anserina, the size of the effect varied with the amount and type of restricted nutrient. Natural genetic variation for the DR response exists. Whereas a switch to carbon restriction up to halfway through the lifetime resulted in extreme lifespan extension for wild-type P. anserina, all autophagy-deficient strains had a shorter time window in which ageing could be delayed by DR. Under nitrogen limitation, only PaAtg1 and PaAtg8 mediate the effect of lifespan extension; the other autophagy-deficient mutants PaPspA and PaUth1 had a similar response as wild-type. Our results thus show that the ageing process impinges on the DR response and that this at least in part involves the genetic regulation of autophagy.

Continuous high-resolution microscopic observation of replicative aging in budding yeast.

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 µm). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

Construction and use of a microfluidic dissection platform for long-term imaging of cellular processes in budding yeast.

This protocol describes the production and operation of a microfluidic dissection platform for long-term, high-resolution imaging of budding yeast cells. At the core of this platform is an array of micropads that trap yeast cells in a single focal plane. Newly formed daughter cells are subsequently washed away by a continuous flow of fresh culture medium. In a typical experiment, 50-100 cells can be tracked during their entire replicative lifespan. Apart from aging-related research, the microfluidic platform can also be a valuable tool for other studies requiring the monitoring of single cells over time. Here we provide step-by-step instructions on how to fabricate the silicon wafer mold, how to produce and operate the microfluidic device and how to analyze the obtained data. Production of the microfluidic dissection platform and setting up an aging experiment takes ~7 h.

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.

A flux-sensing mechanism could regulate the switch between respiration and fermentation.

The yeast Saccharomyces cerevisiae can show different metabolic phenotypes (e.g. fermentation and respiration). Based on data from the literature, we argue that the substrate uptake rate is the core variable in the system that controls the global metabolic phenotype. Consequently the metabolic phenotype that the cell expresses is not dependent on the type of the sugar or its concentration, but only on the rate at which the sugar enters the cell. As this requires the cells to 'measure' metabolic flux, we discuss the existing clues toward a flux-sensing mechanism in this organism and also outline several aspects of the involved flux-dependent regulation system. It becomes clear that the sensing and regulation system that divides the taken up carbon flux into the respiratory or fermentative pathways is complex with many molecular components interacting on multiple levels. To obtain a true understanding about how the global metabolic phenotype of S. cerevisiae is controlled by the glucose uptake rate, different tools and approaches from systems biology will be required.

The moonlighting function of pyruvate carboxylase resides in the non-catalytic end of the TIM barrel.

Pyruvate carboxylase is a highly conserved enzyme that functions in replenishing the tricarboxylic acid cycle with oxaloacetate. In the yeast Hansenulapolymorpha, the pyruvate carboxylase protein is also required for import and assembly of the peroxisomal enzyme alcohol oxidase. This additional role, which is unrelated to the enzyme activity, represents an example of a special form of multifunctionality called moonlighting. We have performed a detailed site-directed mutagenesis approach to elucidate which region(s) of H. polymorpha pyruvate carboxylase are involved in its second function. This resulted in the identification of three amino acids that are essential for the moonlighting function. Mutating these residues in a single mutant protein fully inactivated the moonlighting function, but not the enzyme activity of pyruvate carboxylase because the strain was prototrophic. A 3D homology model revealed that all three residues are positioned at the side of a TIM barrel where the N-terminal ends of the beta-strands are located. This is a novel observation as the TIM barrel proteins invariably are enzymes and have their catalytic side at the C-terminal end of the beta-sheets. Our finding implies that a TIM barrel fold can also fulfill a non-enzymatic function and that this function can reside at the N-terminal end of the barrel.

Moonlighting proteins: an intriguing mode of multitasking.

Proteins are macromolecules, which perform a large variety of functions. Most of them have only a single function, but an increasing number of proteins are being identified as multifunctional. Moonlighting proteins form a special class of multifunctional proteins. They perform multiple autonomous and often unrelated functions without partitioning these functions into different domains of the protein. Striking examples are enzymes, which in addition to their catalytic function are involved in fully unrelated processes such as autophagy, protein transport or DNA maintenance. In this contribution we present an overview of our current knowledge of moonlighting proteins and discuss the significant implications for biomedical and fundamental research.

Calorie restriction causes healthy life span extension in the filamentous fungus Podospora anserina.

Although most fungi appear to be immortal, some show systemic senescence within a distinct time frame. Podospora anserina for example shows an irreversible growth arrest within weeks of culturing associated with a destabilization of the mitochondrial genome. Here, we show that calorie restriction (CR), a regimen of under-nutrition without malnutrition, increases not only life span but also forestalls the aging-related decline in fertility. Similar to respiratory chain deficiencies the life span extension is associated with lower levels of intracellular H(2)O(2) measurements and a stabilization of the mitochondrial genome. Unlike respiratory chain deficiencies, CR cultures have a wild-type-like OXPHOS machinery similar to that of well-fed cultures as shown by native electrophoresis of mitochondrial protein complexes. Together, these data indicate that life span extension via CR is fundamentally different from that via respiratory chain mutations: Whereas the latter can be seen as a pathology, the former promotes healthy life span extension and may be an adaptive response.

Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding.

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37+/-3 A at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.