Integrating fluorescent biosensor data using computational models.

This book chapter provides a tutorial on how to construct computational models of signaling networks for the integration and interpretation of FRET-based biosensor data. A model of cAMP production and PKA activation is presented to provide an example of the model building process. The computational model is defined using hypothesized signaling network structure and measured kinetic parameters and then simulated in Virtual Cell software. Experimental acquisition and processing of FRET biosensor data is discussed in the context of model validation. This data is then used to fit parameters of the computational model such that the model can more accurately predict experimental data. Finally, this model is used to show how computational experiments can interrogate signaling networks and provide testable hypotheses. This simple, yet detailed, tutorial on how to use computational models provides biologists that use biosensors a powerful tool to further probe and evaluate the underpinnings of a biological response.

PKA catalytic subunit compartmentation regulates contractile and hypertrophic responses to ß-adrenergic signaling.

ß-Adrenergic signaling is spatiotemporally heterogeneous in the cardiac myocyte, conferring exquisite control to sympathetic stimulation. Such heterogeneity drives the formation of protein kinase A (PKA) signaling microdomains, which regulate Ca(2+) handling and contractility. Here, we test the hypothesis that the nucleus independently comprises a PKA signaling microdomain regulating myocyte hypertrophy. Spatially-targeted FRET reporters for PKA activity identified slower PKA activation and lower isoproterenol sensitivity in the nucleus (t50=10.6±0.7 min; EC50=89.0 nmol/L) than in the cytosol (t50=3.71±0.25 min; EC50=1.22 nmol/L). These differences were not explained by cAMP or AKAP-based compartmentation. A computational model of cytosolic and nuclear PKA activity was developed and predicted that differences in nuclear PKA dynamics and magnitude are regulated by slow PKA catalytic subunit diffusion, while differences in isoproterenol sensitivity are regulated by nuclear expression of protein kinase inhibitor (PKI). These were validated by FRET and immunofluorescence. The model also predicted differential phosphorylation of PKA substrates regulating cell contractility and hypertrophy. Ca(2+) and cell hypertrophy measurements validated these predictions and identified higher isoproterenol sensitivity for contractile enhancements (EC50=1.84 nmol/L) over cell hypertrophy (EC50=85.9 nmol/L). Over-expression of spatially targeted PKA catalytic subunit to the cytosol or nucleus enhanced contractile and hypertrophic responses, respectively. We conclude that restricted PKA catalytic subunit diffusion is an important PKA compartmentation mechanism and the nucleus comprises a novel PKA signaling microdomain, insulating hypertrophic from contractile ß-adrenergic signaling responses.

A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa.

A novel protein, PA0122, has been identified in Pseudomonas aeruginosa and shown to bind to oxidized low-density lipoprotein (Ox-LDL). The PA0122 gene was recognized based on gene expression pattern differences between two strains of P. aeruginosa isolated from the sputum of an individual with cystic fibrosis (CF). There was an approximately eightfold increase in PA0122 expression in the non-mucoid strain 383, compared to that in the mucoid strain 2192. Quantitative real-time RT-PCR (qRT-PCR) supported PA0122 transcript expression differences between strains 383 and 2192 and revealed growth-phase dependence, with the highest level of expression at early stationary phase (OD(600) 1.5). PA0122 encodes a 136 aa 'conserved hypothetical' protein that has similarity to Aspergillus fumigatus Asp-haemolysin, which is an Ox-LDL-binding protein, and possessed a motif that is homologous to the fungal aegerolysin family of proteins. Antibodies produced to purified recombinant PA0122 recognized a 16 kDa protein band in cell lysates as well as in the supernatant fractions of strain 383. The PA0122 protein expression pattern was growth phase-dependent, with maximal production observed at OD(600) 1.5 that was consistent with the PA0122 transcript expression profile. Subcellular fractionation studies revealed differences in the localization of PA0122 between strains 383 and 2192. In 383, PA0122 was observed in the cytoplasm and in membrane fractions. In 2192, PA0122 was found in the cytoplasm but was not detected in membrane fractions. Surface plasmon resonance revealed that recombinant PA0122 binds with high affinity to Ox-LDL and to its major subcomponent, lysophosphatidylcholine, but not to non-oxidized LDL.

Interaction of the cysteine-rich domain of snake venom metalloproteinases with the A1 domain of von Willebrand factor promotes site-specific proteolysis of von Willebrand factor and inhibition of von Willebrand factor-mediated platelet aggregation.

Snake venom metalloproteinases (SVMPs) have recently been shown to interact with proteins containing von Willebrand factor A (VWA) domains, including the extracellular matrix proteins collagen XII, collagen XIV, matrilins 1, 3 and 4, and von Willebrand factor (VWF) via their cysteine-rich domain. We extended those studies using surface plasmon resonance to investigate the interaction of SVMPs with VWF, and demonstrated that jararhagin, a PIII SVMP containing a metalloproteinase domain followed by disintegrin-like and cysteine-rich domains, catrocollastatin C, a disintegrin-like/cysteine-rich protein, and the recombinant cysteine-rich domain of atrolysin A (A/C) all interacted with immobilized VWF in a dose-dependent fashion. Binding of VWF in solution to immobilized A/C was inhibited by ristocetin and preincubation of platelets with A/C abolished ristocetin/VWF-induced platelet aggregation, indicating that the interaction of A/C with VWF is mediated by the VWA1 domain. Jararhagin cleaved VWF at sites adjacent to the VWA1 domain, whereas atrolysin C, a SVMP lacking the cysteine-rich domain, cleaved VWF at dispersed sites. A/C and catrocollastatin C completely inhibited the digestion of VWF by jararhagin, demonstrating that the specific interaction of jararhagin with VWF via the VWA1 domain is necessary for VWF proteolysis. In summary, we localized the binding site of PIII SVMPs in VWF to the A1 domain. This suggests additional mechanisms by which SVMPs may interfere with the adhesion of platelets at the site of envenoming. Thus, specific interaction of cysteine-rich domain-containing SVMPs with VWF may function to promote the hemorrhage caused by SVMP proteolysis of capillary basements and surrounding stromal extracellular matrix.

Platelet-neutrophil-interactions: linking hemostasis and inflammation.

Platelets are essential for primary hemostasis, but they also play an important pro-inflammatory role. Platelets normally circulate in a quiescent state. Upon activation, platelets can secrete and present various molecules, change their shape as well as the expression pattern of adhesion molecules. These changes are associated with the adhesion of platelets to leukocytes and the vessel wall. The interaction of platelets with neutrophils promotes the recruitment of neutrophils into inflammatory tissue and thus participates in host defense. This interaction of neutrophils with platelets is mainly mediated through P-selectin and beta(2) and beta(3) integrins (CD11b/CD18, CD41/CD61). Platelets can also interact with endothelial cells and monocytes. Adherent platelets promote the 'secondary capture' of neutrophils and other leukocytes. In addition, platelets secrete neutrophil and endothelial activators inducing production of inflammatory cytokines. Thus, platelets are important amplifiers of acute inflammation.

LDL oxidized by hypochlorous acid causes irreversible platelet aggregation when combined with low levels of ADP, thrombin, epinephrine, or macrophage-derived chemokine (CCL22).

The in vitro oxidation of low-density lipoprotein (LDL) by hypochlorous acid produces a modified form (HOCl-LDL) capable of stimulating platelet function. We now report that HOCl-LDL is highly effective at inducing platelet function, causing stable aggregation and alpha-granule secretion. Such stimulation depended on the presence of low levels of primary agonists such as adenosine diphosphate (ADP) and thrombin, or others like epinephrine (EPI) and macrophage-derived chemokine (MDC, CCL22). Agonist levels, which by themselves induced little or reversible aggregation, caused strong stable aggregation when combined with low levels of HOCl-LDL. Platelet activation by HOCl-LDL and ADP (1 microM) caused P-selectin (CD62P) exposure, without serotonin or adenosine triphosphate (ATP) secretion. Intracellular calcium levels rose slowly (from 100 to 200 nM) in response to HOCl-LDL alone and rapidly when combined with ADP to about 300 nM. p38 mitogen-activated protein kinase (MAPK) became phosphorylated in response to HOCl-LDL alone. This phosphorylation was not blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, which reduced the extent of aggregation and calcium increase. However, the p38 MAPK inhibitor SB203580 blocked platelet aggregation and phosphorylation of p38 MAPK. These findings suggest that HOCl-LDL exposed during atherosclerotic plaque rupture, coupled with low levels of primary agonists, can rapidly induce extensive and stable thrombus formation.