Methods and Protocol for Single-Cell Motility Assays under Topological Constraints.

The extracellular environment plays a crucial role in many physiological and pathological processes involving cell motility, such as metastatic invasion in cancer development, by heavily impacting the migration strategies adopted by the cells. The study of how mechanical constraints affect the dynamics of cell migration may be relevant to gain more insight into such processes, and it may prove to be a powerful tool in the hands of biologists. In this chapter, we describe the methods used to investigate the ability of neoplastic cells to migrate through narrowing, rigid microstructures upon chemoattractant stimulation.

Chemotaxis of Large Multinucleate Cells of Dictyostelium Produced by Electric-Pulse Induced Fusion.

Normal-sized cells of Dictyostelium build up a front-tail polarity when they respond to a gradient of chemoattractant. To challenge the polarity-generating system, cells are fused to study the chemotactic response of oversized cells that extend multiple fronts toward the source of attractant. An aspect that can be explored in these cells is the relationship of spontaneously generated actin waves to actin reorganization in response to chemoattractant.

Eukaryotic Chemotaxis under Periodic Stimulation Shows Temporal Gradient Dependence.

When cells of the social amoeba Dictyostelium discoideum are starved of nutrients they start to synthesize and secrete the chemical messenger and chemoattractant cyclic adenosine monophosphate (cAMP). This signal is relayed by other cells, resulting in the establishment of periodic waves. The cells aggregate through chemotaxis toward the center of these waves. We investigated the chemotactic response of individual cells to repeated exposure to waves of cAMP generated by a microfluidic device. For fast-moving waves (short period), the chemotactic ability of the cells was found to increase upon exposure to more waves, suggesting the development of a memory over several cycles. This effect was not significant for slow-moving waves (large period). We show that the experimental results are consistent with a local excitation global inhibition-based model, extended by including a component that rises and decays slowly and that is activated by the temporal gradient of cAMP concentration. The observed enhancement in chemotaxis is relevant to populations in the wild: once sustained, periodic waves of the chemoattractant are established, it is beneficial to cells to improve their chemotactic ability in order to reach the aggregation center sooner.

Neutrophils actively swell to potentiate rapid migration.

While the involvement of actin polymerization in cell migration is well-established, much less is known about the role of transmembrane water flow in cell motility. Here, we investigate the role of water influx in a prototypical migrating cell, the neutrophil, which undergoes rapid, directed movement to sites of injury, and infection. Chemoattractant exposure both increases cell volume and potentiates migration, but the causal link between these processes are not known. We combine single-cell volume measurements and a genome-wide CRISPR screen to identify the regulators of chemoattractant-induced neutrophil swelling, including NHE1, AE2, PI3K-gamma, and CA2. Through NHE1 inhibition in primary human neutrophils, we show that cell swelling is both necessary and sufficient for the potentiation of migration following chemoattractant stimulation. Our data demonstrate that chemoattractant-driven cell swelling complements cytoskeletal rearrangements to enhance migration speed.

Signal integration and adaptive sensory diversity tuning in Escherichia coli chemotaxis.

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

Memoryless chemotaxis with discrete cues.

Biological systems such as axonal growth cones perform chemotaxis at micrometre-level length scales, where chemotactic molecules are sparse. Such systems lie outside the range of validity of existing models, which assume smoothly varying chemical gradients. We investigate the effect of introducing discrete chemoattractant molecules by constructing a minimal dynamical model consisting of a chemotactic cell without internal memory. Significant differences are found in the behaviour of the cell as the chemical gradient is changed from smoothly varying to discrete, including the emergence of a homing radius beyond which chemotaxis is not reliably performed.

[Research progress of chemerin in polycystic ovary syndrome].

As a multifunctional adipokine, chemerin plays a crucial role in various pathophysiological processes through endocrine and paracrine manner. It can bind to three known receptors (ChemR23, GPR1 and CCRL2) and participate in energy metabolism, glucose and lipid metabolism, and inflammation, especially in metabolic diseases. Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases, which seriously affects the normal life of women of childbearing age. Patients with PCOS have significantly increased serum levels of chemerin and high expression of chemerin in their ovaries. More and more studies have shown that chemerin is involved in the occurrence and development of PCOS by affecting obesity, insulin resistance, hyperandrogenism, oxidative stress and inflammatory response. This article mainly reviews the production, subtypes, function and receptors of chemerin protein, summarizes and discusses the research status of chemerin protein in PCOS from the perspectives of metabolism, reproduction and inflammation, and provides theoretical basis and reference for the clinical diagnosis and treatment of PCOS.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Chemoreceptors in Sinorhizobium meliloti require minimal pentapeptide tethers to provide adaptational assistance.

Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl-accepting chemotaxis proteins (MCPs). In Escherichia coli, the adaptation proteins CheR and CheB tether to a conserved C-terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F in Sinorhizobium meliloti chemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C-terminus of pentapeptide-lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate that S. meliloti effectively utilizes a pentapeptide-dependent adaptation system with a minimal number of tethering units to assist pentapeptide-lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB in S. meliloti compared to E. coli allows for ample recruitment of adaptation proteins to the chemosensory array.

Biophysical characterization of the CXC chemokine receptor 2 ligands.

The chemokines of the immune system act as first responders by operating as chemoattractants, directing immune cells to specific locations of inflamed tissues. This promiscuous network is comprised of 50 ligands and 18 receptors where the ligands may interact with the receptors in various oligomeric states i.e., monomers, homodimers, and heterodimers. Chemokine receptors are G-protein coupled receptors (GPCRs) present in the membrane of immune cells. The migration of immune cells occurs in response to a concentration gradient of the ligands. Chemotaxis of neutrophils is directed by CXC-ligand (CXCL) activation of the membrane bound CXC chemokine receptor 2 (CXCR2). CXCR2 plays an important role in human health and is linked to disorders such as autoimmune disorders, inflammation, and cancer. Yet, despite their important role, little is known about the biophysical characteristics controlling ligand:ligand and ligand:receptor interaction essential for biological activity. In this work, we study the homodimers of three of the CXCR2 cognate ligands, CXCL1, CXCL5, and CXCL8. The ligands share high structural integrity but a low sequence identity. We show that the sequence diversity has evolved different binding affinities and stabilities for the CXC-ligands resulting in diverse agonist/antagonist behavior. Furthermore, CXC-ligands fold through a three-state mechanism, populating a folded monomeric state before associating into an active dimer.

Multi-omics Analysis Identifies Hypoxia Subtypes and S100A2 as an Immunosuppressive Factor in Cervical Cancer.

Cervical cancer is a common gynecological oncology. Growing evidence indicates hypoxia plays an important role in tumor progression and immunity. However, no study has examined the hypoxia landscape in cervical cancer. In this study, using hierarchical clustering, we identified three hypoxia subtypes in cervical cancer samples from The Cancer Genome Atlas dataset according to formerly described hypoxia-related genes. The overall survival time, hypoxic features, genomics, and immunological characteristics of these subtypes existed distinct differences. We also created a hypoxia score by principle component analysis for dimension reduction. The hypoxiaScore was an effective prognostic biomarker validated by GSE44001 and was associated with immunotherapy response. Furthermore, combined with single-cell RNA-sequence (scRNA-seq) and experiments, S100A2 was identified as an immunosuppressive factor induced by hypoxia and regulated expression of PD-L1. S100A2 also served as an oncogene promoting the proliferation and migration of cervical cancer cells. These findings depicted a new hypoxia-based classification and identified S100A2 as a potential therapeutic target for cervical cancer, thereby advancing the understanding of immunotherapy resistance mechanisms and cervical cancer genetic markers.

Breaking the feed forward inflammatory cytokine loop in the tumor microenvironment of PDGFB-driven glioblastomas.

Glioblastoma (GBM) tumor-associated macrophages (TAMs) provide a major immune cell population contributing to growth and immunosuppression via the production of proinflammatory factors, including IL-1. In this issue of the JCI, Chen, Giotti, and colleagues investigated loss of ll1b in the immune tumor microenvironment (TME) in GBM models driven by PDGFB expression and Nf1 knockdown. Survival was only improved in PDGFB-driven GBM models, suggesting that tumor cell genotype influenced the immune TME. IL-1ß in the TME increased PDGFB-driven GBM growth by increasing tumor-derived NF-κB, expression of monocyte chemoattractants, and increased infiltration of bone marrow-derived myeloid cells (BMDMs). In contrast, no requirement for IL-1ß was evident in Nf1-silenced tumors due to high basal levels of NF-κB and monocyte chemoattractants and increased infiltration of BMDM and TAMs. Notably, treatment of mice bearing PDGFB-driven GBM with anti-IL-1ß or an IL1R1 antagonist extended survival. These findings suggest that effective clinical immunotherapy may require differential targeting strategies.

Signaling dynamics distinguish high- and low-priority neutrophil chemoattractant receptors.

Human neutrophils respond to multiple chemoattractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils prioritize pathogen- and injury-derived signals over long-range inflammatory signals, such as the leukotriene LTB4, secreted by host cells. Different chemoattractants can also drive qualitatively different modes of migration even though their receptors couple to the same Gαi family of G proteins. Here, we used live-cell imaging to demonstrate that the responses differed in their signaling dynamics. Low-priority chemoattractants caused transient responses, whereas responses to high-priority chemoattractants were sustained. We observed this difference in both primary neutrophils and differentiated HL-60 cells, for downstream signaling mediated by Ca2+, a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. The rapid attenuation of Cdc42 activation in response to LTB4 depended on the phosphorylation sites Thr308 and Ser310 in the carboxyl-terminal tail of its receptor LTB4R in a manner independent of endocytosis. Mutation of these residues to alanine impaired chemoattractant prioritization, although it did not affect chemoattractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes between receptors and contributes to chemoattractant prioritization.

Leukocyte cell-derived chemotaxin 2 correlates with pediatric non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), newly renamed metabolic dysfunction-associated liver disease (MASLD), is a leading cause of liver disease in children and adults. There is a paucity of data surrounding potential biomarkers and therapeutic targets, especially in pediatric NAFLD. Leukocyte cell-derived chemotaxin 2 (LECT2) is a chemokine associated with both liver disease and skeletal muscle insulin resistance. Our aim was to determine associations between LECT2 and common clinical findings of NAFLD in pediatric patients. Enzyme-linked immunosorbent assay (ELISA) was used to measure serum LECT2 concentrations in children (aged 2-17 years) with and without NAFLD. LECT2 concentrations were then correlated to clinical parameters in NAFLD. Mean LECT2 was significantly elevated in children with NAFLD versus healthy controls (n = 63 vs. 42, 5.83 ± 1.98 vs. 4.02 ± 2.02 ng/mL, p < 0.005). Additionally, LECT2 had strong correlations with body mass index (BMI) (Pearson r = 0.301, p = 0.002). A LECT2 concentration of 3.76 mg/mL predicts NAFLD with a sensitivity of 90.5% and specificity of 54.8%. Principal component analysis and logistic regression models further confirmed associations between LECT2 and NAFLD status. This study demonstrates increased serum LECT2 concentrations in pediatric NAFLD, which correlates with BMI and shows strong predictive value within these patients. Our data indicate that LECT2 is a potential diagnostic biomarker of disease and should be further investigated in pediatric as well as adult NAFLD.

A2BAR Antagonism Decreases the Glomerular Expression and Secretion of Chemoattractants for Monocytes and the Pro-Fibrotic M2 Macrophages Polarization during Diabetic Nephropathy.

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

Evaluation of the therapeutic effects of oestradiol on the systemic inflammatory response and on lung injury caused by the occlusion of the proximal descending aorta in male rats.

OBJECTIVES: Ischaemia and reperfusion-induced microvascular dysfunction is a serious problem encountered during a variety surgical procedures, leading to systemic inflammation and affecting remote organs, specially the lungs. 17ß-Oestradiol reduces pulmonary repercussions from various acute lung injury forms. Here, we focused on the 17ß-oestradiol therapeutic effects after aortic ischaemia and reperfusion (I/R) by evaluating lung inflammation. METHODS: Twenty-four Wistar rats were submitted to I/R by insufflation of a 2-F catheter in thoracic aorta for 20 min. Reperfusion took 4 h and 17ß-oestradiol (280 µg/kg, i.v.) was administered after 1 h of reperfusion. Sham-operated rats were controls. Bronchoalveolar lavage was performed and lung samples were prepared for histopathological analysis and tissue culture (explant). Interleukin (IL)-1ß, IL-10 and tumour necrosis factor-α were quantified. RESULTS: After I/R, higher number of leukocytes in bronchoalveolar lavage were reduced by 17ß-oestradiol. The treatment also decreased leukocytes in lung tissue. I/R increased lung myeloperoxidase expression, with reduction by 17ß-oestradiol. Serum cytokine-induced neutrophil chemoattractant 1 and IL-1ß increased after I/R and 17ß-oestradiol decreased cytokine-induced neutrophil chemoattractant 1. I/R increased IL-1ß and IL-10 in lung explants, reduced by 17ß-oestradiol. CONCLUSIONS: Our results showed that 17ß-oestradiol treatment performed in the period of reperfusion, modulated the systemic response and the lung repercussions of I/R by thoracic aortic occlusion. Thus, we can suggest that 17ß-oestradiol might be a supplementary approach leading the lung deterioration after aortic clamping in surgical procedures.

Dynamic subcellular localization of DydA in Dictyostelium cells.

DydA plays an important role in chemotaxis, development, and cell growth as an adaptor protein that connects Ras signaling and cytoskeletal rearrangement. DydA is a downstream effector of RasG and is involved in controlling cell polarity and pseudopodia formation during chemoattractant-directed cell migration. To understand the mechanism by which DydA functions on the cell migration, we investigated the dynamic subcellular localization of DydA in response to chemoattractant stimulation and found that DydA rapidly and transiently translocated to the cell cortex through the RA domain and the PRM region in DydA in response to chemoattractant stimulation. The PRM region appears to play a primary role in the translocation of DydA to the cell cortex and in its localization to the actin foci at the bottom of cells. Colocalization experiments of GFP-PRM with RFP-coronin indicated that GFP-PRM preceded GFP-coronin by 2-3 s in response to chemoattractant stimulation. These results suggest that the PRM region plays an indispensable role in relaying upstream regulators, such as RasG, to downstream effectors by mediating the localization of DydA to the cell cortex upon chemoattractant stimulation.

IL-8 Secreted by Gastric Epithelial Cells Infected with Helicobacter pylori CagA Positive Strains Is a Chemoattractant for Epstein-Barr Virus Infected B Lymphocytes.

Helicobacter pylori and EBV are considered the main risk factors in developing gastric cancer. Both pathogens establish life-lasting infections and both are considered carcinogenic in humans. Different lines of evidence support that both pathogens cooperate to damage the gastric mucosa. Helicobacter pylori CagA positive virulent strains induce the gastric epithelial cells to secrete IL-8, which is a potent chemoattractant for neutrophils and one of the most important chemokines for the bacterium-induced chronic gastric inflammation. EBV is a lymphotropic virus that persists in memory B cells. The mechanism by which EBV reaches, infects and persists in the gastric epithelium is not presently understood. In this study, we assessed whether Helicobacter pylori infection would facilitate the chemoattraction of EBV-infected B lymphocytes. We identified IL-8 as a powerful chemoattractant for EBV-infected B lymphocytes, and CXCR2 as the main IL-8 receptor whose expression is induced by the EBV in infected B lymphocytes. The inhibition of expression and/or function of IL-8 and CXCR2 reduced the ERK1/2 and p38 MAPK signaling and the chemoattraction of EBV-infected B lymphocytes. We propose that IL-8 at least partially explains the arrival of EBV-infected B lymphocytes to the gastric mucosa, and that this illustrates a mechanism of interaction between Helicobacter pylori and EBV.

Competition between chemoattractants causes unexpected complexity and can explain negative chemotaxis.

Negative chemotaxis, where eukaryotic cells migrate away from repellents, is important throughout biology, for example, in nervous system patterning and resolution of inflammation. However, the mechanisms by which molecules repel migrating cells are unknown. Here, we use predictive modeling and experiments with Dictyostelium cells to show that competition between different ligands that bind to the same receptor leads to effective chemorepulsion. 8-CPT-cAMP, widely described as a simple chemorepellent, is inactive on its own and only repels cells when it acts in combination with the attractant cAMP. If cells degrade either competing ligand, the pattern of migration becomes more complex; cells may be repelled in one part of a gradient but attracted elsewhere, leading to populations moving in different directions in the same assay or converging in an arbitrary place. More counterintuitively still, two chemicals that normally attract cells can become repellent when combined. Computational models of chemotaxis are now accurate enough to predict phenomena that have not been anticipated by experiments. We have used them to identify new mechanisms that drive reverse chemotaxis, which we have confirmed through experiments with real cells. These findings are important whenever multiple ligands compete for the same receptors.

Mesenchymal stromal cell-associated migrasomes: a new source of chemoattractant for cells of hematopoietic origin.

BACKGROUND: Multipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell-cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication. RESULTS: Migrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes. CONCLUSION: Overall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment. Video Abstract.