The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence.     

A model of TLR4 signaling and tolerance using a qualitative, particle-event-based method: introduction of spatially configured stochastic reaction chambers (SCSRC)

Introduction

There have been great advances in the examination and characterization of intracellular signaling and synthetic pathways. However, these pathways are generally represented using static diagrams when in reality they exist with considerable dynamic complexity. In addition to the expansion of existing mathematical pathway representation tools (many utilizing systems biology markup language format), there is a growing recognition that spatially explicit modeling methods may be necessary to capture essential aspects of intracellular dynamics. This paper introduces spatially configured stochastic reaction chambers (SCSRC), an agent-based modeling (ABM) framework that incorporates an abstracted molecular ‘event’ rule system with a spatially explicit representation of the relationship between signaling and synthetic compounds. Presented herein is an example of the SCSRC as applied to Toll-like receptor (TLR) 4 signaling and the inflammatory response.

Methods

The underlying rationale for the architecture of the SCSRC is described. A SCSRC model of TLR-4 signaling was developed after a review of the literature regarding TLR-4 signaling and downstream synthetic events. The TLR-4 SCSRC was implemented in the free-ware software platform, Netlogo. A series of in silico experiments were performed to evaluate the response of the TLR-4 SCSRC with respect to response to simulated administration of lipopolysaccharide (LPS). The pro-inflammatory response was represented by simulated secretion of tumor necrosis factor (TNF). Subsequent in silico experiments examined the response to of the TLR-4 SCSRC in terms of a simulated preconditioning effect represented as tolerance of pro-inflammatory signaling to a second dose of LPS.

Results

The SCSRC produces simulated dynamics of TLR-4 signaling in response to LPS stimulation that are qualitatively similar to that reported in the literature. The expression of various components of the signaling cascade demonstrated stochastic noise, consistent with molecular expression data reported in the literature. There is a dose dependent pro-inflammatory response effect seen with increasing initial doses of LPS, and there was also a dose dependent response with respect to preconditioning effect and the establishment of tolerance. Both of these dynamics are consistent with published responses to LPS.

Conclusions

The particle-based, spatially oriented SCSRC model of TLR-4 signaling captures the essential dynamics of the TLR-4 signal transduction cascade, including stochastic signal behavior, dose dependent response, negative feedback control, and preconditioning effect. This is accomplished even given a high degree of molecular event abstraction. The component detail of the SCSRC may allow for sequential parsing of various preconditioning effects, something not possible without computational modeling and simulation, and may give insight into the expected consequences and responses resulting from manipulation of one or many of these modulating factors. The SCSRC is admittedly a work in evolution, and future work will sequentially incorporate additional regulatory mechanisms, both intracellular and paracrine/autocrine, and improved mapping between the spatial chamber configuration and molecular event rules, and experimentally define biochemical reaction rate constants. However, the SCSRC has promise as a highly modular and flexible modeling method that is suited to the dynamic knowledge representation of intracellular processes.     

Histone deacetylases: salesmen and customers in the post‐translational modification market

HDACs (histone deacetylases) are enzymes that remove the acetyl moiety from N‐?‐acetylated lysine residues in histones and non‐histone proteins. In recent years, it has turned out that HDACs themselves are also subject to post‐translational modification. Such structural alterations can determine the stability, localization, activity and protein—protein interactions of HDACs. This subsequently affects the modification of their substrates and the co‐ordination of cellular signalling networks. Intriguingly, physiologically relevant non‐histone proteins are increasingly found to be deacetylated by HDACs, and aberrant deacetylase activity contributes to several severe human diseases. Targeting the catalytic activity of these enzymes and their post‐translational modifications are therefore attractive targets for therapeutical intervention strategies. To achieve this ambitious goal, details on the molecular mechanisms regulating post‐translational modifications of HDACs are required. This review summarizes aspects of the current knowledge on the biological role and enzymology of the phosphorylation, acetylation, ubiquitylation and sumoylation of HDACs.     

The structure of the integrin αIIbβ3 transmembrane complex explains integrin transmembrane signalling

Heterodimeric integrin adhesion receptors regulate cell migration, survival and differentiation in metazoa by communicating signals bi‐directionally across the plasma membrane. Protein engineering and mutagenesis studies have suggested that the dissociation of a complex formed by the single‐pass transmembrane (TM) segments of the α and β subunits is central to these signalling events. Here, we report the structure of the integrin αIIbβ3 TM complex, structure‐based site‐directed mutagenesis and lipid embedding estimates to reveal the structural event that underlies the transition from associated to dissociated states, that is, TM signalling. The complex is stabilized by glycine‐packing mediated TM helix crossing within the extracellular membrane leaflet, and by unique hydrophobic and electrostatic bridges in the intracellular leaflet that mediate an unusual, asymmetric association of the 24‐ and 29‐residue αIIb and β3 TM helices. The structurally unique, highly conserved integrin αIIbβ3 TM complex rationalizes bi‐directional signalling and represents the first structure of a heterodimeric TM receptor complex.     

Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration

G protein‐coupled receptors (GPCRs) have critical functions in intercellular communication. Although a wide range of different receptors have been identified in the same cells, the mechanism by which signals are integrated remains elusive. The ability of GPCRs to form dimers or larger hetero‐oligomers is thought to generate such signal integration. We examined the molecular mechanisms responsible for the GABA_B receptor‐mediated potentiation of the mGlu receptor signalling reported in Purkinje neurons. We showed that this effect does not require a physical interaction between both receptors. Instead, it is the result of a more general mechanism in which the βγ subunits produced by the Gi‐coupled GABA_B receptor enhance the mGlu‐mediated Gq response. Most importantly, this mechanism could be generally applied to other pairs of Gi‐ and Gq‐coupled receptors and the signal integration varied depending on the time delay between activation of each receptor. Such a mechanism helps explain specific properties of cells expressing two different Gi‐ and Gq‐coupled receptors activated by a single transmitter, or properties of GPCRs naturally coupled to both types of the G protein.     

The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina

Guanylate cyclase activating proteins (GCAPs) serve as neuronal Ca²⁺-sensor proteins in vertebrate rod and cone photoreceptor cells. Zebrafish express in their retina a variety of six different GCAPs, of which four are specific for cone cells. One isoform, zGCAP4, is mainly expressed in double cones and long single cones. We cloned the zGCAP4 gene, purified non-myristoylated and myristoylated forms of the protein after heterologous expression in Escherichia coli and studied its properties: zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520–570 nM free Ca²⁺ concentration. Furthermore, the Ca²⁺-sensitive activation properties of non-myristoylated and myristoylated zGCAP4 were similar, indicating no influence of the myristoyl moiety on Ca²⁺-sensor function. Myristoylated zGCAP4 showed low affinity for membranes and did not exhibit a Ca²⁺–myristoyl switch, a feature typical of some but not all neuronal Ca²⁺-sensor proteins. However, tryptophan fluorescence studies and Ca²⁺-dependent differences in protease accessibility revealed Ca²⁺-induced conformational changes in myristoylated and non-myristoylated zGCAP4, indicating the operation as a Ca²⁺ sensor. Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca²⁺-sensitive regulator of guanylate cyclase activity in cone vision.     

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9-1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid-liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.     

Experimental study of intracellular ice growth in human umbilical vein endothelial cells

Study of the intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high resolution cryo-imaging technology, the transient intracellular ice formation and growth processes of the attached human umbilical vein endothelial cells (HUVEC) were successfully captured during freezing. It was found that the intracellular ice nucleation site was on the cell membrane closer to the nucleus. The ice growth was directional and toward the nucleus, which covered the whole nucleus before growing into the cytoplasm. The crystal growth rate in the nucleus was much larger than that in the cytoplasm, and its morphology was influenced by the cooling rate. During the thawing process, small crystals fused into larger ones inside the nucleus. Moreover, the cumulative fraction of the HUVEC with IIF was mainly dependent on the cooling rate not the confluence of the cells attached.