Chemotactic Response Research Articles

Inhibition of the chemokine signal regulator FROUNT by disulfiram ameliorates crescentic glomerulonephritis

Activated monocytes/macrophages promote glomerular injury, including crescent formation, in anti-glomerular basement membrane (GBM) glomerulonephritis. Disulfiram, an alcohol-aversion drug, inhibits monocyte/macrophage migration by inhibiting FROUNT, a cytosolic protein that enhances chemokine receptor signaling. Our study found that disulfiram at a human equivalent dose successfully blocked albuminuria and crescent formation with podocyte loss, and later stage kidney fibrotic lesions, in a rat model of anti-GBM glomerulonephritis. A disulfiram derivative, DSF-41, with more potent FROUNT inhibition activity, inhibited glomerulonephritis at a lower dose than disulfiram. Disulfiram markedly reduced the number of monocytes or macrophages at the early stage of glomerulonephritis and that of CD3+ and CD8+ lymphocytes at the established stage. Impaired pseudopodia formation was observed in the glomerular monocytes/macrophages of the disulfiram group; consistent with the invitro observation that disulfiram blocked chemokine-dependent pseudopodia formation and chemotaxis of bone marrow-derived monocytes/macrophages. Furthermore, disulfiram suppressed macrophage activation as revealed by reduced expression of inflammatory cytokines and chemokines (TNF-α, CCL2, and CXCL9) and reduced CD86 and MHC class II expressions in monocytes/macrophages during glomerulonephritis. The dramatic reduction in monocyte/macrophage number might have resulted from disulfiram suppression of both the chemotactic response of monocytes/macrophages and their subsequent activation to produce cytokines and chemokines, which further recruit monocytes. Additionally, FROUNT was expressed in CD68+ monocytes/macrophages infiltrating the crescentic glomeruli in human anti-GBM glomerulonephritis. Thus, disulfiram can be a highly effective and safe drug for the treatment of glomerulonephritis by blocking the chemotactic responses of monocytes/macrophages and their activation status in the glomerulus.

An atypical MAPK regulates translocation of a GATA transcription factor in response to chemoattractant stimulation.

The Dictyostelium atypical mitogen-activated protein kinase (MAPK) Erk2 is required for chemotactic responses to cAMP as amoeba undergo multicellular development. In this study, Erk2 was found to be essential for the cAMP-stimulated translocation of the GATA transcription factor GtaC as indicated by the distribution of a GFP-GtaC reporter. Erk2 was also found to be essential for the translocation of GtaC in response to external folate, a foraging signal that directs the chemotaxis of amoeba to bacteria. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor Far1 or the coupled G-protein subunit Gα4. Loss of GtaC function resulted in enhanced chemotactic movement to folate, suggesting that GtaC suppresses responses to folate. The alteration of four Erk2-preferred phosphorylation sites in GtaC impacted the translocation of GFP-GtaC in response to folate and the GFP-GtaC-mediated rescue of aggregation and development of gtaC- cells. The ability of different chemoattractants to stimulate Erk2-regulated GtaC translocation suggests that atypical MAPK-mediated regulation of transcription factors can contribute to different cell fates.

The use of microfluidic devices in studies of differential sperm chemotaxis

Differential sperm chemotaxis describes differences among male-female pairings in chemotactic responses of sperm to egg (or female)-derived chemical attractants. Microfluidic devices provide powerful platforms in which to study this complex gamete interaction. Here, we describe key challenges and potential solutions in applying this state-of-the-art technique to differential sperm chemotaxis.

Nonequilibrium phenomena in driven and active Coulomb field theories

The classical Coulomb gas model has served as one of the most versatile frameworks in statistical physics, connecting a vast range of phenomena across many different areas. Nonequilibrium generalisations of this model have so far been studied much more scarcely. With the abundance of contemporary research into active and driven systems, one would naturally expect that such generalisations of systems with long-ranged Coulomb-like interactions will form a fertile playground for interesting developments. Here, we present two examples of novel macroscopic behaviour that arise from nonequilibrium fluctuations in long-range interacting systems, namely (1) unscreened long-ranged correlations in strong electrolytes driven by an external electric field and the associated fluctuation-induced forces in the confined Casimir geometry, and (2) out-of-equilibrium critical behaviour in self-chemotactic models that incorporate the particle polarity in the chemotactic response of the cells. Both of these systems have nonlocal Coulomb-like interactions among their constituent particles, namely, the electrostatic interactions in the case of the driven electrolyte, and the chemotactic forces mediated by fast-diffusing signals in the case of self-chemotactic systems. The results presented here hint to the rich phenomenology of nonequilibrium effects that can arise from strong fluctuations in Coulomb interacting systems, and a rich variety of potential future directions, which are discussed.

Role of hyperpolarization-activated cyclic nucleotide-gated channel HCN2 in embryonic neural stem cell proliferation and differentiation

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels) are involved in spontaneous activity in many electrically active cell types such as cardiomyocytes and neurons. In this study, the role of HCN channels in proliferation and migration of Nestin and Sox2 expressing embryonic neural progenitor cells (NPC) originating from the subventricular zone (SVZ) was examined. Immunostaining and PCR data showed that the HCN2 subtype was highly expressed in these cells. Patch clamp recordings revealed a hyperpolarization-activated current, which was sensitive to inhibitors of HCN channels. Using the fluorescence dye bis-(1,3-dibutylbarbituric acid)-trimethineoxonol (DiBAC(4)(3)) we found that a prompt reduction of the extracellular K+ concentration, or exposing the cells to acute hypoxia, induced an instant hyperpolarization in the whole cell population. Recovery from low K+ induced hyperpolarization after extracellular calcium removal, or by re-oxygenation of hypoxic cells, was sensitive to ZD7288, a HCN channel inhibitor. Treatment of neurosphere cultures from the SVZ with ZD7288 caused a significant and reversible inhibition of neurosphere formation from single cells indicating that proliferation of progenitor cells was reduced. Furthermore, the migration of neuronal cells from neurospheres was considerably retarded in the presence of ZD7288. The results suggest that HCN2 channels are involved in controlling the proliferation of NPC and that HCN2 channel-induced spontaneous electrical activity may trigger the motility response of neurosphere-derived neurons in concert with other ion channels. Furthermore, the response to hypoxia suggests that HCN2 channels may trigger the chemotactic response of NPC to ischemic brain regions seen in many studies.

Abstract 2690: Loss of E-cadherin induces IGF1R activation revealing a targetable pathway in invasive lobular breast carcinoma

Abstract Invasive Ductal Carcinoma (IDC) and Invasive Lobular Carcinoma (ILC) are two major subtypes of breast cancer with significant differences in their histological and molecular underpinnings. ILC has a unique loss of E-cadherin (CDH1) which we have previously demonstrated as a negative regulator of the Insulin-like Growth Factor 1 receptor (IGF1R) through a comprehensive analysis of cell line models and tumor samples. We propose that loss of E-cadherin in ILC sensitizes cells to growth factor signaling and thus alters their sensitivity to growth factor signaling inhibitors. To investigate this, we used CRISPR to generate CDH1 knockout (KO) IDC cell lines (MCF7, T47D, ZR75.1) to uncover the mechanism by which E-cadherin loss activates the IGF pathway while also assessing its targetability. CDH1 KO cells exhibited anchorage independent growth in suspension culture and altered p120 catenin localization as observed in ILC tumors. Through in vitro studies, we show increased signaling sensitivity to IGF/insulin ligands and enhanced signaling duration in CDH1 KO cells. In addition, we observed a higher migratory potential of CDH1 KO cells compared to wild type (WT) cells, which was further enhanced as a chemotactic response to IGF1 or serum. Further, this phenotype could be reversed with an IGF1R inhibitor, BMS-754807. We additionally identified an increase in Collagen I haptotaxis in the CDH1 KO cells, which was also translated into a novel invasive phenotype towards serum in the T47D CDH1 KO cells. Despite no significant differences in membranous IGF1R levels between WT and CDH1 KO cells, higher ligand-receptor interaction was observed with CDH1 KO cells, demonstrating an increased ligand-receptor complex formation upon stimulation. Our results suggest that loss of CDH1 results in an increase in IGF1 receptor availability for ligand binding which in turn allows for an enhanced downstream signaling activation. Interestingly, a physical repression of E-cadherin on IGF1R could not be demonstrated, suggesting spatial changes on the membrane following E-cadherin loss may control ligand binding. Critically, increased sensitivity to IGF1R, PI3K, AKT and MEK inhibitors was observed in CDH1 KO cells suggesting that these targets should be further explored in ILC and that CDH1 loss may be exploited as a biomarker of response, or for patient stratification to inhibitors targeting these pathways. Citation Format: Ashuvinee Elangovan, Laura Savariau, Megan E. Yates, Jagmohan Hooda, Alison M. Nagle, Steffi Oesterreich, Jennifer M. Atkinson, Adrian V. Lee. Loss of E-cadherin induces IGF1R activation revealing a targetable pathway in invasive lobular breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2690.

Features of water extracts sample preparation of solar panels components for biotesting

THE PURPOSE. Detection of the dependences between the toxicity index of the components of crystalline solar panels EVA and Tedlar® and the factors of sample preparation of water extracts used in biotesting on the chemotactic reaction of Paramecium caudatum test objects using Biotester-2M as an analyzer. These factors included: the temperature of the extract, the exposure time of the extract of the studied objects in distilled water, and the use of otherkind of extract.METHODS. In the course of the study, the biotesting method for the chemotactic reaction of Paramecium caudatum was used. In the experiments, an aqueous extract was prepared in accordance with PND F T 16.3.16-10. The resulting extracts were analyzed on a Biotester-2M device. For each experiment number, a study was carried out in three cuvettes, from each of which the device took 10 values. To assess the influence of extract temperature factors and exposure time, an experiment was carried out for temperatures of 4°C and 35°C, which allow taking into account the temperature at solid waste landfills at different times of the year. The extraction times were chosen to be 1, 7, 21, 28, 42 and 56 days. Data analysis was carried out using two-way replicated analysis of variance implemented using MatLab® tools. To assess the effect of the extraction medium, an experiment was carried out with the replacement of distilled water as an extract with mineral water of the Bonaqua brand and 1% acetone solution.RESULTS. The error probability of the hypothesis about the influence of temperature and exposure time factors of the extract on the toxicity index was calculated at given significance levels. A comparative analysis of the average toxicity indices for the studied extraction media with distilled water, which is used as an extraction medium in approved biotesting methods, was carried out.CONCLUSION. For complex organic compounds such as EVA and Tedlar® the toxicity indices will depend on a number of factors: the temperature of the extract, the residence time of the components in the extract, and the extraction medium.

Pseudomonas syringae pv. tomato infection of tomato plants is mediated by GABA and l-Pro chemoperception.

Foliar bacterial pathogens have to penetrate the plant tissue and access the interior of the apoplast in order to initiate the pathogenic phase. The entry process is driven by chemotaxis towards plant‐derived compounds in order to locate plant openings. However, information on plant signals recognized by bacterial chemoreceptors is scarce. Here, we show that the perception of GABA and l‐Pro, two abundant components of the tomato apoplast, through the PsPto‐PscC chemoreceptor drives the entry of Pseudomonas syringae pv. tomato into the tomato apoplast. The recognition of both compounds by PsPto‐PscC caused chemoattraction to both amino acids and participated in the regulation of GABA catabolism. Mutation of the PsPto‐PscC chemoreceptor caused a reduced chemotactic response towards these compounds which in turn impaired entry and reduced virulence in tomato plants. Interestingly, GABA and l‐Pro levels significantly increase in tomato plants upon pathogen infection and are involved in the regulation of the plant defence response. This is an example illustrating how bacteria respond to plant signals produced during the interaction as cues to access the plant apoplast and to ensure efficient infection.

Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell–bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Chemotactic response of Escherichia coli to polymer solutions

Polymers are important components of the complex fluid environment for microorganisms. The mechanical effects on bacterial motile behavior due to the viscous or viscoelastic properties of polymers were extensively studied, whereas possible chemical effects on bacterial motility through bacterial chemoreception of the polymers were unclear. Here we studied the chemotactic response of Escherichia coli to polymeric solutions by combining the bead assay and FRET measurements. We found that the wild-type E. coli strain exhibited an attractant response to widely used polymers such as Ficoll 400, polyethylene glycol (PEG) 20000 and polyvinyl pyrrolidone (PVP) 360000, and the response amplitude from chemoreception was much larger than that from the load-dependence of motor switching due to viscosity change. The chemotactic response depended on the type of receptors and the chain length of the polymers. Our findings here provided important new ingredients for further studies of bacterial motile behavior in complex fluids.

Cells responding to chemoattractant on a structured substrate

Cell migration on an adhesive substrate surface comprises actin-based protrusion at the front and retraction of the tail in combination with coordinated adhesion to, and detachment from, the substrate. To study the effect of cell-to-substrate adhesion on the chemotactic response of Dictyostelium discoideum cells, we exposed the cells to patterned substrate surfaces consisting of adhesive and inert areas, and forced them by a gradient of chemoattractant to enter the border between the two areas. Wild-type as well as myosin II-deficient cells stop at the border of an adhesive area. They do not detach with their rear part, while on the nonadhesive area they protrude pseudopods at their front toward the source of chemoattractant. Avoidance of the nonadhesive area may cause a cell to move in tangential direction relative to the attractant gradient, keeping its tail at the border of the adhesive surface.

Indole modulates cooperative protein-protein interactions in the flagellar motor.

Indole is a major component of the bacterial exometabolome, and the mechanisms for its wide-ranging effects on bacterial physiology are biomedically significant, although they remain poorly understood. Here, we determined how indole modulates the functions of a widely conserved motility apparatus, the bacterial flagellum. Our experiments in Escherichia coli revealed that indole influences the rotation rates and reversals in the flagellum’s direction of rotation via multiple mechanisms. At concentrations higher than 1 mM, indole decreased the membrane potential to dissipate the power available for the rotation of the motor that operates the flagellum. Below 1 mM, indole did not dissipate the membrane potential. Instead, experiments and modeling indicated that indole weakens cooperative protein interactions within the flagellar complexes to inhibit motility. The metabolite also induced reversals in the rotational direction of the motor to promote a weak chemotactic response, even when the chemotaxis response regulator, CheY, was lacking. Experiments further revealed that indole does not require the transporter Mtr to cross the membrane and influence motor functions. Based on these findings, we propose that indole modulates intra- and inter-protein interactions in the cell to influence several physiological functions.

A single-CRD C-type lectin from Haliotis discus hannai acts as pattern recognition receptor enhancing hemocytes opsonization

C-type lectins (CTLs), as a member of the Ca2+-dependent carbohydrate recognition protein superfamily, play multiple roles in non-self recognition and the elimination of invading pathogens. In this study, a C-type lectin was identified and characterized from the Pacific abalone Haliotis discus hannai (designed as HdClec), and its open reading frame (ORF) encoded a polypeptide of 163 amino acids containing a typical signal peptide and only one carbohydrate-recognition domain (CRD). The deduced amino acid sequence of CRD in HdClec shared identities ranging from 22.4% to 39.8% with that of other identified CRDs of CTLs. A novel NPN motif was found in Ca2+-binding site 2 of HdClec. The mRNA transcripts of HdClec were detectable in all the examined tissues of non-stimulated abalones, with the highest expression in hepatopancreas (224.13-fold of that in gills). The expression of HdClec mRNA in hemocytes was significantly up-regulated after Vibrio harveyi challenge. Recombinant HdClec protein (rHdClec) could bind lipopolysaccharide (LPS) and peptidoglycan (PGN) in vitro in the presence of Ca2+. Coinciding with the PAMPs binding assay, rHdClec displayed broad agglutination activities towards Gram-negative bacteria V. splendidus, V. anguillarum, V. parahaemolyticus, V. harveyi, Escherichia coli, and Gram-positive bacteria Micrococcus luteus. Moreover, rHdClec could significantly elicit the chemotactic response of hemocytes in vitro. And the phagocytosis and encapsulation ability of hemocytes could be significantly enhanced by rHdClec. All these results showed that HdClec could function as pattern recognition receptors (PRRs) and further enhance the opsonization of hemocytes, which might play a crucial role in the innate immune responses of Pacific abalone.

α1B‐Adrenoceptor is Required for Chemokine (C‐X‐C Motif) Receptor 4 Mediated Chemotaxis in THP1 Cells

IntroductionChemokine (C‐X‐C motif) receptor 4 (CXCR4) is a key regulator of leukocyte trafficking under physiological conditions and in numerous inflammatory disease processes. Previously, we demonstrated that CXCR4 hetero‐oligomerizes with α1B‐adrenoceptors (ARs), through which the receptors cross‐talk. The existence and function of these hetero‐oligomers in leukocytes, however, is unknown. Thus, we tested whether such heteromeric complexes are detectable in the human monocytic cell line THP1 and in freshly isolated human monocytes, and evaluated how α1B‐AR knockdown by CRISPR/Cas9 gene editing in THP1 cells affects CXCR4‐mediated chemotaxis.MethodsPeripheral blood mononuclear cells were isolated by density gradient centrifugation from human whole blood; monocytes were then isolated via negative selection using MACS LS, an indirect magnetic labeling system. Proximity ligation assays (PLA) were used to visualize individual receptors and receptor‐receptor interactions in THP1 cells and freshly isolated monocytes. CRISPR/Cas9 gene editing was utilized to create a α1B‐AR knockout strain (ADRA1BKO); wild type THP1 cells were used as a negative control (ADRA1Bwt). Cell migration toward the cognate CXCR4 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) was tested employing a 96 well Boyden chamber. Transmigrated cells were counted automatically utilizing high contrast bright field and post‐imaging particle analyses. The chemotactic index (ChI) was calculated as the ratio of cells that transmigrated in the presence versus the absence of CXCL12. Data were analyzed with ANOVA/Tukey’s post‐hoc. Data are mean ± SE from n=4 experiments performed on different days. A 2‐tailed p<0.05 was considered significant.ResultsPLA confirmed the presence of endogenously and constitutively expressed CXCR4:α1B‐AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α1B‐AR and CXCR4:α1B‐AR heteromers were not detectable in ADRA1BKO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1Bwtcells showed a bell‐shaped chemotactic dose response to CXCL12 with a maximal ChI of 9.1±1.1 at 100 nM. CRISPR/Cas9 knockout of α1B‐AR reduced the chemotactic response to CXCL12 by 89±52% (p<0.05). ADRA1BKO results were replicated in an additional α1B‐AR knockout strain to rule out clonal effects.ConclusionOur observations suggest that CXCR4 heteromerizes with α1B‐AR in human monocytes and that CXCL12‐mediated chemotaxis depends on the formation of CXCR4:α1B‐AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4‐mediated chemotaxis in monocytes/leukocytes is controlled.

Anaerobic Bacterial Biofilms Inhibit Neutrophil Responses via the Release of Stable Small‐Molecule Effectors

Background and ObjectiveFusobacterium and Porphyromonasare two genera of opportunistic pathogens that often aggregate in complex communities known as biofilms. Host innate immune cells are frequently frustrated when they encounter biofilm bacteria, resulting in ineffectual clearance and persistent infection. Anaerobic pathogens generate or accumulate organic molecules and use them as defense against environmental pressures such as oxidative stress or attack from host immune cells. Porphyromonasspecies utilize protoporphyrin IX (PPIX) to protect against neutrophil oxidative‐killing. The objective was to develop a model of mixed‐species biofilm generation and to evaluate bovine neutrophil responses to bioactive molecules released from either biofilm or planktonic bacteria. We hypothesized that biofilm‐conditioned growth medium would contain more PPIX than planktonic medium and therefore inhibit the functional responses of peripheral neutrophils.MethodsNeutrophils were isolated from whole blood via hypotonic lysis and bacterial biofilms were generated using previously validated methods for the cultivation of mixed‐species biofilms. Spent media from biofilm and overnight planktonic cultures were collected, centrifuged, and filtered to remove viable bacteria. Supernatants were fractionated via <3kDa‐cutoff filtration and exposed directly to neutrophils. Neutrophil responses to the supernatants were assessed with a fluorescent microplate assay for reactive oxygen species (ROS) and chemotaxis was measured with a Transwell® transmigration assay. The specific mechanism of neutrophil activation was investigated by employing NF‐κB and NADPH oxidase inhibitors.ResultsNeutrophils exposed to planktonic supernatant showed significantly elevated oxidative and chemotactic responses compared to biofilm products from identical bacteria. Ultra‐filtration revealed that a <3kDa molecule is differentially expressed in the supernatants and allows biofilm‐bacteria to inhibit neutrophil responses. Intensive heat, nuclease, lipase, and protease treatment of the <3kDa fractions did not alter neutrophil functional responses. Preliminary experiments suggest that the accumulation of PPIX in the biofilm supernatant attenuates neutrophil chemotaxis and ROS production. In addition, chemical inhibition experiments indicated that neutrophils respond to planktonic supernatants independently of NF‐κB signaling.ConclusionsExperiments reveal a minimal neutrophil response to molecules released from biofilm bacteria and may explain the inadequate host response to biofilm‐mediated infections. The phenotypic changes in the biofilm mode of growth allow for the generation and accumulation of PPIX, providing protection from host immune clearance mechanisms. Understanding the mechanism of immune avoidance is a crucial step to reduce the persistent inflammation and negative clinical outcomes associated with chronic biofilm‐mediated disease.

Genetic Evidences of Biosurfactant Production in Two Bacillus subtilis Strains MB415 and MB418 Isolated From Oil Contaminated Soil.

Biosurfactants are a diverse group of amphiphilic compounds obtained from microbes. In the present study, the genomic analysis of biosurfactant-producing Bacillus subtilis MB415 and MB418 obtained from oil-contaminated soil was performed. Initially, the strains were screened for biosurfactant production by hemolytic assay, emulsification index, and oil displacement. Further FTIR analysis of extracted biosurfactants revealed the presence of lipopeptides. The sequenced genomes of MB415 and MB418 were of 4.2 Mbps with 43% GC content. Among more than 4,500 protein-coding genes, many were involved in virulence, metal/multidrug resistances, flagella assembly, chemotactic response, and aromatic ring hydroxylating dioxygenases. An annotation analysis revealed that both genomes possessed non-ribosomal synthetase gene clusters for the lipopeptide synthetases srf and fen responsible for surfactin and fengycin production. Comparative studies of both genomes highlighted variability in gene operons mainly for surfactin biosynthesis.

The Effect of the Second Messenger c-di-GMP on Bacterial Chemotaxis in Escherichia coli

c-di-GMP is a ubiquitous bacterial second messenger that plays a central regulatory role in diverse biological processes. c-di-GMP was known to regulate chemotaxis in multiple bacterial species, but its effect on Escherichia coli chemotaxis remained unclear. As an effector of c-di-GMP in E. coli, YcgR when bound with c-di-GMP interacts with the flagellar motor to reduce its speed and its probability of rotating clockwise (CW bias). Here, we found that a significant fraction of the c-di-GMP::YcgR dynamically exchange between the motor and the cytosol. Through fluorescent measurements, we found that there was no competitive binding between the chemotaxis response regulator CheY-P and c-di-GMP::YcgR to the motor. To test the influence of elevated c-di-GMP levels on the chemotaxis pathway, we measured the chemotactic responses of E. coli cells using a FRET assay, finding that elevated c-di-GMP levels had no effect on the upstream part of chemotaxis pathway down to the level of CheY-P concentration. This suggested that the possible effect of elevated c-di-GMP levels on chemotactic motion was through regulation of motor speed and CW bias. Using stochastic simulations of chemotactic swimming, we showed that the effects of reducing motor speed and decreasing CW bias on chemotactic drift velocity are compensating for each other, resulting in minimal effect of elevated c-di-GMP levels on E. coli chemotaxis. Therefore, elevated c-di-GMP levels promote the transition from motile to sedentary forms of bacterial life by reducing the bacterial swimming speed and CW bias, while still maintaining a nearly intact chemotaxis capability in E. coli. IMPORTANCE The ubiquitous bacterial second messenger c-di-GMP was known to regulate chemotaxis in many bacterial species, but its effect on E. coli chemotaxis was unclear. Here we studied the effect of elevated c-di-GMP levels on chemotaxis in E. coli. We found that the binding of c-di-GMP::YcgR (its effector) and the chemotaxis response regulator CheY-P to the flagellar motor are noncompetitive, and elevated c-di-GMP levels do not affect the upstream part of the chemotaxis pathway down to the level of CheY-P concentration. Elevated c-di-GMP levels exert direct effects on the flagellar motor by reducing its speed and CW bias, but the resulting effects on chemotaxis performance are compensating for each other. Our findings here showed that elevated c-di-GMP levels maintain a nearly intact chemotaxis capability when promoting the transition from motile to sedentary forms of bacterial life in E. coli.

Gene Therapy for Repair and Proliferation of Human Dental Pulp Stem Cells – An In-Vitro Study

Regulatory cellular process of mesenchymal stem cell remains to be explicated in diseased condition. On the other hand, platelet-derived growth factors (PDGFs) are known to shape the migratory movements of the mesenchymal cells during stressful conditions. The objective of the current study is to understand the role of Recombinant Platelet-Derived Growth Factors-BB (rhPDGF-BB) towards proliferation and migration of Human Dental Pulp Stem Cells (hDPSC). The cytotoxicity of rhPDGF-BB was assessed at various doses and its chemotactic response was measured by experimental wound scratch test. The recombinant protein did not show cytotoxicity at higher doses. It was found that rhPDGF-BB is a strong inducer of indiscriminate as well as directional migration of hDPSCs that persisted upon 24hrs. Thus, the experiment demonstrates critical use of rhPDGF-BB in regenerative process during dental pulp stem injury.

The Dictyostelium Model for Mucolipidosis Type IV.

Mucolipidosis type IV, a devastating neurological lysosomal disease linked to mutations in the transient receptor potential channel mucolipin 1, TRPML1, a calcium permeable channel in the membranes of vesicles in endolysosomal system. TRPML1 function is still being elucidated and a better understanding of the molecular pathogenesis of Mucolipidosis type IV, may facilitate development of potential treatments. We have created a model to study mucolipin function in the eukaryotic slime mould Dictyostelium discoideum by altering expression of its single mucolipin homologue, mcln. We show that in Dictyostelium mucolipin overexpression contributes significantly to global chemotactic calcium responses in vegetative and differentiated cells. Knockdown of mucolipin also enhances calcium responses in vegetative cells but does not affect responses in 6–7 h developed cells, suggesting that in developed cells mucolipin may help regulate local calcium signals rather than global calcium waves. We found that both knocking down and overexpressing mucolipin often, but not always, presented the same phenotypes. Altering mucolipin expression levels caused an accumulation or increased acidification of Lysosensor Blue stained vesicles in vegetative cells. Nutrient uptake by phagocytosis and macropinocytosis were increased but growth rates were not, suggesting defects in catabolism. Both increasing and decreasing mucolipin expression caused the formation of smaller slugs and larger numbers of fruiting bodies during multicellular development, suggesting that mucolipin is involved in initiation of aggregation centers. The fruiting bodies that formed from these smaller aggregates had proportionately larger basal discs and thickened stalks, consistent with a regulatory role for mucolipin-dependent Ca2+ signalling in the autophagic cell death pathways involved in stalk and basal disk differentiation in Dictyostelium. Thus, we have provided evidence that mucolipin contributes to chemotactic calcium signalling and that Dictyostelium is a useful model to study the molecular mechanisms involved in the cytopathogenesis of Mucolipidosis type IV.

Macrophage network dynamics depend on haptokinesis for optimal local surveillance

Macrophages are key immune cells with important roles for tissue surveillance in almost all mammalian organs. Cellular networks made up of many individual macrophages allow for optimal removal of dead cell material and pathogens in tissues. However, the critical determinants that underlie these population responses have not been systematically studied. Here, we investigated how cell shape and the motility of individual cells influences macrophage network responses in 3D culture settings and in mouse tissues. We show that surveying macrophage populations can tolerate lowered actomyosin contractility, but cannot easily compensate for a lack of integrin-mediated adhesion. Although integrins were dispensable for macrophage chemotactic responses, they were crucial to control cell movement and protrusiveness for optimal surveillance by a macrophage population. Our study reveals that β1 integrins are important for maintaining macrophage shape and network sampling efficiency in mammalian tissues, and sets macrophage motility strategies apart from the integrin-independent 3D migration modes of many other immune cell subsets.