A nonequilibrium allosteric model for receptor-kinase complexes: The role of energy dissipation in chemotaxis signaling.

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

A new class of protein sensor links spirochete pleomorphism, persistence, and chemotaxis.

IMPORTANCE: A new class of bacterial protein sensors monitors intracellular levels of S-adenosylmethionine to modulate cell morphology, chemotaxis, and biofilm formation. Simultaneous regulation of these behaviors enables bacterial pathogens to survive within their niche. This sensor, exemplified by Treponema denticola CheWS, is anchored to the chemotaxis array and its sensor domain is located below the chemotaxis rings. This position may allow the sensor to directly interact with the chemotaxis histidine kinase CheA. Collectively, these data establish a critical role of CheWS in pathogenesis and further illustrate the impact of studying non-canonical chemotaxis proteins.

Computational design of dynamic receptor-peptide signaling complexes applied to chemotaxis.

Engineering protein biosensors that sensitively respond to specific biomolecules by triggering precise cellular responses is a major goal of diagnostics and synthetic cell biology. Previous biosensor designs have largely relied on binding structurally well-defined molecules. In contrast, approaches that couple the sensing of flexible compounds to intended cellular responses would greatly expand potential biosensor applications. Here, to address these challenges, we develop a computational strategy for designing signaling complexes between conformationally dynamic proteins and peptides. To demonstrate the power of the approach, we create ultrasensitive chemotactic receptor-peptide pairs capable of eliciting potent signaling responses and strong chemotaxis in primary human T cells. Unlike traditional approaches that engineer static binding complexes, our dynamic structure design strategy optimizes contacts with multiple binding and allosteric sites accessible through dynamic conformational ensembles to achieve strongly enhanced signaling efficacy and potency. Our study suggests that a conformationally adaptable binding interface coupled to a robust allosteric transmission region is a key evolutionary determinant of peptidergic GPCR signaling systems. The approach lays a foundation for designing peptide-sensing receptors and signaling peptide ligands for basic and therapeutic applications.

A microfluidic device inspired by leaky tumor vessels for hematogenous metastasis mechanism research.

Endothelial intercellular pores of tumor vessels generally lead to enhanced interstitial flow and may facilitate the migration of tumor cells. The permeability of tumor vessels causes a concentration gradient of growth factors (CGGF) from blood vessels to tumor tissues, which is opposite to the direction of interstitial flow. In this work, exogenous chemotaxis under the CGGF is demonstrated as a mechanism of hematogenous metastasis. A bionic microfluidic device inspired by endothelial intercellular pores of tumor vessels has been designed to study the mechanism. A porous membrane vertically integrated into the device using a novel compound mold is utilized to mimic the leaky vascular wall. The formation mechanism of the CGGF caused by endothelial intercellular pores is numerically analyzed and experimentally verified. The migration behavior of U-2OS cells is studied in the microfluidic device. The device is divided into three regions of interest (ROI): primary site, migration zone, and tumor vessel. The number of cells in the migration zone increases significantly under the CGGF, but decreases under no CGGF, indicating tumor cells may be guided to the vascellum by exogenous chemotaxis. Transendothelial migration is subsequently monitored, demonstrating the successful replication of the key steps in vitro in the metastatic cascade by the bionic microfluidic device.

Role of senescent tumor cells in building a cytokine shield in the tumor microenvironment: mathematical modeling.

Cellular senescence can induce dual effects (promotion or inhibition) on cancer progression. While immune cells naturally respond and migrate toward various chemotactic sources from the tumor mass, various factors including senescent tumor cells (STCs) in the tumor microenvironment may affect this chemotactic movement. In this work, we investigate the mutual interactions between the tumor cells and the immune cells that either inhibit or facilitate tumor growth by developing a mathematical model that consists of taxis-reaction-diffusion equations and receptor kinetics for the key players in the interaction network. We apply a mathematical model to a transwell Boyden chamber invasion assay used in the experiments to illustrate that STCs can play a pivotal role in negating immune attack through tight regulation of intra- and extra-cellular signaling molecules. In particular, we show that senescent tumor cells in cell cycle arrest can block intratumoral infiltration of CD8+ T cells by secreting a high level of CXCL12, which leads to significant reduction its receptors, CXCR4, on T cells, and thus impaired chemotaxis. The predictions of nonlinear responses to CXCL12 were in good agreement with experimental data. We tested several hypotheses on immune-tumor interactions under various biochemical conditions in the tumor microenvironment and developed new concepts for anti-tumor strategies targeting senescence induced immune impairment.

A New Chemotactic Mechanism Governs Long-Range Angiogenesis Induced by Patching an Arterial Graft into a Vein.

Chemotaxis, the migration of cells in response to chemical stimulus, is an important concept in the angiogenesis model. In most angiogenesis models, chemotaxis is defined as the migration of a sprout tip in response to the upgradient of the VEGF (vascular endothelial growth factor). However, we found that angiogenesis induced by performing arterial patch grafting on rabbits occurred under the decreasing VEGFA gradient. Data show that the VEGFA concentration peaked at approximately 0.3 to 0.5 cm away from the arterial patch and decreased as the measurement approaches the patch. We also observed that the new blood vessels formed are twisted and congested in some areas, in a distinguishable manner from non-pathological blood vessels. To explain these observations, we developed a mathematical model and compared the results from numerical simulations with the experimental data. We introduced a new chemotactic velocity using the temporal change in the chemoattractant gradient to govern the sprout tip migration. We performed a hybrid simulation to illustrate the growth of new vessels. Results indicated the speed of growth of new vessels oscillated before reaching the periphery of the arterial patch. Crowded and congested blood vessel formation was observed during numerical simulations. Thus, our numerical simulation results agreed with the experimental data.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

Transient post-operative overexpression of CXCR2 on monocytes of traumatic brain injury patients drives monocyte chemotaxis toward cerebrospinal fluid and enhances monocyte-mediated immunogenic cell death of neurons in vitro.

BACKGROUND: After traumatic brain injury (TBI), peripheral monocytes infiltrate into the central nervous system due to disruption of the blood-brain barrier, and play an important role in neuroinflammation. However, the mechanisms regulating the movement and function of peripheral monocytes after TBI have not been fully investigated. METHODS: TBI patients who underwent surgery at our hospital were recruited. CXCR2 expression in CD14+ monocytes from peripheral blood and cerebrospinal fluid (CSF) of TBI patients around surgery was analyzed by flow cytometry and compared with that of patients who suffered TBI 2-24 months prior and underwent cranioplasty. In vitro, serum or CSF from TBI/non-TBI patients were used to treat peripheral monocytes isolated from healthy volunteers to evaluate their effect on CXCR2 expression. Transwell experiments were performed to analyze the role of CXCR2 in monocyte chemotaxis toward the CSF. The role of CXCR2 in monocyte-mediated immunogenic cell death (ICD) of nerve cells was explored in an indirect co-culture system. RESULTS: Transient CXCR2 upregulation in monocytes from the peripheral blood and CSF of TBI patients was detected soon after surgery and was associated with unfavorable outcomes. TBI serum and CSF promoted CXCR2 expression in monocytes, and dexamethasone reversed this effect. Peripheral monocytes from TBI patients showed enhanced chemotaxis toward the CSF and increased inflammatory cytokine secretion. The CXCR2 antagonist SB225002 decreased monocyte chemotaxis toward TBI CSF, and lowered pro-inflammatory cytokine secretion in monocytes treated with TBI serum. SB225002 also relieved ICD in nerve cells co-cultured with TBI serum-treated monocytes. CONCLUSIONS: CXCR2 is transiently overexpressed in the peripheral monocytes of TBI patients post-surgery, and drives peripheral monocyte chemotaxis toward CSF and monocyte-mediated ICD of nerve cells. Therefore, CXCR2 may be a target for monocyte-based therapies for TBI.

A novel role for ATP2B in ascidians: Ascidian-specific mutations in ATP2B contribute to sperm chemotaxis.

Sperm chemotaxis, in which sperms are attracted to conspecific eggs via species-specific attractants, plays an important role in fertilization. This phenomenon has been observed in various animals and species-specific sperm attractants have been reported in some species. However, the mechanisms involved in the reception and recognition of the species-specific attractant by the sperms is poorly studied. Previously, we found that the plasma membrane-type Ca2+ /ATPase (PMCA) is the receptor for the sperm-activating and -attracting factor (SAAF) in the ascidian Ciona intestinalis. To determine the role of PMCA in species-specific sperm chemotaxis, we identified the amino acid sequences of PMCAs derived from six Phlebobranchia species. The testis-specific splice variant of PMCA was found to be present in all the species investigated and the ascidian-specific sequence was detected near the 3'-terminus. Moreover, dN/dS analysis revealed that the extracellular loops 1, 2, and 4 in ascidian PMCA underwent a positive selection. These findings suggest that PMCA recognizes the species-specific structure of SAAF at the extracellular loops 1, 2, and 4, and its testis-specific C-terminal region is involved in the activation and chemotaxis of ascidian sperms.

VEGF-A165 -Induced Endothelial Cells Chemotactic Migration and Invasion Assays.

In vitro assays of endothelial cell migration have led to critical insights into the mechanisms of angiogenesis. The transwell assay, or modified Boyden chamber assay was developed to investigate chemotaxis, which corresponds to the directional migration of cells in response to a chemoattractant gradient. It is a reliable and convenient assay that does not require expensive equipment.In the modified Boyden chamber assay, two compartments are separated with a porous membrane through which cells can migrate. The lower compartment contains the chemoattractant, creating a gradient by diffusing into the upper chamber containing the cells. Adherent cells will migrate through the membrane and remain on the lower side of the membrane, where they can finally be fixed, stained, and counted.

NOD1 splenic activation confers ferroptosis protection and reduces macrophage recruitment under pro-atherogenic conditions.

The bioavailability and regulation of iron is essential for central biological functions in mammals. The role of this element in ferroptosis and the dysregulation of its metabolism contribute to diseases, ranging from anemia to infections, alterations in the immune system, inflammation and atherosclerosis. In this sense, monocytes and macrophages modulate iron metabolism and splenic function, while at the same time they can worsen the atherosclerotic process in pathological conditions. Since the nucleotide-binding oligomerization domain 1 (NOD1) has been linked to numerous disorders, including inflammatory and cardiovascular diseases, we investigated its role in iron homeostasis. The iron content was measured in various tissues of Apoe-/- and Apoe-/-Nod1-/- mice fed a high-fat diet (HFD) for 4 weeks, under normal or reduced splenic function after ligation of the splenic artery. In the absence of NOD1 the iron levels decreased in spleen, heart and liver regardless the splenic function. This iron decrease was accompanied by an increase in the recruitment of F4/80+-macrophages in the spleen through a CXCR2-dependent signaling, as deduced by the reduced recruitment after administration of a CXCR2 inhibitor. CXCR2 mediates monocyte/macrophage chemotaxis to areas of inflammation and accumulation of leukocytes in the atherosclerotic plaque. Moreover, in the absence of NOD1, inhibition of CXCR2 enhanced atheroma progression. NOD1 activation increased the levels of GPX4 and other iron and ferroptosis regulatory proteins in macrophages. Our findings highlight the preeminent role of NOD1 in iron homeostasis and ferroptosis. These results suggest promising avenues of investigation for the diagnosis and treatment of iron-related diseases directed by NOD1.

Tumor spheroid invasion in epidermal growth factor gradients revealed by a 3D microfluidic device.

Epidermal growth factor (EGF), a potent cytokine, is known to promote tumor invasion bothin vivoandin vitro. Previously, we observed that single breast tumor cells (MDA-MB-231 cell line) embedded within a 3D collagen matrix displayed enhanced motility but no discernible chemotaxis in the presence of linear EGF gradients using a microfluidic platform. Inspired by a recent theoretical development that clustered mammalian cells respond differently to chemical gradients than single cells, we studied tumor spheroid invasion within a 3D extracellular matrix (ECM) in the presence of EGF gradients. We found that EGF gradients promoted tumor cell detachment from the spheroid core, and the position of the tumor spheroid core showed a mild chemotactic response towards the EGF gradients. For those tumor cells detached from the spheroids, they showed an enhanced motility response in contrast to previous experimental results using single cells embedded within an ECM. No discernible chemotactic response towards the EGF gradients was found for the cells outside the spheroid core. This work demonstrates that a cluster of tumor cells responds differently than single tumor cells towards EGF gradients and highlights the importance of a tumor spheroid platform for tumor invasion studies.

Macrophages and Bone Marrow-Derived Mesenchymal Stem Cells Work in Concert to Promote Fracture Healing: A Brief Review.

Bone marrow-derived mesenchymal stem cell (BMSC)-based and macrophage-based cell therapy are regarded as promising strategies to promote fracture healing because of incredible osteogenic potential of BMSCs and typical immunomodulatory function of macrophages. Apart from their respective key roles, accumulative evidence has also demonstrated the importance of cross talk between these two cell types in fracture healing process. This review takes a deep insight into the recent research progress of the synergic performance of BMSCs and macrophages by discussing not only the cells own functions but also the relevant impact factors and mechanisms (ambient microenvironment stimulus, miRNAs, etc). The aim of this review is to provide some valuable cues and technique support for the macrophage- and BMSC-related research, which will be helpful to propel BMSC/macrophage-based combined cell therapy for bone fracture treatment.

Under-Agarose Chemotaxis and Migration Assays for Dictyostelium.

Chemotaxis-directional cell movement steered by chemical gradients-involved in many biological processes including embryonic morphogenesis and immune cell function. Eukaryotic cells, in response to external gradients of attractants, use conserved mechanisms to achieve chemotaxis by regulating the actin cytoskeleton at their fronts and myosin II at their rears. Dictyostelium discoideum, an amoeba that is widely used to study chemotaxis, uses chemotaxis to move up gradients of folate to identify and locate its bacterial prey. Similarly, when starved, Dictyostelium cells synthesize and secrete cyclic AMP (cAMP) while simultaneously expressing cAMP receptors. This allows them to chemotax toward their neighbors and aggregate together. The chemotactic behavior of cells can be studied using several techniques. One such, under-agarose chemotaxis, is a robust, easy, and inexpensive assay that allows direct quantification of chemotactic parameters such as speed and directionality. With the use of high-resolution imaging, for example confocal microscopy, detailed examination of the distribution of actin and membrane proteins in migrating wild type and mutant cells can be performed. In this chapter, we describe simple and optimized methods for studying folate and cAMP chemotaxis in Dictyostelium cells under agarose.

Hyperactive neutrophil chemotaxis contributes to anti-tumor necrosis factor-α treatment resistance in inflammatory bowel disease.

BACKGROUND AND AIM: Anti-tumor necrosis factor-α (anti-TNF-α) agents have been used for inflammatory bowel disease; however, it has up to 30% nonresponse rate. Identifying molecular pathways and finding reliable diagnostic biomarkers for patient response to anti-TNF-α treatment are needed. METHODS: Publicly available transcriptomic data from inflammatory bowel disease patients receiving anti-TNF-α therapy were systemically collected and integrated. In silico flow cytometry approaches and Metascape were applied to evaluate immune cell populations and to perform gene enrichment analysis, respectively. Genes identified within enrichment pathways validated in neutrophils were tracked in an anti-TNF-α-treated animal model (with lipopolysaccharide-induced inflammation). The receiver operating characteristic curve was applied to all genes to identify the best prediction biomarkers. RESULTS: A total of 449 samples were retrieved from control, baseline, and after primary anti-TNF-α therapy or placebo. No statistically significant differences were observed between anti-TNF-α treatment responders and nonresponders at baseline in immune microenvironment scores. Neutrophil, endothelial cell, and B-cell populations were higher in baseline nonresponders, and chemotaxis pathways may contribute to the treatment resistance. Genes related to chemotaxis pathways were significantly upregulated in lipopolysaccharide-induced neutrophils, but no statistically significant changes were observed in neutrophils treated with anti-TNF-α. Interleukin 13 receptor subunit alpha 2 (IL13RA2) is the best predictor (receiver operating characteristic curve: 80.7%, 95% confidence interval: 73.8-87.5%), with a sensitivity of 68.13% and specificity of 84.93%, and significantly higher in nonresponders compared with responders (P < 0.0001). CONCLUSIONS: Hyperactive neutrophil chemotaxis influences responses to anti-TNF-α treatment, and IL13RA2 is a potential biomarker to predict anti-TNF-α treatment response.

Quantitative modeling of regular retinal microglia distribution.

Microglia are resident immune cells in the central nervous system, showing a regular distribution. Advancing microscopy and image processing techniques have contributed to elucidating microglia's morphology, dynamics, and distribution. However, the mechanism underlying the regular distribution of microglia remains to be elucidated. First, we quantitatively confirmed the regularity of the distribution pattern of microglial soma in the retina. Second, we formulated a mathematical model that includes factors that may influence regular distribution. Next, we experimentally quantified the model parameters (cell movement, process formation, and ATP dynamics). The resulting model simulation from the measured parameters showed that direct cell-cell contact is most important in generating regular cell spacing. Finally, we tried to specify the molecular pathway responsible for the repulsion between neighboring microglia.

Autotaxin impedes anti-tumor immunity by suppressing chemotaxis and tumor infiltration of CD8+ T cells.

Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Basal and IL-1ß enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKß NF-κB activating kinases.

IKKα and IKKß are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKß KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1ß stimulated conditions. We find that in their response to IL-1ß stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1ß dependent increases. Our results suggest that IKKα could be a novel OA disease target.

Forty-five years of cGMP research in Dictyostelium: understanding the regulation and function of the cGMP pathway for cell movement and chemotaxis.

In Dictyostelium, chemoattractants induce a fast cGMP response that mediates myosin filament formation in the rear of the cell. The major cGMP signaling pathway consists of a soluble guanylyl cyclase sGC, a cGMP-stimulated cGMP-specific phosphodiesterase, and the cGMP-target protein GbpC. Here we combine published experiments with many unpublished experiments performed in the past 45 years on the regulation and function of the cGMP signaling pathway. The chemoattractants stimulate heterotrimeric Gαßγ and monomeric Ras proteins. A fraction of the soluble guanylyl cyclase sGC binds with high affinity to a limited number of membrane binding sites, which is essential for sGC to become activated by Ras and Gα proteins. sGC can also bind to F-actin; binding to branched F-actin in pseudopods enhances basal sGC activity, whereas binding to parallel F-actin in the cortex reduces sGC activity. The cGMP pathway mediates cell polarity by inhibiting the rear: in unstimulated cells by sGC activity in the branched F-actin of pseudopods, in a shallow gradient by stimulated cGMP formation in pseudopods at the leading edge, and during cAMP oscillation to erase the previous polarity and establish a new polarity axis that aligns with the direction of the passing cAMP wave.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.