Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint

The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial.     

β‐carotene suppresses Porphyromonas gingivalis lipopolysaccharide‐mediated cytokine production in THP‐1 monocytes cultured with high glucose condition

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of β‐carotene on production of Pg LPS‐induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP‐1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF‐α, IL‐6, and MCP‐1, cell supernatants were collected for ELISA. To examine the effects of NF‐kB signals on cytokine production, Bay11‐7082 was used. HG enhanced Pg LPS‐induced production of TNF‐α, IL‐6, and MCP‐1 via NF‐kB signals in THP‐1. β‐carotene suppressed the enhancement of the Pg LPS‐induced cytokine production in THP‐1 via NF‐κB inactivation. Our results suggest that β‐carotene might be a potential anti‐inflammatory nutrient for circulating Pg LPS‐mediated cytokine production in diabetic patients with periodontitis.     

Evaluation of a high-density oligonucleotide array for characterization of grlA, grlB, gyrA and gyrB mutations in fluoroquinolone resistant Staphylococcus aureus isolates

Using high-density oligonucleotide array technology, 30 Staphylococcus aureus strains were studied for the presence of mutations in genes involved in fluoroquinolone resistance: grlA, gyrA, grlB and gyrB. For the two most important genes, gyrA and grlA, correlation with sequencing reached 95.1%. If all genes were considered, correlation was 88.8%.     

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.Many diseases, including rheumatoid arthritis, pulmonary fibrosis, adult respiratory distress syndrome, and inflammatory bowel disease,^{1, 2, 3, 4} are commonly marked by impaired resolution of inflammation that is linked to defects in the phagocytic clearance of apoptotic cells.^{5, 6, 7} Apoptotic cell (AC) clearance normally eliminates a plethora of pro-inflammatory stimuli,^{8, 9} and the recognition of ACs by phagocytes¹⁰ limits progression to necrosis,¹¹ suppresses pro-inflammatory mediator production, and induces IL-10 and TGF-β release.^{12, 13} As defective clearance of ACs is associated with the development of inflammatory disease and autoimmunity,^{14, 15} new therapeutic approaches designed to increase the capacity of phagocytes to remove ACs could effectively promote the resolution of inflammation.Phagocytosis of ACs can be regulated by soluble mediators, including cytokines,^{16, 17} prostaglandins and lipoxins,^{17, 18, 19} serum proteins,²⁰ agonists of Liver X receptors (LXRs),^{17, 21} and glucocorticoids (GC).^{17, 22} In particular, LXR agonists and GCs promote phagocytosis of ACs predominantly via a Tyro3/Axl/Mer (TAM) receptor tyrosine kinase (RTK)-dependent pathway.^{17, 21, 23} There are two established ligands for the TAM RTKs, Protein S (gene name Pros1), which activates Tyro3 and Mer, and Gas6, which activates all three TAMs,^{24, 25} although other ligands have been suggested.^{26, 27} The amino terminal Gla domains of Protein S and Gas6 bind to phosphatidylserine (PtdSer) on the plasma membrane of ACs,²⁸ a potent ‘eat-me'' signal by which ACs are recognized by phagocytes.²⁹ TAM receptors bind to the carboxy terminal domains of Protein S and Gas6, which effectively act as molecular ‘bridges'' between PtdSer on the AC and TAM receptors on the phagocyte.^{17, 30, 31} TAM receptor- and ligand-deficient mice exhibit defective phagocytic pruning of photoreceptor outer segments by retinal pigment epithelial (RPE) cells of the eye,^{32, 33, 34} defective clearance of apoptotic germ cells by Sertoli cells of the testis,³⁵ and defective clearance of ACs by macrophages/dendritic cells in lymphoid organs.³⁶ These phenotypes are also detectable in Mer (gene name Mertk) single knockouts.³⁷ In addition to phagocytic clearance, TAM signaling also has a pivotal role in controlling the innate immune response to pathogenic stimuli.^{13, 17, 38}Although the importance of Mer in the internalization of ACs by macrophages is now well-established, this receptor has been thought not to have a significant role in the initial ‘tethering'' of ACs to the macrophage surface.^{36, 39} In their studies, Scott et al.³⁶ used peritoneal macrophages for which tethering of ACs has now been shown to be mediated by T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM4).³⁹ Subsequent internalization of tethered ACs is then mediated by either integrin αvβ3- or Mer-mediated signaling.^{39, 40} Similarly, for RPE cells, the initial capture of photoreceptor outer segments by RPE cells required the integrin αvβ5,⁴¹ with Mer-dependent signaling necessary for subsequent internalization. To further probe the mechanistic role of Mer in AC recognition and engulfment, we have now examined macrophages that predominantly use a Mer-dependent AC phagocytosis mechanism.^{17, 23} We show that in these cells, which do not express TIM4, Mer has the capacity to serve a unique dual role in mediating both tethering of ACs to the macrophage surface as well as subsequent AC engulfment.     

An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells

Oscillations in cytosolic-free Ca²⁺ concentration ([Ca2+]_i) have been proposed to encode information that controls stomatal closure. [Ca2+]_i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca2+]_i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca2+]_i and, with the accompanying oscillations in voltage, drive the K⁺ and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.Stomata in the leaf epidermis are the main pathway both for CO₂ entry for photosynthesis and for foliar water loss by transpiration. Guard cells surround the stomatal pore and regulate the aperture, balancing the often conflicting demands for CO₂ and water conservation. Guard cells open and close the pore by expanding and contracting through the uptake and loss, respectively, of osmotic solutes, notably of K⁺, Cl⁻, and malate²⁻ (Mal²⁻; Pandey et al., 2007; Kim et al., 2010; Roelfsema and Hedrich, 2010; Lawson and Blatt, 2014). These transport processes comprise the final effectors of a regulatory network that coordinates transport across the plasma membrane and tonoplast, and maintains the homeostasis of the guard cell. A number of well-defined signals—including light, CO₂, drought and the water stress hormone abscisic acid (ABA)—act on this network, altering transport, solute content, turgor and cell volume, and ultimately stomatal aperture.Much research has focused on stomatal closure, underscoring both Ca²⁺-independent and Ca²⁺-dependent signaling. Of the latter, elevated cytosolic-free Ca²⁺ concentration ([Ca2+]_i) inactivates inward-rectifying K⁺ channels (I_K,in) to prevent K⁺ uptake and activates Cl⁻ (anion) channels (I_Cl) at the plasma membrane to depolarize the membrane and engage K⁺ efflux through outward-rectifying K⁺ channels (I_K,out; Keller et al., 1989; Blatt et al., 1990; Thiel et al., 1992; Lemtiri-Chlieh and MacRobbie, 1994). ABA, and most likely CO₂ (Kim et al., 2010), elevate [Ca2+]_i by facilitating Ca²⁺ entry at the plasma membrane to trigger Ca²⁺ release from endomembrane stores, a process often described as Ca²⁺-induced Ca²⁺ release (Grabov and Blatt, 1998, 1999). The hormone promotes Ca²⁺ influx by activating Ca²⁺ channels (I_Ca) at the plasma membrane, even in isolated membrane patches (Hamilton et al., 2000, 2001), which is linked to reactive oxygen species (Kwak et al., 2003; Wang et al., 2013). In parallel, cADP-ribose and nitric oxide promote endomembrane Ca²⁺ release and [Ca2+]_i elevation (Leckie et al., 1998; Neill et al., 2002; Garcia-Mata et al., 2003; Blatt et al., 2007). Best estimates indicate that endomembrane release accounts for more than 95% of the Ca²⁺ entering the cytosol to raise [Ca2+]_i (Chen et al., 2012; Wang et al., 2012).One feature of stomatal response to ABA, and indeed to a range of stimuli both hormonal as well as external, is its capacity for oscillations both in membrane voltage and [Ca2+]_i. Guard cell [Ca2+]_i at rest is typically around 100 to 200 nm, as it is in virtually all living cells. In response to ABA, [Ca2+]_i can rise above 1 μm—and locally, most likely above 10 μm—often in cyclic transients of tens of seconds to several minutes’ duration in association with oscillations in voltage and stomatal closure (Gradmann et al., 1993; McAinsh et al., 1995; Webb et al., 1996; Grabov and Blatt, 1998, 1999; Staxen et al., 1999; Allen et al., 2001). In principle, cycling in voltage and [Ca2+]_i arises as closure is accelerated with a controlled release of K⁺, Cl⁻, and Mal²⁻ from the guard cell and is subject to extracellular ion concentrations (Gradmann et al., 1993; Chen et al., 2012). However, it has been proposed that these, and similar oscillations in a variety of plant cell models, serve as physiological signals in their own right (McAinsh et al., 1995; Ehrhardt et al., 1996; Taylor et al., 1996). In support of such a signaling role, experiments designed to impose [Ca2+]_i (and voltage) oscillations in guard cells have yielded an optimal frequency for closure with a period near 10 min (Allen et al., 2001). Nonetheless, the studies offer no mechanistic explanation for this optimum that could validate a causal role in signaling, and none has been forthcoming since. Here we address questions of how such optimal frequencies in [Ca2+]_i oscillation arise and their relevance for stomatal closure, using quantitative systems analysis of guard cell transport and homeostasis. Our findings indicate that oscillations in voltage and [Ca2+]_i, and their optima associated with stomatal closure, are most simply explained as emerging from the interactions between ion transporters that drive stomatal closure. Thus, we conclude that these oscillations do not control, but are a by-product of the transport that determines stomatal aperture.     

The conceptual approach to quantitative modeling of guard cells

Much of the 70% of global water usage associated with agriculture passes through stomatal pores of plant leaves. The guard cells, which regulate these pores, thus have a profound influence on photosynthetic carbon assimilation and water use efficiency of plants. We recently demonstrated how quantitative mathematical modeling of guard cells with the OnGuard modeling software yields detail sufficient to guide phenotypic and mutational analysis. This advance represents an all-important step toward applications in directing “reverse-engineering” of guard cell function for improved water use efficiency and carbon assimilation. OnGuard is nonetheless challenging for those unfamiliar with a modeler’s way of thinking. In practice, each model construct represents a hypothesis under test, to be discarded, validated or refined by comparisons between model predictions and experimental results. The few guidelines set out here summarize the standard and logical starting points for users of the OnGuard software.