LYST deficiency impairs autophagic lysosome reformation in neurons and alters lysosome number and size.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder caused by biallelic mutations in the lysosomal trafficking regulator (LYST) gene. Even though enlarged lysosomes and/or lysosome-related organelles (LROs) are the typical cellular hallmarks of CHS, they have not been investigated in human neuronal models. Moreover, how and why the loss of LYST function causes a lysosome phenotype in cells has not been elucidated. We report that the LYST-deficient human neuronal model exhibits lysosome depletion accompanied by hyperelongated tubules extruding from enlarged autolysosomes. These results have also been recapitulated in neurons differentiated from CHS patients' induced pluripotent stem cells (iPSCs), validating our model system. We propose that LYST ensures the correct fission/scission of the autolysosome tubules during autophagic lysosome reformation (ALR), a crucial process to restore the number of free lysosomes after autophagy. We further demonstrate that LYST is recruited to the lysosome membrane, likely to facilitate the fission of autolysosome tubules. Together, our results highlight the key role of LYST in maintaining lysosomal homeostasis following autophagy and suggest that ALR dysregulation is likely associated with the neurodegenerative CHS phenotype.

Increased Mitochondrial Biogenesis and Reactive Oxygen Species Production Accompany Prolonged CD4+ T Cell Activation.

Activation of CD4+ T cells to proliferate drives cells toward aerobic glycolysis for energy production while using mitochondria primarily for macromolecular synthesis. In addition, the mitochondria of activated T cells increase production of reactive oxygen species, providing an important second messenger for intracellular signaling pathways. To better understand the critical changes in mitochondria that accompany prolonged T cell activation, we carried out an extensive analysis of mitochondrial remodeling using a combination of conventional strategies and a novel high-resolution imaging method. We show that for 4 d following activation, mouse CD4+ T cells sustained their commitment to glycolysis facilitated by increased glucose uptake through increased expression of GLUT transporters. Despite their limited contribution to energy production, mitochondria were active and showed increased reactive oxygen species production. Moreover, prolonged activation of CD4+ T cells led to increases in mitochondrial content and volume, in the number of mitochondria per cell and in mitochondrial biogenesis. Thus, during prolonged activation, CD4+ T cells continue to obtain energy predominantly from glycolysis but also undergo extensive mitochondrial remodeling, resulting in increased mitochondrial activity.

Phosphorylation of chemoattractant receptors regulates chemotaxis, actin reorganization and signal relay.

Migratory cells, including mammalian leukocytes and Dictyostelium, use G-protein-coupled receptor (GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G-protein pathways, but activating other pathways. However, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production and cytoskeletal actin response. Phosphorylation defects thus promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of the function of GPCR phosphorylation, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils and other migratory cells.

Cutting edge: NKG2D-dependent cytotoxicity is controlled by ligand distribution in the target cell membrane.

Although the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution on APC and target cells. Detergent-resistant membrane domains, into which GPI-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation, we fused the extracellular domain of ULBP1 to the transmembrane domain of CD45. Introduction of this transmembrane domain eliminated the association of ULBP1 with the detergent-resistant membrane fraction and caused a significant reduction of cytotoxicity and degranulation by NK cells. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. These results show that the partitioning of receptor ligands in discrete membrane domains of target cells is an important determinant of NK cell activation.

Distinct external signals trigger sequential release of apical organelles during erythrocyte invasion by malaria parasites.

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.

A vesicle surface tyrosine kinase regulates phagosome maturation.

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.

Tethering of intercellular adhesion molecule on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization.

Alpha(L)beta(2) integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion but also polarization of cytolytic granules in NK cells. In this study, we tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adaptor molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, although NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM, promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes.

Kinetic Tracking of Plasmodium falciparum Antigens on Infected Erythrocytes with a Novel Reporter of Protein Insertion and Surface Exposure.

Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11-amino acid (aa) HiBit fragment of NanoLuc inserted into a target protein and detects surface exposure through high-affinity complementation to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein's trafficking and insertion at the host erythrocyte membrane. The RISE assay will enable study of surface antigens from divergent intracellular pathogens. IMPORTANCE Malaria parasites invade and replicate within red blood cells of their human or animal hosts to avoid immune detection. At the same time, these parasites insert their own proteins into the host membrane to scavenge plasma nutrients, facilitate immune evasion, and perform other essential activities. As there is broad interest in developing vaccines and antimalarial therapies against these surface-exposed antigens, robust methods are needed to examine how and when parasite proteins insert at the host membrane. We therefore developed and used Reporter of Insertion and Surface Exposure (RISE) to track parasite antigen exposure. Using RISE, we followed the time course of membrane insertion for CLAG3, a conserved protein linked to a nutrient uptake channel on infected erythrocytes. We found that CLAG3 insertion occurs at specific parasite stages and that this insertion is required for the formation of the nutrient uptake channel. We also varied the size and charge of the extracellular domain to define constraints on protein insertion at the host membrane. Single-cell imaging revealed that some cells continued to export CLAG3 even with large extracellular loops, suggesting sophisticated strategies used by malaria parasites to control their interactions with host plasma.

Live-Cell FRET Reveals that Malaria Nutrient Channel Proteins CLAG3 and RhopH2 Remain Associated throughout Their Tortuous Trafficking.

Malaria parasites increase their host erythrocyte's permeability to various nutrients, fueling intracellular pathogen development and replication. The plasmodial surface anion channel (PSAC) mediates this uptake and is linked to the parasite-encoded RhopH complex, consisting of CLAG3, RhopH2, and RhopH3. While interactions between these subunits are well established, it is not clear whether they remain associated from their synthesis in developing merozoites through erythrocyte invasion and trafficking to the host membrane. Here, we explored protein-protein interactions between RhopH subunits using live-cell imaging and Förster resonance energy transfer (FRET) experiments. Using the green fluorescent protein (GFP) derivatives mCerulean and mVenus, we generated single- and double-tagged parasite lines for fluorescence measurements. While CLAG3-mCerulean served as an efficient FRET donor for RhopH2-mVenus within rhoptry organelles, mCerulean targeted to this organelle via a short signal sequence produced negligible FRET. Upon merozoite egress and reinvasion, these tagged RhopH subunits were deposited into the new host cell's parasitophorous vacuole; these proteins were then exported and trafficked to the erythrocyte membrane, where CLAG3 and RhopH2 remained fully associated. Fluorescence intensity measurements identified stoichiometric increases in exported RhopH protein when erythrocytes are infected with two parasites; whole-cell patch-clamp revealed a concomitant increase in PSAC functional copy number and a dose effect for RhopH contribution to ion and nutrient permeability. These studies establish live-cell FRET imaging in human malaria parasites, reveal that RhopH subunits traffic to their host membrane destination without dissociation, and suggest quantitative contribution to PSAC formation.IMPORTANCE Malaria parasites grow within circulating red blood cells and uptake nutrients through a pore on their host membrane. Here, we used gene editing to tag CLAG3 and RhopH2, two proteins linked to the nutrient pore, with fluorescent markers and tracked these proteins in living infected cells. After their synthesis in mature parasites, imaging showed that both proteins are packaged into membrane-bound rhoptries. When parasites ruptured their host cells and invaded new red blood cells, these proteins were detected within a vacuole around the parasite before they migrated and inserted in the surface membrane of the host cell. Using simultaneous labeling of CLAG3 and RhopH2, we determined that these proteins interact tightly during migration and after surface membrane insertion. Red blood cells infected with two parasites had twice the protein at their surface and a parallel increase in the number of nutrient pores. Our work suggests that these proteins directly facilitate parasite nutrient uptake from human plasma.

How human leukocytes track down and destroy pathogens: lessons learned from the model organism Dictyostelium discoideum.

Human leukocytes, including macrophages and neutrophils, are phagocytic immune cells that capture and engulf pathogens and subsequently destroy them in intracellular vesicles. To accomplish this vital task, these leukocytes utilize two basic cell behaviors-chemotaxis for chasing down infectious pathogens and phagocytosis for destroying them. The molecular mechanisms controlling these behaviors are not well understood for immune cells. Interestingly, a soil amoeba, Dictyostelium discoideum, uses these same behaviors to pursue and injest its bacterial food source and to organize its multi-cellular development. Consequently, studies of this model system have provided and will continue to provide us with mechanistic insights into the chemotaxis and phagocytosis of immune cells. Here, we review recent research in these areas that have been conducted in the Chemotaxis Signal Section of NIAID's Laboratory of Immunogenetics.

Nonadaptive regulation of ERK2 in Dictyostelium: implications for mechanisms of cAMP relay.

It is assumed that ERK2 in Dictyostelium is subject to adaptive regulation in response to constant extracellular ligand stimulation. We now show, to the contrary, that ERK2 remains active under continuous stimulation, differing from most ligand-activated pathways in chemotactically competent Dictyostelium and other cells. We show that the upstream phosphorylation pathway, responsible for ERK2 activation, transiently responds to receptor stimulation, whereas ERK2 dephosphorylation (deactivation) is inhibited by continuous stimulation. We argue that the net result of these two regulatory actions is a persistently active ERK2 pathway when the extracellular ligand (i.e., cAMP) concentration is held constant and that oscillatory production/destruction of secreted cAMP in chemotaxing cells accounts for the observed oscillatory activity of ERK2. We also show that pathways controlling seven-transmembrane receptor (7-TMR) ERK2 activation/deactivation function independently of G proteins and ligand-induced production of intracellular cAMP and the consequent activation of PKA. Finally, we propose that this regulation enables ERK2 to function both in an oscillatory manner, critical for chemotaxis, and in a persistent manner, necessary for gene expression, as secreted ligand concentration increases during later development. This work redefines mechanisms of ERK2 regulation by 7-TMR signaling in Dictyostelium and establishes new implications for control of signal relay during chemotaxis.

Imaging Protein-Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells.

This updated unit compares three methods to acquire Förster Resonance Energy Transfer (FRET) data in living cells using a confocal microscope: Acceptor photobleaching, Acceptor-sensitized emission FRET, and Donor fluorescence lifetime imaging. Detailed protocols for live cell husbandry, image acquisition, and data analysis are provided. In addition to providing instructions for manufacturer's analysis tool sets, we provide an easy-to-use, MATLAB-based code to calculate FRET efficiency from data obtained using the Acceptor photobleaching or Acceptor-sensitized emission method, which can be freely downloaded. © 2018 by John Wiley & Sons, Inc.

Galpha-mediated inhibition of developmental signal response.

BACKGROUND: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (desensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function/gain-of-function studies that the novel heterotrimeric Galpha9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for the cAMP signal response. RESULTS: galpha9 null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type cells. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of galpha9 null cells. Cells expressing constitutively activated Galpha9 are defective in cAMP signaling center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of the cAMP signal response. Finally, we demonstrate that galpha9 null cells, which have been codeveloped with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non-autonomously direct wild-type cells to adopt a galpha9 null-like phenotype. CONCLUSIONS: We suggest that Galpha9 functions in an inhibitory-feedback pathway that regulates cAMP signaling center formation and propagation. Galpha9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor desensitization.

The Plasmodium falciparum rhoptry protein RhopH3 plays essential roles in host cell invasion and nutrient uptake.

Merozoites of the protozoan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invade erythrocytes. Invasion involves discharge of rhoptries, specialized secretory organelles. Once intracellular, parasites induce increased nutrient uptake by generating new permeability pathways (NPP) including a Plasmodium surface anion channel (PSAC). RhopH1/Clag3, one member of the three-protein RhopH complex, is important for PSAC/NPP activity. However, the roles of the other members of the RhopH complex in PSAC/NPP establishment are unknown and it is unclear whether any of the RhopH proteins play a role in invasion. Here we demonstrate that RhopH3, the smallest component of the complex, is essential for parasite survival. Conditional truncation of RhopH3 substantially reduces invasive capacity. Those mutant parasites that do invade are defective in nutrient import and die. Our results identify a dual role for RhopH3 that links erythrocyte invasion to formation of the PSAC/NPP essential for parasite survival within host erythrocytes.

Using quantitative fluorescence microscopy and FRET imaging to measure spatiotemporal signaling events in single living cells.

The mechanisms that mediate how migratory eukaryotic cells amplify a shallow, extracellular chemoattractant gradient into a steep intracellular gradient of signaling components to guide chemotaxis remains unknown. To unravel these mechanisms, it is essential to quantitatively measure the spatiotemporal patterns of chemoattractant gradients, the dynamic movement of intracellular signaling pathway molecules, and the localized activation of these molecules in single living cells. Recent developments in live-cell fluorescence microscopy have permitted direct visualization and quantitative measurement of signal transduction events with high temporal and spatial resolution. Here, we outline fluorescence imaging methods to simultaneously visualize and quantitatively measure spatiotemporal changes in chemoattractant concentration by using the fluorescent tracer dye Alexa 594. Next, we provide a method to correlate the dynamic changes in ligand to the spatiotemporal changes in the second messenger phosphatidylinositol 3,4,5-triphosphate (PIP3) along the inner surface of the plasma membrane in live cells. Finally, we describe a fluorescence resonance energy transfer (FRET) method to determine the extent of heterotrimeric G protein activation in single living cells in response to various chemoattractant fields.

Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum.

Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein-coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB(-)C(-)) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB(-)C(-) cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain-containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB(-)C(-) cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.

A Gßγ effector, ElmoE, transduces GPCR signaling to the actin network during chemotaxis.

Activation of G protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Gα and Gßγ subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gßγ subunits regulate actin assembly and migration, but the protein(s) linking Gßγ to the actin cytoskeleton remains unknown. Here, we identified a Gßγ effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE associates with Gßγ and Dock-like proteins to activate the small GTPase Rac, in a GPCR-dependent manner, and also associates with Arp2/3 complex and F-actin. Thus, ElmoE serves as a link between chemoattractant GPCRs, G-proteins and the actin cytoskeleton. The pathway, consisting of GPCR, Gßγ, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis.

Coupling mechanism of a GPCR and a heterotrimeric G protein during chemoattractant gradient sensing in Dictyostelium.

The coupling of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) with G proteins is fundamental for GPCR signaling; however, the mechanism of coupling is still debated. Moreover, how the proposed mechanisms affect the dynamics of downstream signaling remains unclear. Here, through experiments involving fluorescence recovery after photobleaching and single-molecule imaging, we directly measured the mobilities of cyclic adenosine monophosphate (cAMP) receptor 1 (cAR1), a chemoattractant receptor, and a G protein ßγ subunit in live cells. We found that cAR1 diffused more slowly in the plasma membrane than did Gßγ. Upon binding of ligand to the receptor, the mobility of cAR1 was unchanged, whereas the speed of a fraction of the faster-moving Gßγ subunits decreased. Our measurements showed that cAR1 was relatively immobile and Gßγ diffused freely, suggesting that chemoattractant-bound cAR1 transiently interacted with G proteins. Using models of possible coupling mechanisms, we computed the temporal kinetics of G protein activation. Our fluorescence resonance energy transfer imaging data showed that fully activated cAR1 induced the sustained dissociation of G protein α and ßγ subunits, which indicated that ligand-bound cAR1 activated G proteins continuously. Finally, simulations indicated that a high-affinity coupling of ligand-bound receptors and G proteins was essential for cAR1 to translate extracellular gradient signals into directional cellular responses. We suggest that chemoattractant receptors use a ligand-induced coupling rather than a precoupled mechanism to control the activation of G proteins during chemotaxis.

Monitoring dynamic GPCR signaling events using fluorescence microscopy, FRET imaging, and single-molecule imaging.

How a eukaryotic cell translates a small concentration difference of a chemoattractant across the length of its surface into highly polarized intracellular responses is a fundamental question in chemotaxis. Chemoattractants are detected by G-protein-coupled receptors (GPCRs). Binding of chemoattractants to GPCRs induces the dissociation of heterotrimeric G-proteins into G alpha and G betagamma subunits, which in turn, activate downstream signaling networks. To fully understand the molecular mechanisms of chemotaxis, it is essential to quantitatively measure the dynamic changes of chemoattractant concentrations around cells, activation of heterotrimeric G-proteins, and the mobility of GPCR and G-protein subunits in the cell membrane. Here, we outline fluorescence imaging methods including Förster resonance energy transfer (FRET) imaging and a single-molecule analysis that allow us to measure the dynamic properties of GPCR signaling in single live cells.