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Bacterial motility relying on flagella is characterized by several modes, including swimming, swarming, twitching, and gliding. This motility allows bacteria to adapt remarkably well to hostile environments. More than 50% of bacteria naturally contain flagella, which are crucial for bacterial chemotaxis motility. Chemotaxis can be either positive, where bacteria move towards a chemical source, or negative, known as chemorepulsion, where bacteria move away from the source. Although much is known about the mechanisms driving chemotaxis towards attractants, the molecular mechanisms underlying chemorepulsion remain elusive. Chemotaxis plays an important role in the colonization of the rhizosphere by rhizobacteria. Recently, researchers have systematically studied the identification and recognition mechanisms of chemoattractants. However, the mechanisms underlying chemorepellents remain unclear. Systematically sorting and analyzing research on chemorepellents could significantly enhance our understanding of how these compounds help probiotics evade harmful environments or drive away pathogens.

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Little is known about eukaryotic chemorepulsion. The enzymes phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion.

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Background: Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive arthritis, and differences in neutrophil physiological responses such as a faster response to the chemorepellent SLIGKV. Little is known about the basis of sex-based differences in human neutrophils. Methods: Starting with human neutrophils from healthy donors, we used RNA-seq to examine total mRNA profiles, mRNAs not associated with ribosomes and thus not being translated, mRNAs in monosomes, and mRNAs in polysomes and thus heavily translated. We used mass spectrometry systems to identify proteins and phosphoproteins. Results: There were sex-based differences in the translation of 24 mRNAs. There were 132 proteins with higher levels in male neutrophils; these tended to be associated with RNA regulation, ribosome, and phosphoinositide signaling pathways, whereas 30 proteins with higher levels in female neutrophils were associated with metabolic processes, proteosomes, and phosphatase regulatory proteins. Male neutrophils had increased phosphorylation of 32 proteins. After exposure to SLIGKV, male neutrophils showed a faster response in terms of protein phosphorylation compared to female neutrophils. Conclusions: Male neutrophils have higher levels of proteins and higher phosphorylation of proteins associated with RNA processing and signaling pathways, while female neutrophils have higher levels of proteins associated with metabolism and proteolytic pathways. This suggests that male neutrophils might be more ready to adapt to a new environment, and female neutrophils might be more effective at responding to pathogens. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.

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During developmental and immune responses, cells move towards or away from some signals. Although much is known about chemoattraction, chemorepulsion (the movement of cells away from a stimulus) remains poorly understood. Proliferating Dictyostelium discoideum cells secrete a chemorepellent protein called AprA. Examining existing knockout strains, we previously identified proteins required for AprA-induced chemorepulsion, and a genetic screen suggested that the enzyme phosphatidylinositol phosphate kinase A (PIPkinA, also known as Pik6) might also be needed for chemorepulsion. Here, we show that cells lacking PIPkinA are not repelled by AprA, and that this phenotype is rescued by expression of PIPkinA. To bias cell movement, AprA inhibits Ras activation at the side of the cell closest to the source of AprA, and we find that PIPkinA is required for AprA to inhibit Ras activation. PIPkinA decreases levels of phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3], and possibly because of these effects, potentiates phagocytosis and inhibits cell proliferation. Cells lacking PIPkinA show normal AprA binding, suggesting that PIPkinA regulates chemorepulsion at a step between the AprA receptor and AprA inhibition of Ras activation.

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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.

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Acute respiratory distress syndrome (ARDS) involves damage to lungs causing an influx of neutrophils from the blood into the lung airspaces, and the neutrophils causing further damage, which attracts more neutrophils in a vicious cycle. There are ∼190,000 cases of ARDS per year in the US, and because of the lack of therapeutics, the mortality rate is ∼40%. Repelling neutrophils out of the lung airspaces, or simply preventing neutrophil entry, is a potential therapeutic. In this minireview, we discuss how our lab noticed that a protein called AprA secreted by growing Dictyostelium cells functions as a repellent for Dictyostelium cells, causing cells to move away from a source of AprA. We then found that AprA has structural similarity to a human secreted protein called dipeptidyl peptidase IV (DPPIV), and that DPPIV is a repellent for human neutrophils. In animal models of ARDS, inhalation of DPPIV or DPPIV mimetics blocks neutrophil influx into the lungs. To move DPPIV or DPPIV mimetics into the clinic, we need to know how this repulsion works to understand possible drug interactions and side effects. Combining biochemistry and genetics in Dictyostelium to elucidate the AprA signal transduction pathway, followed by drug studies in human neutrophils to determine similarities and differences between neutrophil and Dictyostelium chemorepulsion, will hopefully lead to the safe use of DPPIV or DPPIV mimetics in the clinic.

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Crucial to an animal's movement through their environment and to the maintenance of their homeostatic physiology is the integration of sensory information. This is achieved by axons communicating from organs, muscle spindles and skin that connect to the sensory ganglia composing the peripheral nervous system (PNS), enabling organisms to collect an ever-constant flow of sensations and relay it to the spinal cord. The sensory system carries a wide spectrum of sensory modalities - from sharp pain to cool refreshing touch - traveling from the periphery to the spinal cord via the dorsal root ganglia (DRG). This review covers the origins and development of the DRG and the cells that populate it, and focuses on how sensory connectivity to the spinal cord is achieved by the diverse developmental and molecular processes that control axon guidance in the trunk sensory system. We also describe convergences and differences in sensory neuron formation among different vertebrate species to gain insight into underlying developmental mechanisms.

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We previously identified a unique population of interstitial muscle progenitors, marked by expression of the Twist2 transcription factor, which fuses specifically to type IIb/x fast-twitch myofibers. Tw2+ progenitors are distinct from satellite cells, a muscle progenitor that expresses Pax7 and contributes to all myofiber types. Through RNA sequencing and immunofluorescence, we identify the membrane receptor, Nrp1, as a marker of Tw2+ cells but not Pax7+ cells. We also found that Sema3a, a chemorepellent ligand for Nrp1, is expressed by type I and IIa myofibers but not IIb myofibers. Using stripe migration assays, chimeric cell-cell fusion assays, and a Sema3a transgenic mouse model, we identify Sema3a-Nrp1 signaling as a major mechanism for Tw2+ cell fiber-type specificity. Our findings reveal an extracellular signaling mechanism whereby a cell-surface receptor for a chemorepellent confers specificity of intercellular fusion of a specific muscle progenitor with its target tissue.

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Microbial communities are the simplest possible model of multicellular tissues, allowing studies of cell-cell interactions to be done with as few extraneous factors as possible. For instance, the eukaryotic microbe Dictyostelium discoideum proliferates as single cells, and when starved, the cells aggregate together and form structures of ~20,000 cells. The cells use a variety of signals to direct their movement, inform each other of their local cell density and whether they are starving, and organize themselves into groups of ~20,000 cells. Mathematical models and computational approaches have been a key check on, and guide of, the experimental work. In this minireview, I will discuss diffusion calculations and Monte Carlo simulations that were used for Dictyostelium studies that offer general paradigms for several aspects of cell-cell communication. For instance, computational work showed that diffusible secreted cell-density sensing (quorum) factors can diffuse away so quickly from a single cell that the local concentration will not build up to incorrectly cause the cell to sense that it is in the presence of a high density of other cells secreting that signal. In another example, computation correctly predicted a mechanism that allows a group of cells to break up into subgroups. These are thus some examples of the power and necessity of computational work in biology.

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The facial nerve is necessary for our ability to eat, speak, and make facial expressions. Both the axons and cell bodies of the facial nerve undergo a complex embryonic developmental pattern involving migration of the cell bodies caudally and tangentially through rhombomeres, and simultaneously the axons projecting to exit the hindbrain to form the facial nerve. Our goal in this study was to test the functions of the chemorepulsive receptors Robo1 and Robo2 in facial neuron migration and axon projection by analyzing genetically marked motor neurons in double-mutant mouse embryos through the migration time course, E10.0-E13.5. In Robo1/2 double mutants, axon projection and cell body migration errors were more severe than in single mutants. Most axons did not make it to their motor exit point, and instead projected into and longitudinally within the floor plate. Surprisingly, some facial neurons had multiple axons exiting and projecting into the floor plate. At the same time, a subset of mutant facial cell bodies failed to migrate caudally, and instead either streamed dorsally toward the exit point or shifted into the floor plate. We conclude that Robo1 and Robo2 have redundant functions to guide multiple aspects of the complex cell migration of the facial nucleus, as well as regulating axon trajectories and suppressing formation of ectopic axons.

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In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum AprA is a chemorepellent for and inhibits the proliferation of D. discoideum We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells (grlH¯/grlHOE ) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideumIMPORTANCE Little is known about how eukaryotic cells can count themselves and thus regulate the size of a tissue or density of cells. In addition, little is known about how eukaryotic cells can sense a repellant signal and move away from the source of the repellant, for instance, to organize the movement of cells in a developing embryo or to move immune cells out of a tissue. In this study, we found that a eukaryotic microbe uses G protein-coupled receptors to mediate both cell density sensing and chemorepulsion.

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Neural activity plays a key role in pruning aberrant synapses in various neural systems, including the mammalian cortex, where low-frequency (0.01 Hz) calcium oscillations refine topographic maps. However, the activity-dependent molecular mechanisms remain incompletely understood. Activity-dependent pruning also occurs at embryonic Drosophila neuromuscular junctions (NMJs), where low-frequency Ca2+ oscillations are required for synaptic refinement and the response to the muscle-derived chemorepellant Sema2a. We examined embryonic growth cone filopodia in vivo to directly observe their exploration and to analyze the episodic Ca2+ oscillations involved in refinement. Motoneuron filopodia repeatedly contacted off-target muscle fibers over several hours during late embryogenesis, with episodic Ca2+ signals present in both motile filopodia as well as in later-stabilized synaptic boutons. The Ca2+ transients matured over several hours into regular low-frequency (0.03 Hz) oscillations. In vivo imaging of intact embryos of both sexes revealed that the formation of ectopic filopodia is increased in Sema2a heterozygotes. We provide genetic evidence suggesting a complex presynaptic Ca2+-dependent signaling network underlying refinement that involves the phosphatases calcineurin and protein phosphatase-1, as well the serine/threonine kinases CaMKII and PKA. Significantly, this network influenced the neuron's response to the muscle's Sema2a chemorepellant, critical for the removal of off-target contacts.SIGNIFICANCE STATEMENT To address the question of how synaptic connectivity is established during development, we examined the behavior of growth cone filopodia during the exploration of both correct and off-target muscle fibers in Drosophila embryos. We demonstrate that filopodia repeatedly contact off-target muscles over several hours, until they ultimately retract. We show that intracellular signals are observed in motile and stabilized "ectopic" contacts. Several genetic experiments provide insight in the molecular pathway underlying network refinement, which includes oscillatory calcium signals via voltage-gated calcium channels as a key component. Calcium orchestrates the activity of several kinases and phosphatases, which interact in a coordinated fashion to regulate chemorepulsion exerted by the muscle.

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During development, neurons establish inappropriate connections as they seek out their synaptic partners, resulting in supernumerary synapses that must be pruned away. The removal of miswired synapses usually involves electrical activity, often through a Hebbian spike-timing mechanism. A novel form of activity-dependent refinement is used by Drosophila that may be non-Hebbian, and is critical for generating the precise connectivity observed in that system. In Drosophila, motoneurons use both glutamate and the biogenic amine octopamine for neurotransmission, and the muscle fibers receive multiple synaptic inputs. Motoneuron growth cones respond in a time-regulated fashion to multiple chemotropic signals arising from their postsynaptic partners. Central to this mechanism is a very low frequency (<0.03 Hz) oscillation of presynaptic cytoplasmic calcium, that regulates and coordinates the action of multiple downstream effectors involved in the withdrawal from off-target contacts. Low frequency calcium oscillations are widely observed in developing neural circuits in mammals, and have been shown to be critical for normal connectivity in a variety of neural systems. In Drosophila these mechanisms allow the growth cone to sample widely among possible synaptic partners, evaluate opponent chemotropic signals, and withdraw from off-target contacts. It is possible that the underlying molecular mechanisms are conserved widely among invertebrates and vertebrates.

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The removal of miswired synapses is a fundamental prerequisite for normal circuit development, leading to clinical problems when aberrant. However, the underlying activity-dependent molecular mechanisms involved in synaptic pruning remain incompletely resolved. Here the dynamic properties of intracellular calcium oscillations and a role for cAMP signaling during synaptic refinement in intact Drosophila embryos were examined using optogenetic tools. We provide In vivo evidence at the single gene level that the calcium-dependent adenylyl cyclase rutabaga, the phosphodiesterase dunce, the kinase PKA, and Protein Phosphatase 1 (PP1) all operate within a functional signaling pathway to modulate Sema2a-dependent chemorepulsion. It was found that presynaptic cAMP levels were required to be dynamically maintained at an optimal level to suppress connectivity defects. It was also proposed that PP1 may serve as a molecular link between cAMP signaling and CaMKII in the pathway underlying refinement. The results introduced an in vivo model where presynaptic cAMP levels, downstream of electrical activity and calcium influx, act via PKA and PP1 to modulate the neuron's response to chemorepulsion involved in the withdrawal of off-target synaptic contacts. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 39-60, 2017.

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Semaphorins are a large family of proteins characterized by sema domains and play a key role not only in the formation of neural circuits, but in the immune system, angiogenesis, tumor progression, and bone metabolism. To date, 15 semaphorins have been reported to be involved in the formation of the peripheral nervous system (PNS) in higher vertebrates. A number of experiments have revealed their functions in the PNS, where they act mainly as axonal guidance cues (as repellents or attractants). Semaphorins also play an important role in the migration of neurons and formation of sensory-motor connections in the PNS. This review summarizes recent knowledge regarding the functions of higher vertebrate semaphorins in the formation of the PNS.

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Effector functions and cellular properties of natural killer (NK) cells are regulated by cellular and extracellular factors shaped by the microenvironments. NK cells express specific chemokine and non-chemokine receptors to aid preferential migrations or localizations in tissues. Good understanding of how NK-cell migratory properties are regulated in physiological and pathological microenvironments will provide further insights into the development of NK cell-based therapeutic approaches. In contrast to the commonly used conventional in vitro migration assays such as Trans-well assays that measure movements of a population of the migratory cells, microfluidic-based devices support live-cell imaging of cell migrations under a well-defined chemical gradient(s) at microscale. Subsequent analyses at single-cell level provide quantitative measurements of cell-migration parameters such as speed and Chemotactic Index, and permit distinguishing chemotaxis, chemokinesis, and chemo-repulsion. Our recent work established the use of a Y-shaped microfluidic device to study NK cell migrations in vitro. In this chapter, we described the detailed method of acquiring and analyzing NK cell migration in the microfluidic devices.

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Boundary cap cells (BCC) are a transient, neural-crest-derived population found at the motor exit point (MEP) and dorsal root entry zone (DREZ) of the embryonic spinal cord. These cells contribute to the central/peripheral nervous system (CNS/PNS) boundary, and in their absence neurons and glia from the CNS migrate into the PNS. We found Netrin5 (Ntn5), a previously unstudied member of the netrin gene family, to be robustly expressed in BCC. We generated Ntn5 knockout mice and examined neurodevelopmental and BCC-related phenotypes. No abnormalities in cranial nerve guidance, dorsal root organization, or sensory projections were found. However, Ntn5 mutant embryos did have ectopic motor neurons (MNs) that migrated out of the ventral horn and into the motor roots. Previous studies have implicated semaphorin6A (Sema6A) in BCC signaling to plexinA2 (PlxnA2)/neuropilin2 (Nrp2) in MNs in restricting MN cell bodies to the ventral horn, particularly in the caudal spinal cord. In Ntn5 mutants, ectopic MNs are likely to be a different population, as more ectopias were found rostrally. Furthermore, ectopic MNs in Ntn5 mutants were not immunoreactive for NRP2. The netrin receptor deleted in colorectal cancer (DCC) is a potential receptor for NTN5 in MNs, as similar ectopic neurons were found in Dcc mutant mice, but not in mice deficient for other netrin receptors. Thus, Ntn5 is a novel netrin family member that is expressed in BCC, functioning to prevent MN migration out of the CNS.

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Migration and localization of NK cells into peripheral tissues are tightly regulated under normal and pathological conditions. The physiological importance of NK cell-DC crosstalk has been well documented. However, the ways in which DCs regulate the migratory properties of NK cells (such as chemotaxis, chemokinesis, chemo-repulsion) are not fully defined in vitro. Here, we employed a microfluidic platform to examine, at the single-cell level, C57BL/6 NK-cell migrations in a stable chemical gradient. We observed that soluble factors released by the immature and LPS-activated mature DCs induced a high level of chemotactic movement of IL-2-activated NK cells in vitro. We confirmed these findings in a standard trans-well migration assay, and identified CXCR3 as a key receptor on the NK cells that mediated the migration. More interestingly, we revealed a novel function of granulocyte macrophage colony-stimulating factor in repulsing NK-cell migrations. The future uses of such microfluidic device in the systematic evaluations of NK-cell migratory responses in NK cell-DC crosstalk will provide new insights into the development of DC-based NK-cell therapies against tumor and infections.

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Many of the models of neurodevelopmental processes such as cell migration, axon outgrowth, and dendrite arborization involve cell adhesion and chemoattraction as critical physical or mechanical aspects of the mechanism. However, the prevention of adhesion or attraction is under-appreciated as a necessary, active process that balances these forces, insuring that the correct cells are present and adhering in the correct place at the correct time. The phenomenon of not adhering is often viewed as the passive alternative to adhesion, and in some cases this may be true. However, it is becoming increasingly clear that active signaling pathways are involved in preventing adhesion. These provide a balancing force during development that prevents overly exuberant adhesion, which would otherwise disrupt normal cellular and tissue morphogenesis. The strength of chemoattractive signals may be similarly modulated. Recent studies, described here, suggest that Down Syndrome Cell Adhesion Molecule (DSCAM), and closely related proteins such as DSCAML1, may play an important developmental role as such balancers in multiple systems.