high-throughput-robotically-assisted-isolation-temperature-sensitive

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in <em>Chlamydomonas reinhardtii</em>

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

The Rockefeller University

High throughput assays, as well as advances in computational approaches, have recently allowed the acquisition of vast amounts of genetic interaction (GI) data in several organisms. Since GIs are a functional measure that reports on the effect of a mutation in one gene on the phenotype of a mutation in another, they can serve as a powerful tool to study both the function of individual genes and the wiring of biological networks. Therefore, these data hold much promise for advancing our understanding of cellular systems. In this review we focus on the methodologies currently available for using and interpreting large datasets of GIs for functional gene groups (GI maps), and elaborate on the challenges ahead. In addition, we discuss potential applications for the study of evolution and disease mechanisms, and highlight the need for comprehensive integrative analysis to extract the wealth of information found in these maps.

Explorations in topology-delving underneath the surface of genetic interaction maps.

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is an ATP-fueled machine that assists protein folding. It consists of two back-to-back stacked rings formed by eight different subunits that are arranged in a fixed permutation. The different subunits of CCT are believed to possess unique substrate binding specificities that are still mostly unknown. Here, we used high-throughput microscopy analysis of yeast cells to determine changes in protein levels and localization as a result of a Glu to Asp mutation in the ATP binding site of subunits 3 (CCT3) or 6 (CCT6). The mutation in subunit CCT3 was found to induce cytoplasmic foci termed P-bodies where mRNAs, which are not translated, accumulate and can be degraded. Analysis of the changes in protein levels and structural modeling indicate that P-body formation in cells with the mutation in CCT3 is linked to the specific interaction of this subunit with Gln/Asn-rich segments that are enriched in many P-body proteins. An in vitro gel-shift analysis was used to show that the mutation in subunit CCT3 interferes with the ability of CCT to bind a Gln/Asn-rich protein aggregate. More generally, the strategy used in this work can be used to unravel the substrate specificities of other chaperone systems.

Interactions of subunit CCT3 in the yeast chaperonin CCT/TRiC with Q/N-rich proteins revealed by high-throughput microscopy analysis.

The endomembrane system of yeast contains different tail-anchored proteins that are post-translationally targeted to membranes via their C-terminal transmembrane domain. This hydrophobic segment could be hazardous in the cytosol if membrane insertion fails, resulting in the need for energy-dependent chaperoning and the degradation of aggregated tail-anchored proteins. A cascade of GET proteins cooperates in a conserved pathway to accept newly synthesized tail-anchored proteins from ribosomes and guide them to a receptor at the endoplasmic reticulum, where membrane integration takes place. It is, however, unclear how the GET system reacts to conditions of energy depletion that might prevent membrane insertion and hence lead to the accumulation of hydrophobic proteins in the cytosol. Here we show that the ATPase Get3, which accommodates the hydrophobic tail anchor of clients, has a dual function: promoting tail-anchored protein insertion when glucose is abundant and serving as an ATP-independent holdase chaperone during energy depletion. Like the generic chaperones Hsp42, Ssa2, Sis1 and Hsp104, we found that Get3 moves reversibly to deposition sites for protein aggregates, hence supporting the sequestration of tail-anchored proteins under conditions that prevent tail-anchored protein insertion. Our findings support a ubiquitous role for the cytosolic GET complex as a triaging platform involved in cellular proteostasis.

Get3 is a holdase chaperone and moves to deposition sites for aggregated proteins when membrane targeting is blocked.

Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.

Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.

Uncovering the mechanisms underlying robust responses of cells to stress is crucial for our understanding of cellular physiology. Indeed, vast amounts of data have been collected on transcriptional responses in Saccharomyces cerevisiae. However, only a handful of pioneering studies describe the dynamics of proteins in response to external stimuli, despite the fact that regulation of protein levels and localization is an essential part of such responses. Here we characterized unprecedented proteome plasticity by systematically tracking the localization and abundance of 5,330 yeast proteins at single-cell resolution under three different stress conditions (DTT, H2O2, and nitrogen starvation) using the GFP-tagged yeast library. We uncovered a unique "fingerprint" of changes for each stress and elucidated a new response arsenal for adapting to radical environments. These include bet-hedging strategies, organelle rearrangement, and redistribution of protein localizations. All data are available for download through our online database, LOQATE (localization and quantitation atlas of yeast proteome).

A novel single-cell screening platform reveals proteome plasticity during yeast stress responses.

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH.

Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called 'LoQAtE' (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H₂O₂ and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations.

LoQAtE--Localization and Quantitation ATlas of the yeast proteomE. A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast.

During the lifetime of a cell proteins can change their localization, alter their abundance and undergo modifications, all of which cannot be assayed by tracking mRNAs alone. Methods to study proteomes directly are coming of age, thereby opening new perspectives on the role of post-translational regulation in stabilizing the cellular milieu. Proteomics has undergone a revolution, and novel technologies for the systematic analysis of proteins have emerged. These methods can expand our ability to acquire information from single proteins to proteomes, from static to dynamic measures and from the population level to the level of single cells. Such approaches promise that proteomes will soon be studied at a similar level of dynamic resolution as has been the norm for transcriptomes.

The emergence of proteome-wide technologies: systematic analysis of proteins comes of age.

Proteolysis by aspartyl intramembrane proteases such as presenilin and signal peptide peptidase (SPP) underlies many cellular processes in health and disease. Saccharomyces cerevisiae encodes a homolog that we named yeast presenilin fold 1 (Ypf1), which we verify to be an SPP-type protease that localizes to the endoplasmic reticulum (ER). Our work shows that Ypf1 functionally interacts with the ER-associated degradation (ERAD) factors Dfm1 and Doa10 to regulate the abundance of nutrient transporters by degradation. We demonstrate how this noncanonical branch of the ERAD pathway, which we termed "ERAD regulatory" (ERAD-R), responds to ligand-mediated sensing as a trigger. More generally, we show that Ypf1-mediated posttranslational regulation of plasma membrane transporters is indispensible for early sensing and adaptation to nutrient depletion. The combination of systematic analysis alongside mechanistic details uncovers a broad role of intramembrane proteolysis in regulating secretome dynamics.

The yeast ER-intramembrane protease Ypf1 refines nutrient sensing by regulating transporter abundance.

Cell-to-cell variation in gene expression levels (noise) generates phenotypic diversity and is an important phenomenon in evolution, development and disease. TATA-box binding protein (TBP) is an essential factor that is required at virtually every eukaryotic promoter to initiate transcription. While the presence of a TATA-box motif in the promoter has been strongly linked with noise, the molecular mechanism driving this relationship is less well understood. Through an integrated analysis of multiple large-scale data sets, computer simulation and experimental validation in yeast, we provide molecular insights into how noise arises as an emergent property of variable binding affinity of TBP for different promoter sequences, competition between interaction partners to bind the same surface on TBP (to either promote or disrupt transcription initiation) and variable residence times of TBP complexes at a promoter. These determinants may be fine-tuned under different conditions and during evolution to modulate eukaryotic gene expression noise.

Michal Breker

Laboratory of Cell Cycle Genetics

The Rockefeller University

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii