This study aimed at investigating the antitumor effect and immune response induced by local high-temperature hyperthermia at different thermal doses in B16 murine melanoma. The screened optimal thermal dose (50°C, 15 min) which was demonstrated to be the most effective in immune response activation was applied to the treatment of lung metastasis. The optimal thermal dose was determined by evaluating the tumor volume change, survival period of tumor-bearing mice, and immune indices including interleukin (IL)-2, interferon (IFN)-γ and TNF-α mRNA expression in the spleen of mice subjected to local hyperthermia at various thermal doses. The activation of the immune response was further investigated by rechallenging the cured mice 60 days after hyperthermia treatment. The screened optimal thermal dose combined with immunoadjuvant compound 48/80 was applied for melanoma lung metastasis. While local hyperthermia effectively inhibited B16 melanoma tumor growth and prolonged the survival period of tumor-bearing mice, the antitumor immunity was significantly enhanced and the effect was thermal dose-dependent. Higher temperatures (≥50°C) induced a significant effect even with a short treatment time (≤15 min). No tumor regrowth was observed for rechallenged B16 melanoma in mice following treatment with local hyperthermia at a higher temperature. Local hyperthermia by optimal thermal dose in combination with immunoadjuvant compound 48/80 is an effective approach for the treatment of B16 melanoma lung metastasis. This study indicated that the use of a local high-temperature hyperthermia protocol inhibits tumor growth and stimulates a favorable antitumor immune response against malignant melanoma. The results of these experiments may have clinical significance for the treatment of melanoma.
local hyperthermia; B16 melanoma; lung metastasis; thermal dose; immunity-response
Chemotaxis, chemoattractant-guided directional cell migration, plays major roles in human innate immunity and in development of a model organism Dictyostelium discoideum. Human leukocytes and D. disscoideum share remarkable similarities in the molecular mechanisms that control chemotaxis. These cells use G-Protein-Coupled Receptors (GPCRs), such as chemokine receptors, to control a signaling network that carries out chemotactic gradient sensing and directs cell migration. Diverse chemokines bind to their receptors to activate small G protein Rac through an evolutionarily conserved mechanism. Elmo and Dock180 proteins form ELMO/Dock180 complexes functioning as guanine nucleotide exchange factors (GEFs) for Rac activation. However, the linkage between GPCR to Elmo/Dock180 for Rac activation that controls F-actin dynamics remained unclear. Recently, we discovered a novel function of an ELMO protein in Dictyostelium discoideum linking GPCR signaling from Gβ to actin dynamics through regulating Rac activation during chemotaxis.
ELMO protein; G-protein-coupled receptor; Gβ subunit; Rac; chemotaxis; guanine nucleotide exchange factor (GEF); heterotrimeric G protein; small G protein; the actin cytoskeleton
AIM: To investigate the signaling mechanism of anti-oxidative action by curcumin and its impact on glucose disposal.
METHODS: Male C57BL/6J mice were fed with either a normal diet (n = 10) or a high fat diet (HFD) (n = 20) to induce obesity and insulin resistance. After 16 wk, 10 HFD-fed mice were further treated with daily curcumin oral gavage at the dose of 50 mg/kg body weight (BW) (HFD + curcumin group). After 15 d of the curcumin supplementation, an intraperitoneal glucose tolerance test was performed. Fasting blood samples were also collected for insulin and glucose measurements. Insulin-sensitive tissues, including muscle, adipose tissue and the liver, were isolated for the assessments of malondialdehyde (MDA), reactive oxygen species (ROS) and nuclear factor erythroid-2-related factor-2 (Nrf2) signaling.
RESULTS: We show here that in a HFD mouse model, short-term curcumin gavage attenuated glucose intolerance without affecting HFD-induced BW gain. Curcumin also attenuated HFD-induced elevations of MDA and ROS in the skeletal muscle, particularly in its mitochondrial fraction, but it had no such an effect in either adipose tissue or the liver of HFD-fed mice. Correspondingly, in skeletal muscle, the levels of total or nuclear content of Nrf2, as well as its downstream target, heme oxygenase-1, were reduced by HFD-feeding. Curcumin intervention dramatically reversed these defects in Nrf2 signaling. Further analysis of the relationship of oxidative stress with glucose level by a regression analysis showed a positive and significant correlation between the area under the curve of a glucose tolerance test with MDA levels either in muscle or muscular mitochondria.
CONCLUSION: These findings suggest that the short-term treatment of curcumin in HFD-fed mice effectively ameliorates muscular oxidative stress by activating Nrf2 function that is a novel mechanism for its effect in improving glucose intolerance.
Oxidative stress; Insulin resistance; Glucose tolerance; Nuclear factor erythroid-2-related factor-2; Curcumin; Mitochondria
Redox balance is fundamentally important for physiological homeostasis. Pathological factors that disturb this dedicated balance may result in oxidative stress, leading to the development or aggravation of a variety of diseases, including diabetes mellitus, cardiovascular diseases, metabolic syndrome as well as inflammation, aging and cancer. Thus, the capacity of endogenous free radical clearance can be of patho-physiological importance; in this regard, the major reactive oxygen species defense machinery, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system needs to be precisely modulated in response to pathological alterations. While oxidative stress is among the early events that lead to the development of insulin resistance, the activation of Nrf2 scavenging capacity leads to insulin sensitization. Furthermore, Nrf2 is evidently involved in regulating lipid metabolism. Here we summarize recent findings that link the Nrf2 system to metabolic homeostasis and insulin action and present our view that Nrf2 may serve as a novel drug target for diabetes and its complications.
Nuclear factor (erythroid-derived 2)-like 2; Oxidative stress; Insulin resistance; Metabolism; Diabetic drug
Following DNA damage, human cells undergo arrests in the G1 and G2 phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN+/+ cells recapitulated the lack of size checkpoint control seen in PTEN−/− cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.
Chemotaxis is crucial for many physiological processes including the recruitment of leukocytes to sites of infection, trafficking of lymphocytes in the human body, and metastasis of cancer cells. A family of small proteins, chemokines, serves as the signals, and a family of G-protein coupled receptors (GPCRs) detects chemokines and direct cell migration. One of the basic questions in chemotaxis of eukaryotes is how a GPCR transduces signals to control the assembly of the actin network that generates directional force for cell migration. Over the past decade, a variety of signaling components have been implicated to transduce the GPCR signaling to the actin cytoskeleton. Studies in a lower eukaryotic organism, Dictyostelium discoideum, have allowed us to discover evolutionary conversed components involved in the GPCR-controlled actin network during chemotaxis. However, complete pathways linking GPCR to the actin network are still far from clear. Here we first summarize the previous studies on these components, and then update with our finding showing a new pathway, consisting of a GPCR, Gβγ, Elmo/Dock, Rac and Arp2/3 and actin. We suggest that this pathway serves as a direct linkage between the GPCR/G-protein, the chemoattractant sensing machinery, and the actin cytoskeleton, the machinery of cell movement during chemotaxis of eukaryotic cells.
Dictyostelium; Dock; Elmo; GPCR; actin; chemotaxis; cytoskeleton; signaling
Magnetic fluid hyperthermia (MFH) induced by a magnetic field has become a new heating technology for the treatment of malignant tumors due to its ability to heat the tumor tissue precisely and properly, and due to its significant therapeutic effects. In this study, MFH induced by radiofrequency capacitive field (RCF) for the treatment of transplanted subcutaneous tumors in rats, was investigated. A total of 50 rats bearing subcutaneous tumors were randomly divided into five groups, including i) a pseudo-treatment (PT) control group, ii) magnetic fluid (MF) group, iii) pure hyperthermia (PH) group, iv) magnetic fluid hyperthermia 1 (MFH1) group, and v) magnetic fluid hyperthermia 2 (MFH2) group. Tumors were irradiated for 30 min in the MFH1 group 24 h following injection of MF. Tumors were irradiated for 30 min in the MFH2 group 24 h following injection of MF, and irradiation was repeated for 30 min 72 h following injection of MF. Tumor volumes, tumor volume inhibition ratios and survival times in the rat model were examined. Temperatures of tumor cores and rims both rapidly reached the desired temperature (∼50°C) for tumor treatment within 5 to 10 min in the MFH1 and MFH2 groups, and we maintained this temperature level by manually adjusting the output power (70–130 W). Tumor volumes of the MFH1 and MFH2 groups were reduced compared to those of the PT, MF and PH groups. The inhibitory effect on tumor growth in the MFH2 group (91.57%) was higher compared to that in the MFH1 group (85.21%) and the other groups. The survival time of the MFH2 group (51.62±2.28 days) and MFH1 group (43.10±1.57 days) was increased compared to that of the PH, MF and PT groups. The results obtained show that MFH induced by RCF may serve as a potential and promising method for the treatment of tumors.
magnetic fluid hyperthermia; radiofrequency capacitive field; transplanted subcutaneous tumor; rat
AIM: To investigate whether the transactivator of the proglucagon gene (Gcg), Cdx-2, synergizes with other transcription factors in stimulating Gcg expression and the trans-differentiation of Gcg-expressing cells.
METHODS: We conducted affinity chromatography to identify proteins that interact with Cdx-2, using GST-tagged Cdx-2 against cell lysates from pancreatic InR1-G9 and intestinal GLUTag cell lines. This was followed by a mass-spectrometry analysis. From a potential Cdx-2 interaction protein identified, we examined its expression in pancreatic and gut endocrine cells, confirmed its interaction with Cdx-2 by GST-pull down and determined its effect in provoking Gcg expression in cell lines that do not express endogenous Gcg.
RESULTS: We identified 18 potential Cdx-2 interacting proteins. One of them is Nkx6.2. This homeodomain (HD) protein is expressed in pancreatic α and intestinal endocrine L cells but not in insulin producing cell lines, including In111. Nkx6.2, but not Nkx6.1, was shown to interact with Cdx-2, detected by GST-pull down. Furthermore, Nkx6.2 was found to synergize with Cdx-2 in provoking Gcg expression when they were ectopically expressed in the In111 cell line. Finally, when Cdx-2 and Nkx6.2 were co-transfected into the undifferentiated rat intestinal IEC-6 cell line, it produced detectable amount of Gcg mRNA.
CONCLUSION: Cdx-2 recruits Nkx6.2 in exerting its effect in stimulating Gcg expression. Our observations further support the notion that multiple HD proteins, including Cdx-2 and Nkx6.2, are involved in the regulation of Gcg expression and the genesis of Gcg-producing cells.
Cdx-2; Nkx6.2; Homeodomain; Proglucagon; Affinity chromatograph
IL-17 and its receptor are founding members of a novel inflammatory cytokine family. To date, only one IL-17 receptor subunit has been identified, termed IL-17RA. All known cytokine receptors consist of a complex of multiple subunits. Although IL-17-family cytokines exist as homodimers, the configuration and stoichiometry of the IL-17R complex remain unknown. We used fluorescence resonance energy transfer (FRET) to determine whether IL-17RA subunits multimerize, and, if so, whether they are preassembled in the plasma membrane. HEK293 cells coexpressing IL-17RA fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate FRET before and after IL-17A or IL-17F treatment. In the absence of ligand, IL-17RA molecules exhibited significant specific FRET efficiency, demonstrating that they exist in a multimeric, preformed receptor complex. Strikingly, treatment with IL-17A or IL-17F markedly reduced FRET efficiency, suggesting that IL-17RA subunits within the IL-17R complex undergo a conformational change upon ligand binding.
IL-17 is the hallmark cytokine of the newly described “Th17” lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a “pre-ligand assembly domain” (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.
Cigarette smoking is the leading preventable cause of death in the US. Although smoking behavior has a significant genetic determination, the specific genes and associated mechanisms underlying smoking behavior are largely unknown. Here, we performed a genome-wide association study on smoking behavior in 840 Caucasians, including 417 males and 423 females, in which we examined ∼380,000 SNPs. We found that a cluster of nine SNPs upstream from the IL15 gene were associated with smoking status in males, with the most significant SNP, rs4956302, achieving a p value (8.80×10−8) of genome-wide significance. Another SNP, rs17354547, that is highly conserved across multiple species, achieved a p value of 5.65×10−5. These two SNPs, together with two additional SNPs (rs1402812 and rs4956396) were selected from the above nine SNPs for replication in an African-American sample containing 1,251 subjects, including 412 males and 839 females. The SNP rs17354547 was successfully replicated in the male subgroup of the replication sample; it was associated with smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerstrom Test for Nicotine Dependence (FTND), with p values of 0.031, 0.0046 and 0.019, respectively. In addition, a haplotype formed by rs17354547, rs1402812 and rs4956396 was also associated with SQ, HSI and FTND, achieving p values of 0.039, 0.0093 and 0.0093, respectively. To further confirm our findings, we performed an in silico replication study of the nine SNPs in a Framingham Heart Study sample containing 7,623 Caucasians from 1,731 families, among which, 3,491 subjects are males and 4,132 are females. Again, male-specific association with smoking status was observed, for which seven of the nine SNPs achieved significant p values (p<0.05) and two achieved marginally significant p values (p<0.10) in males. Several of the nine SNPs, including the highly conserved one across species, rs17354547, are located at potential transcription factor binding sites, suggesting transcription regulation as a possible function for these SNPs. Through this function, the SNPs may modulate gene expression of IL15, a key cytokine regulating immune function. As the immune system has long been recognized to influence drug addiction behavior, our association findings suggest a novel mechanism for smoking addiction involving immune modulation via the IL15 pathway.
smoking; nicotine addiction; IL15; genomewide association; genetics
Cell migration is involved in diverse physiological processes including embryogenesis, immunity, and diseases such as cancer and chronic inflammatory disease. The movement of many cell types is directed by extracellular gradients of diffusible chemicals. This phenomenon, referred to as "chemotaxis", was first described in 1888 by Leber who observed the movement of leukocytes toward sites of inflammation. We now know that a large family of small proteins, chemokines, serves as the extracellular signals and a family of G-protein-coupled receptors (GPCRs), chemokine receptors, detects gradients of chemokines and guides cell movement in vivo. Currently, we still know little about the molecular machineries that control chemokine gradient sensing and migration of immune cells. Fortunately, the molecular mechanisms that control these fundamental aspects of chemotaxis appear to be evolutionarily conserved, and studies in lower eukaryotic model systems allowed us to form concepts, uncover molecular components, develop new techniques, and test models of chemotaxis. These studies have helped our current understanding of this complicated cell behavior. In this review, we wish to mention landmark discoveries in the chemotaxis research field that shaped our current understanding of this fundamental cell behavior and lay out key questions that remain to be addressed in the future.
chemotaxis; chemokine; GPCR; actin; inflammation
The Elmo protein family members are important mediators of small G protein activity, regulating actin-mediated processes such as chemotaxis and engulfment. Until recently,1 Elmo function has not been explored in professional phagocytes such as Dictyostelium discoideum. We discuss the significance of this family with respect to pathways that regulate Rac signaling, we present a comparison of Elmo proteins between representative taxa, and discuss our findings on ElmoA, one of six Elmo proteins found in D. discoideum.
Elmo; TIRFM; Dictyostelium; actin; myosin; small G-protein; chemotaxis
Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation.
Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5STA and CCR5Δ4.
Taken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.
Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating ∼380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72×10−7. The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62×10−3 and 2.44×10−3, respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10−5 in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only ∼0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66×10−3 (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF.
Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.
Gprotein–coupled receptor (GPCR) signaling mediates a balance of excitatory and inhibitory activities that regulate Dictyostelium chemosensing to cAMP. The molecular nature and kinetics of these inhibitors are unknown. We report that transient cAMP stimulations induce PIP3 responses without a refractory period, suggesting that GPCR-mediated inhibition accumulates and decays slowly. Moreover, exposure to cAMP gradients leads to asymmetric distribution of the inhibitory components. The gradients induce a stable accumulation of the PIP3 reporter PHCrac-GFP in the front of cells near the cAMP source. Rapid withdrawal of the gradient led to the reassociation of G protein subunits, and the return of the PIP3 phosphatase PTEN and PHCrac-GFP to their pre-stimulus distribution. Reapplication of cAMP stimulation produces a clear PHCrac-GFP translocation to the back but not to the front, indicating that a stronger inhibition is maintained in the front of a polarized cell. Our study demonstrates a novel spatiotemporal feature of currently unknown inhibitory mechanisms acting locally on the PI3K activation pathway.
Valproic acid (VPA) is used to treat epilepsy and bipolar disorder and to prevent migraine. It is also undergoing trials for cancer therapy. However, the biochemical and molecular biological actions of VPA are poorly understood. Using the social amoeba Dictyostelium discoideum, we show that an acute effect of VPA is the inhibition of chemotactic cell movement, a process partially dependent upon phospholipid signaling. Analysis of this process shows that VPA attenuates the signal-induced translocation of PHCrac-green fluorescent protein from cytosol to membrane, suggesting the inhibition of phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) production. Direct labeling of lipids in vivo also shows a reduction in PIP and PIP2 phosphorylation following VPA treatment. We further show that VPA acutely reduces endocytosis and exocytosis—processes previously shown to be dependent upon PIP3 production. These results suggest that in Dictyostelium, VPA rapidly attenuates phospholipid signaling to reduce endocytic trafficking. To examine this effect in a mammalian model, we also tested depolarization-dependent neurotransmitter release in rat nerve terminals, and we show that this process is also suppressed upon application of VPA and an inhibitor of phosphatidylinositol 3-kinase. Although a more comprehensive analysis of the effect of VPA on lipid signaling will be necessary in mammalian systems, these results suggest that VPA may function to reduce phospholipid signaling processes and thus may provide a novel therapeutic effect for this drug.
The signaling network underlying eukaryotic chemosensing is a complex combination of receptor-mediated transmembrane signals, lipid modifications, protein translocations, and differential activation/deactivation of membrane-bound and cytosolic components. As such, it provides particularly interesting challenges for a combined computational and experimental analysis. We developed a novel detailed molecular signaling model that, when used to simulate the response to the attractant cyclic adenosine monophosphate (cAMP), made nontrivial predictions about Dictyostelium chemosensing. These predictions, including the unexpected existence of spatially asymmetrical, multiphasic, cyclic adenosine monophosphate–induced PTEN translocation and phosphatidylinositol-(3,4,5)P3 generation, were experimentally verified by quantitative single-cell microscopy leading us to propose significant modifications to the current standard model for chemoattractant-induced biochemical polarization in this organism. Key to this successful modeling effort was the use of “Simmune,” a new software package that supports the facile development and testing of detailed computational representations of cellular behavior. An intuitive interface allows user definition of complex signaling networks based on the definition of specific molecular binding site interactions and the subcellular localization of molecules. It automatically translates such inputs into spatially resolved simulations and dynamic graphical representations of the resulting signaling network that can be explored in a manner that closely parallels wet lab experimental procedures. These features of Simmune were critical to the model development and analysis presented here and are likely to be useful in the computational investigation of many aspects of cell biology.
Cells can orient their migration in response to small local differences in the concentration of extracellular chemicals (chemoattractants). Understanding this process (chemosensing) requires analyzing the time and position-dependent behavior of the signaling molecules within the responding cell, making it an especially interesting challenge for both experimental and computational investigation. Here, the authors report the development and testing of a new detailed molecular model of the chemosensing apparatus of the amoeba Dictyostelium discoidium reacting to the chemoattractant cyclic adenosine monophosphate. Computer simulations performed using this model predicted unexpected and previously unreported patterns of changes in the concentration and location of two important intracellular signaling molecules. These predictions were experimentally verified using microscopy, suggesting the need for modifications to the current “standard” model of eukaryotic chemosensing. The high degree of detail in their model was made possible by a new software suite called “Simmune,” which allows biologists to enter information about molecular interactions using a graphical interface. Without requiring the user to write any equations, the software automatically constructs the overall reaction network, simulates the model, and provides several ways to view the biochemistry of simulated cells. This new tool should help biologists to translate qualitative representations of cell biological processes into quantitative, predictive models.
Ligand binding to a chemokine receptor triggers signaling events through heterotrimeric G-proteins. The mechanisms underlying receptor-mediated G-protein activation in the heterogeneous microenvironments of the plasma membrane are unclear. Here, using live-cell fluorescence resonance energy transfer imaging to detect the proximity between CXCR1-cyan fluorescent protein (CFP) and fluorescence probes that label lipid raft or non-lipid raft microdomains and using fluorescence recovery after photobleaching analysis to measure the lateral diffusion of CXCR1-CFP, we found that interleukin-8 induces association between the receptors and lipid raft microenvironments. Disruption of lipid rafts impaired G-protein-dependent signaling, such as Ca2+ responses and phosphatidylinositol 3-kinase activation, but had no effect on ligand-binding function and did not completely abolish ligand-induced receptor phosphorylation. Our results suggest a novel mechanism by which ligand binding to CXCR1 promotes lipid raft partitioning of receptors and facilitates activation of heterotrimeric G-proteins.
Activation of G-protein-coupled chemoattractant receptors triggers dissociation of Gα and Gβγ subunits. These subunits induce intracellular responses that can be highly polarized when a cell experiences a gradient of chemoattractant. Exactly how a cell achieves this amplified signal polarization is still not well understood. Here, we quantitatively measure temporal and spatial changes of receptor occupancy, G-protein activation by FRET imaging, and PIP3 levels by monitoring the dynamics of PHCrac-GFP translocation in single living cells in response to different chemoattractant fields. Our results provided the first direct evidence that G-proteins are activated to different extents on the cell surface in response to asymmetrical stimulations. A stronger, uniformly applied stimulation triggers not only a stronger G-protein activation but also a faster adaptation of downstream responses. When naïve cells (which have not experienced chemoattractant) were abruptly exposed to stable cAMP gradients, G-proteins were persistently activated throughout the entire cell surface, whereas the response of PHCrac-GFP translocation surprisingly consisted of two phases, an initial transient and asymmetrical translocation around the cell membrane, followed by a second phase producing a highly polarized distribution of PHCrac-GFP. We propose a revised model of gradient sensing, suggesting an important role for locally controlled components that inhibit PI3Kinase activity.
Oxidative stress and reactive oxygen species (ROS) can elicit and modulate various physiological and pathological processes, including cell death. However, the mechanisms controlling ROS-induced cell death are largely unknown. Data from this study suggest that receptor-interacting protein (RIP) and tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2), two key effector molecules of TNF signaling, are essential for ROS-induced cell death. We found that RIP−/− or TRAF2−/− mouse embryonic fibroblasts (MEF) are resistant to ROS-induced cell death when compared to wild-type cells, and reconstitution of RIP and TRAF2 gene expression in their respective deficient MEF cells restored their sensitivity to H2O2-induced cell death. We also found that RIP and TRAF2 form a complex upon H2O2 exposure, but without the participation of TNFR1. The colocalization of RIP with a membrane lipid raft marker revealed a possible role of lipid rafts in the transduction of cell death signal initiated by H2O2. Finally, our results demonstrate that activation of c-Jun NH2-terminal kinase 1 is a critical event downstream of RIP and TRAF2 in mediating ROS-induced cell death. Therefore, our study uncovers a novel signaling pathway regulating oxidative stress-induced cell death.
Cells display chemotaxis and electrotaxis by migrating directionally in gradients of specific chemicals or electrical potential. Chemotaxis in Dictyostelium discoideum is mediated by G protein–coupled receptors. The unique Gβ is essential for all chemotactic responses, although different chemoattractants use different receptors and Gα subunits. Dictyostelium amoebae show striking electrotaxis in an applied direct current electric field. Perhaps electrotaxis and chemotaxis share similar signaling mechanisms? Null mutation of Gβ and cAMP receptor 1 and Gα2 did not abolish electrotaxis, although Gβ-null mutations showed suppressed electrotaxis. By contrast, G protein signaling plays an essential role in chemotaxis. G protein–coupled receptor signaling was monitored with PHcrac–green fluorescent protein, which translocates to inositol phospholipids at the leading edge of cells during chemotaxis. There was no intracellular gradient of this protein during electrotaxis. However, F-actin was polymerized at the leading edge of cells during electrotaxis. We conclude that reception and transduction of the electrotaxis signal are largely independent of G protein–coupled receptor signaling and that the pathways driving chemotaxis and electrotaxis intersect downstream of heterotrimeric G proteins to invoke cytoskeletal elements.
Dictyostelium; cell migration; electrotaxis; electric fields; G protein–coupled receptor signaling