Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

Progressive heterogeneity of enlarged and irregularly shaped apicoplasts in P. falciparum persister blood stages after drug treatment.

Morphological modifications and shifts in organelle relationships are hallmarks of dormancy in eukaryotic cells. Communications between altered mitochondria and nuclei are associated with metabolic quiescence of cancer cells that can survive chemotherapy. In plants, changes in the pathways between nuclei, mitochondria, and chloroplasts are associated with cold stress and bud dormancy. Plasmodium falciparum parasites, the deadliest agent of malaria in humans, contain a chloroplast-like organelle (apicoplast) derived from an ancient photosynthetic symbiont. Antimalarial treatments can fail because a small fraction of the blood stage parasites enter dormancy and recrudesce after drug exposure. Altered mitochondrial-nuclear interactions in these persisters have been described for P. falciparum, but interactions of the apicoplast remained to be characterized. In the present study, we examined the apicoplasts of dormant persisters obtained after exposure to dihydroartemisinin (a first-line antimalarial drug) followed by sorbitol treatment, or after exposure to sorbitol treatment alone. As previously observed, the mitochondrion of persisters was consistently enlarged and in close association with the nucleus. In contrast, the apicoplast varied from compact and oblate, like those of active ring stage parasites, to enlarged and irregularly shaped. Enlarged apicoplasts became more prevalent later in dormancy, but regular size apicoplasts subsequently predominated when actively replicating parasites recrudesced. All three organelles, nucleus, mitochondrion, and apicoplast, became closer during dormancy. Understanding their relationships in erythrocytic-stage persisters may lead to new strategies to prevent recrudescences and protect the future of malaria chemotherapy.

Mosaic quadrivalent influenza vaccine single nanoparticle characterization.

Recent work by our laboratory and others indicates that co-display of multiple antigens on protein-based nanoparticles may be key to induce cross-reactive antibodies that provide broad protection against disease. To reach the ultimate goal of a universal vaccine for seasonal influenza, a mosaic influenza nanoparticle vaccine (FluMos-v1) was developed for clinical trial (NCT04896086). FluMos-v1 is unique in that it is designed to co-display four recently circulating haemagglutinin (HA) strains; however, current vaccine analysis techniques are limited to nanoparticle population analysis, thus, are unable to determine the valency of an individual nanoparticle. For the first time, we demonstrate by total internal reflection fluorescence microscopy and supportive physical-chemical methods that the co-display of four antigens is indeed achieved in single nanoparticles. Additionally, we have determined percentages of multivalent (mosaic) nanoparticles with four, three, or two HA proteins. The integrated imaging and physicochemical methods we have developed for single nanoparticle multivalency will serve to further understand immunogenicity data from our current FluMos-v1 clinical trial.

SARS-CoV-2 infection of human lung epithelial cells induces TMPRSS-mediated acute fibrin deposition.

Severe COVID-associated lung injury is a major confounding factor of hospitalizations and death with no effective treatments. Here, we describe a non-classical fibrin clotting mechanism mediated by SARS-CoV-2 infected primary lung but not other susceptible epithelial cells. This infection-induced fibrin formation is observed in all variants of SARS-CoV-2 infections, and requires thrombin but is independent of tissue factor and other classical plasma coagulation factors. While prothrombin and fibrinogen levels are elevated in acute COVID BALF samples, fibrin clotting occurs only with the presence of viral infected but not uninfected lung epithelial cells. We suggest a viral-induced coagulation mechanism, in which prothrombin is activated by infection-induced transmembrane serine proteases, such as ST14 and TMPRSS11D, on NHBE cells. Our finding reveals the inefficiency of current plasma targeted anticoagulation therapy and suggests the need to develop a viral-induced ARDS animal model for treating respiratory airways with thrombin inhibitors.

B cell responses to membrane-presented antigens require the function of the mechanosensitive cation channel Piezo1.

The demand for a vaccine for coronavirus disease 2019 (COVID-19) highlighted gaps in our understanding of the requirements for B cell responses to antigens, particularly to membrane-presented antigens, as occurs in vivo. We found that human B cell responses to membrane-presented antigens required the function of Piezo1, a plasma membrane mechanosensitive cation channel. Simply making contact with a glass probe induced calcium (Ca2+) fluxes in B cells that were blocked by the Piezo1 inhibitor GsMTx4. When placed on glass surfaces, the plasma membrane tension of B cells increased, which stimulated Ca2+ influx and spreading of B cells over the glass surface, which was blocked by the Piezo1 inhibitor OB-1. B cell responses to membrane-presented antigens but not to soluble antigens were inhibited both by Piezo1 inhibitors and by siRNA-mediated knockdown of Piezo1. Thus, the activation of Piezo1 defines an essential event in B cell activation to membrane-presented antigens that may be exploited to improve the efficacy of vaccines.

FcγRI FG-loop functions as a pH sensitive switch for IgG binding and release.

Understanding the molecular mechanism underlying the hierarchic binding between FcγRs and IgG antibodies is critical for therapeutic antibody engineering and FcγR functions. The recent determination of crystal structures of FcγRI-Fc complexes, however, resulted in two controversial mechanisms for the high affinity receptor binding to IgG. Here, we describe high resolution structures of a bovine FG-loop variant of FcγRI in complex with the Fc fragment of IgG1 crystallized in three different conditions at neutral pH, confirming the characteristic FG loop-Fc interaction is critical to the high affinity immunoglobulin binding. We showed that the FcγRI D2-domain FG-loop functioned as a pH-sensing switch for IgG binding. Further live cell imaging of FcγRI-mediated internalization of immune complexes showed a pH sensitive temporal-spatial antibody-antigen uptake and release. Taken together, we demonstrate that the structures of FcγRI-Fc crystallized at neutral and acidic pH, respectively, represent the high and low affinity binding states of the receptor for IgG uptake and release. These results support a role for FcγRI in antigen delivery, highlight the importance of Fc glycan in antibody binding to the high affinity receptor and provide new insights to future antibody engineering.

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.

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.

Endosomal mTORC2 Is Required for Phosphoinositide-Dependent AKT Activation in Platelet-Derived Growth Factor-Stimulated Glioma Cells.

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.

Restructured Mitochondrial-Nuclear Interaction in Plasmodium falciparum Dormancy and Persister Survival after Artemisinin Exposure.

Artemisinin and its semisynthetic 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. IMPORTANCE The 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.

A sand fly salivary protein acts as a neutrophil chemoattractant.

Apart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

General Considerations for Acquiring a Three-Color Image by Laser Scanning Confocal Microscopy.

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.

Visualizing Key Signaling Components of Macropinocytosis and Phagocytosis Using Confocal Microscopy in the Model Organism Dictyostelium discoideum.

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.

Studying Neuronal Biology Using Spinning Disc Confocal 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 as a Noninvasive Tool to Study Plasmodium Falciparum Metabolism.

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.

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.

Expression of inhibitory receptors by B cells in chronic human infectious diseases restricts responses to membrane-associated antigens.

Chronic human infectious diseases, including malaria, are associated with a large expansion of a phenotypically and transcriptionally distinct subpopulation of B cells distinguished by their high expression of a variety of inhibitory receptors including FcγRIIB. Because these B cells, termed atypical memory B cells (MBCs), are unable to respond to soluble antigens, it was suggested that they contributed to the poor acquisition of immunity in chronic infections. Here, we show that the high expression of FcγRIIB restricts atypical MBC responses to membrane-associated antigens that function to actively exclude FcγRIIB from the B cell immune synapse and include the co-receptor CD19, allowing B cell antigen receptor signaling and differentiation toward plasma cells. Thus, chronic infectious diseases result in the expansion of B cells that robustly respond to antigens that associate with cell surfaces, such as antigens in immune complexes, but are unable to respond to fully soluble antigens, such as self-antigens.

DPF is a cell-density sensing factor, with cell-autonomous and non-autonomous functions during Dictyostelium growth and development.

BACKGROUND: Cellular functions can be regulated by cell-cell interactions that are influenced by extra-cellular, density-dependent signaling factors. Dictyostelium grow 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 use Dictyostelium in the identification of such a factor. RESULTS: We show that multi-cell developmental aggregation in Dictyostelium is 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 this DPF gene 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. CONCLUSIONS: We have purified a novel secreted protein, DPF, that acts as a density-sensing factor for development and functions to define local collective thresholds for Dictyostelium development 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.