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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 aa NanoLuc fragment inserted into a target protein and detects surface exposure through high-affinity complementation. We tracked insertion of CLAG3, a malaria parasite protein linked to nutrient uptake, throughout the P. falciparum cycle in human erythrocytes. Our approach also 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 surface exposure by up to 50-fold, revealing that both size and charge of the extracellular epitope influence membrane insertion. Insertion of parasite proteins at the host cell surface and antigen accessibility is regulated by multiple factors, enabling intracellular parasite survival and immune evasion under a broad range of conditions.

Artemisinin and its semi-synthetic derivatives (ART) are fast acting, potent antimalarials; however, their use in malaria treatment is frequently confounded by recrudescences from bloodstream Plasmodium parasites that enter into and later reactivate from a dormant persister state. Here we provide evidence that the mitochondria of dihydroartemisinin (DHA)-exposed persisters are dramatically altered and enlarged relative to the mitochondria of young, actively replicating ring forms. Restructured mitochondrial-nuclear associations and an altered metabolic state are consistent with stress from reactive oxygen species. New contacts between the mitochondria and nuclei may support communication pathways of mitochondrial retrograde signaling, resulting in transcriptional changes in the nucleus as a survival response. Further characterization of the organelle communication and metabolic dependencies of persisters may suggest strategies to combat recrudescences of malaria after treatment.IMPORTANCEThe major first-line treatment for malaria, especially the deadliest form caused by Plasmodium falciparum, is combination therapy with an artemisinin-based drug (ART) plus a partner drug to assure complete cure. Without an effective partner drug, ART administration alone can fail because of the ability of small populations of blood-stage malaria parasites to enter into a dormant state and survive repeated treatments for a week or more. Understanding the nature of parasites in dormancy (persisters), and their ability to wake and reestablish actively-propagating parasitemias (recrudesce) after ART exposure, may suggest strategies to improve treatment outcomes and counter the threats posed by parasites that develop resistance to partner drugs. Here we show that persisters have dramatically altered mitochondria and mitochondrial-nuclear interactions associated with features of metabolic quiescence. Restructured associations between the mitochondria and nuclei may support signaling pathways that enable the ART survival responses of dormancy.

Macropinocytosis and phagocytosis are the processes by which eukaryotic cells use their plasma membrane to engulf liquid or a large particle and give rise to an internal compartment called the macropinosomes or phagosome, respectively. Dictyostelium discoideum provides a powerful system to understand the molecular mechanism of these two fundamental cellular processes that impact human health and disease. Recent developments in fluorescence microscopy allow direct visualization of intracellular signaling events with high temporal and spatial resolution. Here, we describe methods to visualize temporospatial activation or localization of key signaling components that are crucial for macropinocytosis and phagocytosis using confocal fluorescence microscopy.

Cytoskeletal integrity is essential for neuronal complexity and functionality. Certain inherited neurological diseases are associated with mutated genes that directly or indirectly compromise cytoskeletal stability. While the large size and complexity of the neurons grown in culture poses certain challenges for imaging, live-cell imaging is an excellent approach to determine the morphological consequences of such mutants. This protocol details the use of spinning disk confocal microscopy and image analysis tools to evaluate branching and neurite length of healthy iPSC-derived glutamatergic neurons that express specific fluorescent proteins. The protocols can be adapted to neuronal cell lines of choice by the investigator.

Fluorescence lifetime imaging (FLIM) measures the characteristic time that a molecule remains in an excited state prior to emitting a photon and returning to the ground state. It is a state-of-the-art and noninvasive technique that has the potential to obtain signature physiological information during malaria blood-stage infection. The use of autofluorescence signals from intrinsic fluorophores obviates the need to tag the cells with synthetic molecules or to modify their gene expression. Furthermore, it permits time-lapse interrogation of the changes that occur from invasion to the point when the parasite takes over the host for its own survival mechanisms, as well as changes in the health of the parasite due to extrinsically applied metabolic disruptors. In this chapter, we present a protocol to investigate the autofluorescence lifetime signals of both normal red blood cells (RBC) and P. falciparum-infected RBCs. The data shared with this protocol reveals that there is a significant overall increase in autofluorescence lifetime in infected erythrocytes compared to the healthy uninfected ones. We include a metabolic experiment that confirms that the signals obtained from this imaging technique are key metabolites in energetics of the parasites. Furthermore, facilitating these protocols makes it possible to identify infected RBC based on FLIM signals alone, which presents a huge potential for the study of energetic effects of antimalarials and fast, noninvasive diagnosing.

Laser scanning confocal microscopy is the workhorse epifluorescence imaging technique used in laboratories worldwide to acquire three-dimensional images of both fixed and live specimens with fine, high-contrast optical sections to discern details that cannot be afforded by standard widefield microscopy. This basic protocol steps the user through a typical three-color imaging experiment using a Zeiss LSM 880 confocal microscope for the example. The extensive Notes section attempts to generalize the method so that concepts and considerations can be applied to other laser scanning confocal systems.

The serine/threonine kinase AKT is a major effector during phosphatidylinositol 3-kinase (PI3K)-driven cell signal transduction in response to extracellular stimuli. AKT activation mechanisms have been extensively studied, however, the mechanism underlying target of rapamycin complex 2 (mTORC2) phosphorylation of AKT at Ser473 in the cellular endomembrane system remains to be elucidated. Here, we demonstrate that endocytosis is required for AKT activation through phosphorylation at Ser473 via mTORC2 using platelet-derived growth factor-stimulated U87MG glioma cells. mTORC2 components are localized to early endosomes during growth factor activation, and the association of mTORC2 with early endosomes is responsible for the local activation of AKT, which is critical for specific signal transduction through glycogen synthase kinase-3 beta and forkhead box O1/O3 phosphorylation. Furthermore, endosomal phosphoinositide, represented by PtdIns(3,4)P2, provides a binding platform for mTORC2 to phosphorylate AKT Ser473 in endosomes through mammalian Sty1/Spc1-interacting protein (mSIN), a pleckstrin homology domain-containing protein, and is dispensable for AKT phosphorylation at Thr308. This PtdIns(3,4)P2-mediated endosomal AKT activation provides a means to integrate PI3K activated by diverse stimuli to mTORC2 assembly. These early endosomal events induced by endocytosis, together with the previously identified AKT activation by PtdIns(3,4,5)P3, contribute to the strengthening of the transduction of AKT signaling through phosphoinositide.

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.

Laser scanning confocal microscopy is the workhorse epifluorescence imaging technique used in laboratories worldwide to acquire three-dimensional images of both fixed and live specimens with fine, high-contrast optical sections to discern details that cannot be afforded by standard widefield microscopy. This basic protocol steps the user through a typical three-color imaging experiment using a Zeiss LSM 880 confocal microscope for the example. The extensive Notes section attempts to generalize the method so that concepts and considerations can be applied to other laser scanning confocal systems.

Cytoskeletal integrity is essential for neuronal complexity and functionality. Certain inherited neurological diseases are associated with mutated genes that directly or indirectly compromise cytoskeletal stability. While the large size and complexity of the neurons grown in culture poses certain challenges for imaging, live-cell imaging is an excellent approach to determine the morphological consequences of such mutants. This protocol details the use of spinning disk confocal microscopy and image analysis tools to evaluate branching and neurite length of healthy iPSC-derived glutamatergic neurons that express specific fluorescent proteins. The protocols can be adapted to neuronal cell lines of choice by the investigator.

Fluorescence lifetime imaging (FLIM) measures the characteristic time that a molecule remains in an excited state prior to emitting a photon and returning to the ground state. It is a state-of-the-art and noninvasive technique that has the potential to obtain signature physiological information during malaria blood-stage infection. The use of autofluorescence signals from intrinsic fluorophores obviates the need to tag the cells with synthetic molecules or to modify their gene expression. Furthermore, it permits time-lapse interrogation of the changes that occur from invasion to the point when the parasite takes over the host for its own survival mechanisms, as well as changes in the health of the parasite due to extrinsically applied metabolic disruptors. In this chapter, we present a protocol to investigate the autofluorescence lifetime signals of both normal red blood cells (RBC) and P. falciparum-infected RBCs. The data shared with this protocol reveals that there is a significant overall increase in autofluorescence lifetime in infected erythrocytes compared to the healthy uninfected ones. We include a metabolic experiment that confirms that the signals obtained from this imaging technique are key metabolites in energetics of the parasites. Furthermore, facilitating these protocols makes it possible to identify infected RBC based on FLIM signals alone, which presents a huge potential for the study of energetic effects of antimalarials and fast, noninvasive diagnosing.

Macropinocytosis and phagocytosis are the processes by which eukaryotic cells use their plasma membrane to engulf liquid or a large particle and give rise to an internal compartment called the macropinosomes or phagosome, respectively. Dictyostelium discoideum provides a powerful system to understand the molecular mechanism of these two fundamental cellular processes that impact human health and disease. Recent developments in fluorescence microscopy allow direct visualization of intracellular signaling events with high temporal and spatial resolution. Here, we describe methods to visualize temporospatial activation or localization of key signaling components that are crucial for macropinocytosis and phagocytosis using confocal fluorescence microscopy.

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., 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.

BackgroundCellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors.Dictyosteliumgrow as individual cells in nutrient-rich sources, but, as nutrients become depleted, they initiate a multi-cell developmental program that is dependent upon a cell-density threshold. We hypothesized that novel secreted proteins may serve as density-sensing factors to promote multi-cell developmental fate decisions at a specific cell-density threshold, and useDictyosteliumin the identification of such a factor.ResultsWe show that multi-cell developmental aggregation inDictyosteliumis lost upon minimal (2-fold) reduction in local cell density. Remarkably, developmental aggregation response at non-permissive cell densities is rescued by addition of conditioned media from high-density, developmentally competent cells. Using rescued aggregation of low-density cells as an assay, we purified a single, 150-kDa extra-cellular protein with density aggregation activity. MS/MS peptide sequence analysis identified the gene sequence, and cells that overexpress the full-length protein accumulate higher levels of a development promoting factor (DPF) activity than parental cells, allowing cells to aggregate at lower cell densities; cells deficient for thisDPFgene lack density-dependent developmental aggregation activity and require higher cell density for cell aggregation compared to WT. Density aggregation activity co-purifies with tagged versions of DPF and tag-affinity-purified DPF possesses density aggregation activity. In mixed development with WT, cells that overexpress DPF preferentially localize at centers for multi-cell aggregation and define cell-fate choice during cytodifferentiation. Finally, we show that DPF is synthesized as a larger precursor, single-pass transmembrane protein, with the p150 fragment released by proteolytic cleavage and ectodomain shedding. The TM/cytoplasmic domain of DPF possesses cell-autonomous activity for cell-substratum adhesion and for cellular growth.ConclusionsWe have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds forDictyosteliumdevelopment and to facilitate cell-cell communication and multi-cell formation. Regions of high DPF expression are enriched at centers for cell-cell signal-response, multi-cell formation, and cell-fate determination. Additionally, DPF has separate cell-autonomous functions for regulation of cellular adhesion and growth.

Significance We have shown in this study that the malaria parasite can rapidly evolve to adapt for loss of an “essential” kinase, PfCDPK1. PfCDPK1 could not be disrupted in the wild-type parasite. However, we were able to disrupt CDPK1 in the transgenic parasites adapted for reduced kinase activity of mutant PfCDPK1. Strategic disruption of PfCDPK1 highlights the importance of understanding the compensatory mechanisms, especially for targets belonging to multigene families. Our study unequivocally demonstrates that PfCDPK1 is critical for mosquito infections and its disruption leads to defective gametogenesis. Our study also suggests involvement of CDPK1 in regulation of sexual stage-specific genes during the asexual proliferation. Targeting PfCDPK1 may be a good strategy for developing transmission-blocking drugs., Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

The malarial parasite within the erythrocyte is surrounded by two membranes. Plasmodium translocon of exported proteins (PTEX) in the parasite vacuolar membrane critically transports proteins from the parasite to the erythrocytic cytosol and membrane to create protein infrastructure important for virulence. The components of PTEX are stored within the dense granule, which is secreted from the parasite during invasion. We now describe a protein, RON3, from another invasion organelle, the rhoptry, that is also secreted during invasion. We find that RON3 is required for the protein transport function of the PTEX and for glucose transport from the RBC cytoplasm to the parasite, a function thought to be mediated by PTEX component EXP2., The survival of Plasmodium spp. within the host red blood cell (RBC) depends on the function of a membrane protein complex, termed the Plasmodium translocon of exported proteins (PTEX), that exports certain parasite proteins, collectively referred to as the exportome, across the parasitophorous vacuolar membrane (PVM) that encases the parasite in the host RBC cytoplasm. The core of PTEX consists of three proteins: EXP2, PTEX150, and the HSP101 ATPase; of these three proteins, only EXP2 is a membrane protein. Studying the PTEX-dependent transport of members of the exportome, we discovered that exported proteins, such as ring-infected erythrocyte surface antigen (RESA), failed to be transported in parasites in which the parasite rhoptry protein RON3 was conditionally disrupted. RON3-deficient parasites also failed to develop beyond the ring stage, and glucose uptake was significantly decreased. These findings provide evidence that RON3 influences two translocation functions, namely, transport of the parasite exportome through PTEX and the transport of glucose from the RBC cytoplasm to the parasitophorous vacuolar (PV) space where it can enter the parasite via the hexose transporter (HT) in the parasite plasma membrane.

Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected on the basis of their ability to bind, gather, and present antigen to T follicular helper (Tfh) cells. Here, we show that human GC B cells have intrinsically higher-affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared with naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to an- tigen affinity– and Tfh cell–dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structures was dictated by the intrinsic antigen affinity thresholds of GC B cells. Low-affinity antigens triggered continuous engagement and disengagement of membrane-associated antigens, whereas high-affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.

A common feature of many chronic human infectious diseases, including malaria, includes the large expansion of a population termed atypical memory B cells (MBCs) in peripheral blood, which are phenotypically and transcriptionally distinct from naïve B cells and classical MBCs. One shared distinguished phenotype among these is their high expression of a variety of inhibitory receptors including FcγRIIB. Despite the potential importance of atypical MBCs, if or how they function in chronic infectious diseases has yet to be clearly known. We reported earlier that in malaria atypical MBCs, in contrast to naïve B cells and classical MBCs, are refractory to activation by soluble antigen. Thus, atypical MBCs have been described as hypo-responsive to antigen-driven B cell activation and believed to be ‘exhausted’ and to contribute to the poor acquisition of immunity in chronic infectious diseases. However, we show the evidence here that atypical MBCs respond robustly to membrane-associated antigen by signaling through the B cell antigen receptor, gathering and internalizing antigen for presentation and initiating differentiation toward plasma cells. We discovered that on antigen-containing membranes the immune synapses of atypical MBCs actively exclude the inhibitory receptor FcγRIIB thus allowing BCR signaling. The high expression of inhibitory receptors by atypical MBCs may serve to restrict B cell responses to antigens presented on cell surfaces, avoiding the potential deleterious effects of chronic soluble antigen exposure.

Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/μm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action., eLife digest Malaria is a deadly disease caused by a parasite transmitted by mosquitoes. The parasite infects red blood cells, causing fever with flu-like symptoms. In some people, particularly pregnant women and children, the disease may be very serious and even lead to death. An effective malaria vaccine is urgently needed because malaria parasites are developing resistance to current drugs. People living in areas where malaria is common develop specific proteins called antibodies that protect them from malaria. Learning more about how the antibodies achieve this, could help to develop better vaccines. Scientists already know some antibodies bind to the malaria parasites and prevent them from entering red blood cells. Some vaccines have been based on these antibodies. Other antibodies bind to infected cells flagging them for destruction by cells of the immune system. Immune cells called natural killer cells can eliminate viruses or cancer cells this way, but it was not clear if they could also eliminate malaria parasite-infected red blood cells. Now, Arora et al. show that natural killer cells can selectively destroy malaria-infected red blood cells flagged with antibodies from people who live in areas where malaria is common. In laboratory experiments, natural killer cells from US volunteers, who were never exposed to malaria, did not kill normal or malaria-infected red blood cells. Adding antibodies collected from malaria-resistant volunteers from Africa allowed these natural killer cells from unexposed people to selectively seek out and destroy malaria-infected cells and leave uninfected red blood cells intact. Arora et al. also found that the antibodies from the malaria-resistant volunteers bound to parasite proteins on the surface of infected blood cells. The experiments suggest that vaccines designed to stimulate the production of antibodies to malaria proteins that are displayed on infected red blood cells, could destroy the parasite in infected people and help prevent disease and save lives.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite–host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

For decades, various Förster resonance energy transfer (FRET) techniques have been developed to measure the distance between interacting molecules. FRET imaging by the sensitized acceptor emission method has been widely applied to study the dynamical association between two molecules at a nanometer scale in live cells. Here, we provide a detailed protocol for FRET imaging by sensitized emission using a confocal laser scanning microscope to analyze the interaction of the B cell receptor (BCR) with the Lyn-enriched lipid microdomain on the plasma membrane of live cells upon antigen binding, one of the earliest signaling events in BCR-mediated B cell activation.

For decades, various Forster resonance energy transfer (FRET) techniques have been developed to measure the distance between interacting molecules. FRET imaging by the sensitized acceptor emission method has been widely applied to study the dynamical association between two molecules at a nanometer scale in live cells. Here, we provide a detailed protocol for FRET imaging by sensitized emission using a confocal laser scanning microscope to analyze the interaction of the B cell receptor (BCR) with the Lyn-enriched lipid microdomain on the plasma membrane of live cells upon antigen binding, one of the earliest signaling events in BCR-mediated B cell activation.

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.

B cell activation is regulated through the interplay of the BCR with the inhibitory coreceptor FcγRIIB and the activating coreceptor CD19. Recent studies suggest that Ag-driven BCR microclusters are efficiently converted to a signaling active state on colocalization with CD19 microclusters. Using total internal reflection fluorescence microscopy–based, high-resolution, high-speed live-cell and molecule imaging approaches, we show that when co-ligated to the BCR, the FcγRIIB can inhibit B cell activation by blocking the colocalization of BCR and CD19 microclusters within the B cell immunological synapse. Remarkably, this inhibitory function of FcγRIIB is dependent not on its well-characterized ITIM-containing cytoplasmic domain, but its transmembrane domain. Indeed, human primary B cells from systemic lupus erythematosus patients homozygous for gene encoding the loss-of-function transmembrane domain mutant FcγRIIB-I232T fail to block the synaptic colocalization of the BCR with CD19, leading to dysregulated recruitment of downstream signaling molecule p-PI3K to membrane proximal signalosome. This inhibitory function of FcγRIIB in impairing the spatial-temporal colocalization of BCR and CD19 microclusters in the B cell immunological synapse may help explain the hyper-reactive features of systemic lupus erythematosus patient B cells in reported studies. These observations may also provide new targets for therapies for systemic autoimmune disease.

Evolutionarily conserved arrestin-like proteins are key components of the cAR1-mediated ERK2 activation that controls cAMP cell–cell signaling during Dictyostelium aggregation. They are also involved in ligand-induced cAR1 internalization, which is required for the switch of cAMP receptors during multicellular development., 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.

In resting and activated B-cells, IgM and IgG B-cell receptors (BCRs) reside in highly heterogeneous protein islands that vary in size and number of BCRs but intrinsically maintain a high frequency of BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation., B-cell activation is initiated by the binding of antigen to the B-cell receptor (BCR). Here we used dSTORM superresolution imaging to characterize the nanoscale spatial organization of immunoglobulin M (IgM) and IgG BCRs on the surfaces of resting and antigen-­activated human peripheral blood B-cells. We provide insights into both the fundamental process of antigen-driven BCR clustering and differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B-cells to antigen. We provide evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in size and number of BCR single-molecule localizations, both resting and activated B-cells intrinsically maintain a high ­frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands after antigen activation. Small, dense BCR clusters likely formed via protein–protein interactions are present on the surface of resting cells, and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein–lipid interactions.

SummaryActivation of G protein-coupled receptors (GPCRs) leads to the dissociation of heterotrimeric G-proteins into Gα and Gβγ subunits, which go on to regulate various effectors involved in a panoply of cellular responses. During chemotaxis, Gβγ subunits regulate actin assembly and migration, but the protein(s) linking Gβγ to the actin cytoskeleton remains unknown. Here, we identified a Gβγ effector, ElmoE in Dictyostelium, and demonstrated that it is required for GPCR-mediated chemotaxis. Remarkably, ElmoE associates with Gβγ 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, Gβγ, Elmo/Dock, Rac, and Arp2/3, spatially guides the growth of dendritic actin networks in pseudopods of eukaryotic cells during chemotaxis.

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.

αLβ2 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.

Immunometabolism is an emerging field that studies the activation-induced reprogramming required to alter immune cell function and meet new energetic demands. In-depth investigations of the metabolism-related cellular changes necessitate high-resolution visualization of mitochondria. However, current strategies for imaging mitochondria are optimized for model cells with high cytoplasm-to-nucleus ratios and are not readily adaptable for many immune cells. Here, we devised a multicolor STED microscopy method to assess B lymphocyte mitochondria in resting and activated states. By staining specimens with TOM-20 and MitoTracker dyes, as well as DAPI and a viability dye, our strategy allowed us to assess the morphology and volumes of mitochondria while efficiently excluding dead cells. Our analyses showed that stimulation of B lymphocytes via toll-like receptor 9 (TLR9) and/or B cell receptor (BCR) led to notable mass and volume increases in individual mitochondria within 24 hours. Furthermore, TLR9-stimulated cells demonstrated more tubular and connected mitochondrial networks consistent with their higher metabolic demands. In addition, collecting images of the same specimens side-by-side on AiryScan and standard confocal laser microscopes showed that STED microscopy performed significantly better in resolving changes in 3D mitochondrial morphology. However, none of the light microscopy imaging strategies could resolve activation-induced structural alterations in cristae or mitochondrial matrix. In conclusion, our novel STED-based multicolor imaging approach offers a powerful super-resolution tool to study immunometabolism and mitochondrial reorganization in lymphocytes at the whole cell level.

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.

We have identified two LIM domain proteins, LimF and ChLim, from Dictyostelium that interact with each other and with the small, Rab5-related, Rab21 GTPase to collectively regulate phagocytosis. To investigate in vivo functions, we generated cell lines that lack or overexpress LimF and ChLim and strains that express activating or inhibiting variants of Rab21. Overexpression of LimF, loss of ChLim, or expression of constitutively active Rab21 increases the rate of phagocytosis above that of wild type. Conversely, loss of LimF, overexpression of ChLim, or expression of a dominant-negative Rab21 inhibits phagocytosis. Our studies using cells carrying multiple mutations in these genes further indicate that ChLim antagonizes the activating function of Rab21-GTP during phagocytosis; in turn, LimF is required for Rab21-GTP function. Finally, we demonstrate that ChLim and LimF localize to the phagocytic cup and phago-lysosomal vesicles. We suggest that LimF, ChLim, and activated Rab21-GTP participate as a novel signaling complex that regulates phagocytic activity.

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.

High-resolution microscopy techniques have advanced our understanding of lymphocyte biology. An emerging focus in the field of immunometabolism on activation-induced metabolic reprogramming and its effects on immune cell function will require in-depth analyses of mitochondria. However, even with newer imaging technologies, obtaining super-resolution images of lymphocyte mitochondria remains challenging due to their small cytoplasmic sizes. Here, we compared standard confocal laser microscopy with three commonly used super-resolution methods to analyze activation-induced mitochondrial changes in B and T cells. Naïve B cells were stimulated in vitro via Toll-like receptor 9 and/or the B cell receptor, and naïve T cells were differentiated in vitro into activated or T regulatory phenotypes. The cells were imaged using standard laser confocal microscopy, AiryScan, stimulated emission depletion (STED) microscopy, and computerized tomography electron microscopy (TEM). Mitochondrial morphology was best resolved by staining both the mitochondrial membrane and matrix simultaneously. In preliminary analyses, we observed activation-induced changes in the distribution of lymphocyte mitochondria. Of the imaging methods used, only TEM allowed for 3D reconstructions of the mitochondria’s internal structure, which revealed swelling of the inner matrix and significant loss of cristae in B cells, but not in T cells. In conclusion, the super-resolution light microscopy techniques we developed appear to be powerful tools for immunometabolism studies of lymphocyte mitochondria, having demonstrated clear visualization even at the molecular level, while TEM answered key questions about intra-mitochondrial morphology.

Neutrophils are the first cells recruited to the site of an infected sand fly bite and interact with Leishmania parasites. Here we investigate the effect of sand fly salivary proteins on neutrophil recruitment. We purified bone marrow neutrophils from C57BL/6 mice or from peripheral blood of healthy human or dog donors. Chemotaxis driven by sand fly saliva of vectors of visceral (Lutzomyia longipalpis) or cutaneous (Phlebotomus duboscqi) disease was measured by modified Boyden chamber assay and by imaging with the EZTAXIScan assay. Neutrophils migrated towards saliva glands of the vectors P. duboscqi and L. longipalpis, in a dose-dependent manner. Proteinase K treatment of saliva completely abrogated neutrophil recruitment, suggesting a protein as the chemotactic factor. To unearth the identity of the chemotactic protein, we generated plasmids coding for the 20 most abundant salivary proteins from P. duboscqi and injected them in the ears of C57BL/6 mice. Neutrophil recruitment was analyzed by flow cytometry staining. We identified 2 different salivary protein-coding plasmids with neutrophil recruitment activity. We produced these 2 recombinant proteins in the HEK293 cell line system and we observed that a family of 40kDa salivary proteins acts as chemokines for neutrophils both in our in vitro and in vivo assays. Moreover, the coinjection of Leishmania major parasites with the recombinant proteins in the ear of C57BL/6 mice exacerbated the infection. We plan to test if neutralization of the salivary chemotactic protein can alter Leishmania infection. We put forward the hypothesis that blockage of this protein will disrupt neutrophil migration early in the Leishmania infection possibly altering the disease outcome.

Eukaryotic cells, whether free-living, single-celled microbes or components of complex metazoa, can sense environmental cues through specialized seven-transmembrane (7-TM) receptors (also called heptahelical or G-protein-coupled receptors). 7-TM receptors detect "inputs" such as light, peptide hormones, neurotransmitters, pheromones, odorants, morphogens and chemoattractants, linking extracellular stimuli to intracellular signaling networks via heterotrimeric G proteins. Recently, this obligatory paradigm has been challenged. A growing body of evidence indicates that 7-TM receptors can also transmit extracellular signals through mechanisms that function independently of G-protein coupling. This review discusses pathways and protein interactions for 7-TM receptors signaling "at zero G" in Dictyostelium and mammalian cells.