Natural sequence variants of yeast environmental sensors confer cell‐to‐cell expression variability

Living systems may have evolved probabilistic bet hedging strategies that generate cell‐to‐cell phenotypic diversity in anticipation of environmental catastrophes, as opposed to adaptation via a deterministic response to environmental changes. Evolution of bet hedging assumes that genotypes segregating in natural populations modulate the level of intraclonal diversity, which so far has largely remained hypothetical. Using a fluorescent P_met17‐GFP reporter, we mapped four genetic loci conferring to a wild yeast strain an elevated cell‐to‐cell variability in the expression of MET17, a gene regulated by the methionine pathway. A frameshift mutation in the Erc1p transmembrane transporter, probably resulting from a release of laboratory strains from negative selection, reduced P_met17‐GFP expression variability. At a second locus, cis‐regulatory polymorphisms increased mean expression of the Mup1p methionine permease, causing increased expression variability in trans. These results demonstrate that an expression quantitative trait locus (eQTL) can simultaneously have a deterministic effect in cis and a probabilistic effect in trans. Our observations indicate that the evolution of transmembrane transporter genes can tune intraclonal variation and may therefore be implicated in both reactive and anticipatory strategies of adaptation.     

Analysis of individual cells identifies cell‐to‐cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.     

Fractional killing arises from cell‐to‐cell variability in overcoming a caspase activity threshold

When cells are exposed to death ligands such as TRAIL, a fraction undergoes apoptosis and a fraction survives; if surviving cells are re‐exposed to TRAIL, fractional killing is once again observed. Therapeutic antibodies directed against TRAIL receptors also cause fractional killing, even at saturating concentrations, limiting their effectiveness. Fractional killing arises from cell‐to‐cell fluctuations in protein levels (extrinsic noise), but how this results in a clean bifurcation between life and death remains unclear. In this paper, we identify a threshold in the rate and timing of initiator caspase activation that distinguishes cells that live from those that die; by mapping this threshold, we can predict fractional killing of cells exposed to natural and synthetic agonists alone or in combination with sensitizing drugs such as bortezomib. A phenomenological model of the threshold also quantifies the contributions of two resistance genes (c‐FLIP and Bcl‐2), providing new insight into the control of cell fate by opposing pro‐death and pro‐survival proteins and suggesting new criteria for evaluating the efficacy of therapeutic TRAIL receptor agonists.     

Temporal switching and cell‐to‐cell variability in Ca2+ release activity in mammalian cells

Genetically identical cells in a uniform external environment can exhibit different phenotypes, which are often masked by conventional measurements that average over cell populations. Although most studies on this topic have used microorganisms, differentiated mammalian cells have rarely been explored. Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca²⁺ increase when ryanodine receptor Ca²⁺ release channels in the endoplasmic reticulum are maximally activated by caffeine. On the other hand, the expression levels of ryanodine receptor showed a unimodal distribution. We showed that the difference in the caffeine sensitivity depends on a critical balance between Ca²⁺ release and Ca²⁺ uptake activities, which is amplified by the regenerative nature of the Ca²⁺ release mechanism. Furthermore, individual cells switched between the caffeine‐sensitive and caffeine‐insensitive states with an average transition time of approximately 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response. These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell‐to‐cell phenotypic variation in mammalian cells.     

Type I interferon promotes cell‐to‐cell spread of Listeria monocytogenes

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1^?/?) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell‐to‐cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin‐based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.     

Host endoplasmic reticulum COPII proteins control cell‐to‐cell spread of the bacterial pathogen Listeria monocytogenes

Listeria monocytogenes is a food‐borne pathogen that uses actin‐dependent motility to spread between human cells. Cell‐to‐cell spread involves the formation by motile bacteria of plasma membrane‐derived structures termed ‘protrusions’. In cultured enterocytes, the secreted Listeria protein InlC promotes protrusion formation by binding and inhibiting the human scaffolding protein Tuba. Here we demonstrate that protrusions are controlled by human COPII components that direct trafficking from the endoplasmic reticulum. Co‐precipitation experiments indicated that the COPII proteins Sec31A and Sec13 interact directly with a Src homology 3 domain in Tuba. This interaction was antagonized by InlC. Depletion of Sec31A or Sec13 restored normal protrusion formation to a Listeria mutant lacking inlC, without affecting spread of wild‐type bacteria. Genetic impairment of the COPII component Sar1 or treatment of cells with brefeldin A affected protrusions similarly to Sec31A or Sec13 depletion. These findings indicated that InlC relieves a host‐mediated restriction of Listeria spread otherwise imposed by COPII. Inhibition of Sec31A, Sec13 or Sar1 or brefeldin A treatment also perturbed the structure of cell–cell junctions. Collectively, these findings demonstrate an important role for COPII in controlling Listeria spread. We propose that COPII may act by delivering host proteins that generate tension at cell junctions.     

Cell‐to‐cell and type‐to‐type heterogeneity of signaling networks: insights from the crowd

Recent technological developments allow us to measure the status of dozens of proteins in individual cells. This opens the way to understand the heterogeneity of complex multi‐signaling networks across cells and cell types, with important implications to understand and treat diseases such as cancer. These technologies are, however, limited to proteins for which antibodies are available and are fairly costly, making predictions of new markers and of existing markers under new conditions a valuable alternative. To assess our capacity to make such predictions and boost further methodological development, we organized the Single Cell Signaling in Breast Cancer DREAM challenge. We used a mass cytometry dataset, covering 36 markers in over 4,000 conditions totaling 80 million single cells across 67 breast cancer cell lines. Through four increasingly difficult subchallenges, the participants predicted missing markers, new conditions, and the time‐course response of single cells to stimuli in the presence and absence of kinase inhibitors. The challenge results show that despite the stochastic nature of signal transduction in single cells, the signaling events are tightly controlled and machine learning methods can accurately predict new experimental data.     

Myosin‐X facilitates Shigella‐induced membrane protrusions and cell‐to‐cell spread

The intracellular pathogen Shigella flexneri forms membrane protrusions to spread from cell to cell. As protrusions form, myosin‐X (Myo10) localizes to Shigella. Electron micrographs of immunogold‐labelled Shigella‐infected HeLa cells reveal that Myo10 concentrates at the bases and along the sides of bacteria within membrane protrusions. Time‐lapse video microscopy shows that a full‐length Myo10 GFP‐construct cycles along the sides of Shigella within the membrane protrusions as these structures progressively lengthen. RNAi knock‐down of Myo10 is associated with shorter protrusions with thicker stalks, and causes a >80% decrease in confluent cell plaque formation. Myo10 also concentrates in membrane protrusions formed by another intracellular bacteria, Listeria, and knock‐down of Myo10 also impairs Listeria plaque formation. In Cos7 cells (contain low concentrations of Myo10), the expression of full‐length Myo10 nearly doubles Shigella‐induced protrusion length, and lengthening requires the head domain, as well as the tail‐PH domain, but not the FERM domain. The GFP‐Myo10‐HMM domain localizes to the sides of Shigella within membrane protrusions and the GFP‐Myo10‐PH domain localizes to host cell membranes. We conclude thatMyo10 generates the force to enhance bacterial‐induced protrusions by binding its head region to actin filaments and its PH tail domain to the peripheral membrane.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Listeria monocytogenes wall teichoic acid decoration in virulence and cell‐to‐cell spread

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral‐virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA‐binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant – which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell‐to‐cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter – suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non‐antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd     

Bacterial cell‐to‐cell signaling promotes the evolution of resistance to parasitic bacteriophages

The evolution of host–parasite interactions could be affected by intraspecies variation between different host and parasite genotypes. Here we studied how bacterial host cell‐to‐cell signaling affects the interaction with parasites using two bacteria‐specific viruses (bacteriophages) and the host bacterium Pseudomonas aeruginosa that communicates by secreting and responding to quorum sensing (QS) signal molecules. We found that a QS‐signaling proficient strain was able to evolve higher levels of resistance to phages during a short‐term selection experiment. This was unlikely driven by demographic effects (mutation supply and encounter rates), as nonsignaling strains reached higher population densities in the absence of phages in our selective environment. Instead, the evolved nonsignaling strains suffered relatively higher growth reduction in the absence of the phage, which could have constrained the phage resistance evolution. Complementation experiments with synthetic signal molecules showed that the Pseudomonas quinolone signal (PQS) improved the growth of nonsignaling bacteria in the presence of a phage, while the activation of las and rhl quorum sensing systems had no effect. Together, these results suggest that QS‐signaling can promote the evolution of phage resistance and that the loss of QS‐signaling could be costly in the presence of phages. Phage–bacteria interactions could therefore indirectly shape the evolution of intraspecies social interactions and PQS‐mediated virulence in P. aeruginosa.     

Cell‐specific responses to the cytokine TGFβ are determined by variability in protein levels

The cytokine TGFβ provides important information during embryonic development, adult tissue homeostasis, and regeneration. Alterations in the cellular response to TGFβ are involved in severe human diseases. To understand how cells encode the extracellular input and transmit its information to elicit appropriate responses, we acquired quantitative time‐resolved measurements of pathway activation at the single‐cell level. We established dynamic time warping to quantitatively compare signaling dynamics of thousands of individual cells and described heterogeneous single‐cell responses by mathematical modeling. Our combined experimental and theoretical study revealed that the response to a given dose of TGFβ is determined cell specifically by the levels of defined signaling proteins. This heterogeneity in signaling protein expression leads to decomposition of cells into classes with qualitatively distinct signaling dynamics and phenotypic outcome. Negative feedback regulators promote heterogeneous signaling, as a SMAD7 knock‐out specifically affected the signal duration in a subpopulation of cells. Taken together, we propose a quantitative framework that allows predicting and testing sources of cellular signaling heterogeneity.