Making cups and rings: the 'stalled-wave' model for macropinocytosis.

Macropinocytosis is a broadly conserved endocytic process discovered nearly 100 years ago, yet still poorly understood. It is prominent in cancer cell feeding, immune surveillance, uptake of RNA vaccines and as an invasion route for pathogens. Macropinocytic cells extend large cups or flaps from their plasma membrane to engulf droplets of medium and trap them in micron-sized vesicles. Here they are digested and the products absorbed. A major problem - discussed here - is to understand how cups are shaped and closed. Recently, lattice light-sheet microscopy has given a detailed description of this process in Dictyostelium amoebae, leading to the 'stalled-wave' model for cup formation and closure. This is based on membrane domains of PIP3 and active Ras and Rac that occupy the inner face of macropinocytic cups and are readily visible with suitable reporters. These domains attract activators of dendritic actin polymerization to their periphery, creating a ring of protrusive F-actin around themselves, thus shaping the walls of the cup. As domains grow, they drive a wave of actin polymerization across the plasma membrane that expands the cup. When domains stall, continued actin polymerization under the membrane, combined with increasing membrane tension in the cup, drives closure at lip or base. Modelling supports the feasibility of this scheme. No specialist coat proteins or contractile activities are required to shape and close cups: rings of actin polymerization formed around PIP3 domains that expand and stall seem sufficient. This scheme may be widely applicable and begs many biochemical questions.

Phosphatidylinositol 3,5-bisphosphate facilitates axonal vesicle transport and presynapse assembly.

Neurons relay information via specialized presynaptic compartments for neurotransmission. Unlike conventional organelles, the specialized apparatus characterizing the neuronal presynapse must form de novo. How the components for presynaptic neurotransmission are transported and assembled is poorly understood. Our results show that the rare late endosomal signaling lipid phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] directs the axonal cotransport of synaptic vesicle and active zone proteins in precursor vesicles in human neurons. Precursor vesicles are distinct from conventional secretory organelles, endosomes, and degradative lysosomes and are transported by coincident detection of PI(3,5)P2 and active ARL8 via kinesin KIF1A to the presynaptic compartment. Our findings identify a crucial mechanism that mediates the delivery of synaptic vesicle and active zone proteins to developing synapses.

Investigating Metabolic and Molecular Ecological Evolution of Opportunistic Pulmonary Fungal Coinfections: Protocol for a Laboratory-Based Cross-Sectional Study.

BACKGROUND: Fungal-bacterial cocolonization and coinfections pose an emerging challenge among patients suspected of having pulmonary tuberculosis (PTB); however, the underlying pathogenic mechanisms and microbiome interactions are poorly understood. Understanding how environmental microbes, such as fungi and bacteria, coevolve and develop traits to evade host immune responses and resist treatment is critical to controlling opportunistic pulmonary fungal coinfections. In this project, we propose to study the coexistence of fungal and bacterial microbial communities during chronic pulmonary diseases, with a keen interest in underpinning fungal etiological evolution and the predominating interactions that may exist between fungi and bacteria. OBJECTIVE: This is a protocol for a study aimed at investigating the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections through determining and characterizing the burden, etiological profiles, microbial communities, and interactions established between fungi and bacteria as implicated among patients with presumptive PTB. METHODS: This will be a laboratory-based cross-sectional study, with a sample size of 406 participants. From each participant, 2 sputa samples (one on-spot and one early morning) will be collected. These samples will then be analyzed for both fungal and bacterial etiology using conventional metabolic and molecular (intergenic transcribed spacer and 16S ribosomal DNA-based polymerase chain reaction) approaches. We will also attempt to design a genome-scale metabolic model for pulmonary microbial communities to analyze the composition of the entire microbiome (ie, fungi and bacteria) and investigate host-microbial interactions under different patient conditions. This analysis will be based on the interplays of genes (identified by metagenomics) and inferred from amplicon data and metabolites (identified by metabolomics) by analyzing the full data set and using specific computational tools. We will also collect baseline data, including demographic and clinical history, using a patient-reported questionnaire. Altogether, this approach will contribute to a diagnostic-based observational study. The primary outcome will be the overall fungal and bacterial diagnostic profile of the study participants. Other diagnostic factors associated with the etiological profile, such as incidence and prevalence, will also be analyzed using univariate and multivariate schemes. Odds ratios with 95% CIs will be presented with a statistical significance set at P<.05. RESULTS: The study has been approved by the Mbarara University Research Ethic Committee (MUREC1/7-07/09/20) and the Uganda National Council of Science and Technology (HS1233ES). Following careful scrutiny, the protocol was designed to enable patient enrollment, which began in March 2022 at Mbarara University Teaching Hospital. Data collection is ongoing and is expected to be completed by August 2023, and manuscripts will be submitted for publication thereafter. CONCLUSIONS: Through this protocol, we will explore the metabolic and molecular ecological evolution of opportunistic pulmonary fungal coinfections among patients with presumptive PTB. Establishing key fungal-bacterial cross-kingdom synergistic relationships is crucial for instituting fungal bacterial coinfecting etiology. TRIAL REGISTRATION: ISRCTN Registry ISRCTN33572982; https://tinyurl.com/caa2nw69. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/48014.

A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes.

Phosphoinositide signaling lipids (PIPs) are key regulators of membrane identity and trafficking. Of these, PI(3,5)P2 is one of the least well-understood, despite key roles in many endocytic pathways including phagocytosis and macropinocytosis. PI(3,5)P2 is generated by the phosphoinositide 5-kinase PIKfyve, which is critical for phagosomal digestion and antimicrobial activity. However PI(3,5)P2 dynamics and regulation remain unclear due to lack of reliable reporters. Using the amoeba Dictyostelium discoideum, we identify SnxA as a highly selective PI(3,5)P2-binding protein and characterize its use as a reporter for PI(3,5)P2 in both Dictyostelium and mammalian cells. Using GFP-SnxA, we demonstrate that Dictyostelium phagosomes and macropinosomes accumulate PI(3,5)P2 3 min after engulfment but are then retained differently, indicating pathway-specific regulation. We further find that PIKfyve recruitment and activity are separable and that PIKfyve activation stimulates its own dissociation. SnxA is therefore a new tool for reporting PI(3,5)P2 in live cells that reveals key mechanistic details of the role and regulation of PIKfyve/PI(3,5)P2.

Formation and closure of macropinocytic cups in Dictyostelium.

Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.

Katnip is needed to maintain microtubule function and lysosomal delivery to autophagosomes and phagosomes.

The efficient delivery of lysosomes is essential for many cell functions, such as the degradation of unwanted intracellular components by autophagy and the killing and digestion of extracellular microbes within phagosomes. Using the amoeba Dictyostelium discoideum, we find that cells lacking Katnip (Katanin interacting protein) have a general defect in lysosomal delivery and although they make autophagosomes and phagosomes correctly, cells are then unable to digest them. Katnip is largely unstudied yet highly conserved across evolution. Previously studies found that Katnip mutations in animals cause defects in cilia structure. Here we show that Katnip plays a more general role in maintaining microtubule function. We find that loss of Katnip has no overall effect on microtubule dynamics or organization, but is important for the transport and degradation of endocytic cargos. Strikingly, Katnip mutants become highly sensitive to GFP-tubulin expression, which leads to microtubule tangles, defective anaphase extension, and slow cell growth. Our findings establish a general role for Katnip in regulating microtubule function, beyond the roles previously described in cilia. We speculate this is via a key function in microtubule repair, needed to maintain endosomal trafficking and lysosomal degradation.

The Amoebal Model for Macropinocytosis.

Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.

A bacterial endosymbiont of the fungus Rhizopus microsporus drives phagocyte evasion and opportunistic virulence.

Opportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.

Moving the Research Forward: The Best of British Biology Using the Tractable Model System Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.

Dynamic Rac1 inhibition by CYRI helps cells drink, but stops them from driving.

In this issue, Le et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202012114) describe a new role for the recently discovered protein CYRI in controlling the protrusions that allow cells to engulf extracellular fluid by macropinocytosis. This study helps explain how these structures are disassembled, but also uncovers a new mechanism linking the ability of cells to drink and their capacity for invasive migration.

The great host-pathogen war: U.K. Cellular microbiology meeting 2020.

In 2019 we started a new annual meeting, aimed at bringing together researchers from across the United Kingdom studying cellular microbiology and the cell biology of host-pathogen interactions. In contrast to large glamourous meetings, featuring the great and the good from across the world, we wanted to create a forum for early career researchers to present their work and enjoy lively discussion. In particular, we hope that focussing on making the meeting accessible, affordable, and informal would help integrate and build the U.K. community working on this exciting topic.

Coordinated Ras and Rac Activity Shapes Macropinocytic Cups and Enables Phagocytosis of Geometrically Diverse Bacteria.

Engulfment of extracellular material by phagocytosis or macropinocytosis depends on the ability of cells to generate specialized cup-shaped protrusions. To effectively capture and internalize their targets, these cups are organized into a ring or ruffle of actin-driven protrusion encircling a non-protrusive interior domain. These functional domains depend on the combined activities of multiple Ras and Rho family small GTPases, but how their activities are integrated and differentially regulated over space and time is unknown. Here, we show that the amoeba Dictyostelium discoideum coordinates Ras and Rac activity using the multidomain protein RGBARG (RCC1, RhoGEF, BAR, and RasGAP-containing protein). We find RGBARG uses a tripartite mechanism of Ras, Rac, and phospholipid interactions to localize at the protruding edge and interface with the interior of both macropinocytic and phagocytic cups. There, we propose RGBARG shapes the protrusion by expanding Rac activation at the rim while suppressing expansion of the active Ras interior domain. Consequently, cells lacking RGBARG form enlarged, flat interior domains unable to generate large macropinosomes. During phagocytosis, we find that disruption of RGBARG causes a geometry-specific defect in engulfing rod-shaped bacteria and ellipsoidal beads. This demonstrates the importance of coordinating small GTPase activities during engulfment of more complex shapes and thus the full physiological range of microbes, and how this is achieved in a model professional phagocyte.

The endocytic pathways of Dictyostelium discoideum.

The formation and processing of vesicles from the cell surface serves many important cellular functions ranging from nutrient acquisition to regulating the turnover of membrane components and signalling. In this article, we summarise the endocytic pathways of the social amoeba Dictyostelium from the clathrin-dependent and independent internalisation of surface components to the engulfment of bacteria or fluid by phagocytosis and macropinocytosis respectively. Due to similarities with the professional phagocytes of the mammalian immune system Dictyostelium has been extensively used to investigate the complex remodelling and trafficking events that occur as phagosomes and macropinosomes transit through the cell. Here we discuss what is known about this maturation process in order to kill any potential pathogens and obtain nutrients for growth. Finally, we aim to put these studies in evolutionary context and highlight some of the many questions that remain in our understanding of these complex and important pathways.

The UK cellular microbiology network: Exploring the host-bacterial interface.

The UK Cellular Microbiology Network held its inaugural conference in February 2019. This stimulating day of scientific exchange will be the first of many, its organisers hope.

The origins and evolution of macropinocytosis.

In macropinocytosis, cells take up micrometre-sized droplets of medium into internal vesicles. These vesicles are acidified and fused to lysosomes, their contents digested and useful compounds extracted. Indigestible contents can be exocytosed. Macropinocytosis has been known for approaching 100 years and is described in both metazoa and amoebae, but not in plants or fungi. Its evolutionary origin goes back to at least the common ancestor of the amoebozoa and opisthokonts, with apparent secondary loss from fungi. The primary function of macropinocytosis in amoebae and some cancer cells is feeding, but the conserved processing pathway for macropinosomes, which involves shrinkage and the retrieval of membrane to the cell surface, has been adapted in immune cells for antigen presentation. Macropinocytic cups are large actin-driven processes, closely related to phagocytic cups and pseudopods and appear to be organized around a conserved signalling patch of PIP3, active Ras and active Rac that directs actin polymerization to its periphery. Patches can form spontaneously and must be sustained by excitable kinetics with strong cooperation from the actin cytoskeleton. Growth-factor signalling shares core components with macropinocytosis, based around phosphatidylinositol 3-kinase (PI3-kinase), and we suggest that it evolved to take control of ancient feeding structures through a coupled growth factor receptor. This article is part of the Theo Murphy meeting issue 'Macropinocytosis'.

Gamma secretase orthologs are required for lysosomal activity and autophagic degradation in Dictyostelium discoideum, independent of PSEN (presenilin) proteolytic function.

Mutations in the γ-secretase complex are strongly associated with familial Alzheimer disease. Both proteolytic and non-proteolytic functions for the γ-secretase complex have been previously described in mammalian model organisms, but their relative contributions to disease pathology remain unclear. Here, we dissect the roles of orthologs of the γ-secretase components in the model system Dictyostelium, focusing on endocytosis, lysosomal activity and autophagy. In this model, we show that the orthologs of PSEN (psenA and psenB), Ncstn (nicastrin) and Aph-1 (gamma-secretase subunit Aph-1), are necessary for optimal fluid-phase uptake by macropinocytosis and in multicellular development under basic pH conditions. Disruption of either psenA/B or Aph-1 proteins also leads to disrupted phagosomal proteolysis as well as decreased autophagosomal acidification and autophagic flux. This indicates a general defect in lysosomal trafficking and degradation, which we show leads to the accumulation of ubiquitinated protein aggregates in cells lacking psenA/B and Aph-1 proteins. Importantly, we find that all the endocytic defects observed in Dictyostelium PSEN ortholog mutants can be fully rescued by proteolytically inactive Dictyostelium psenB and human PSEN1 proteins. Our data therefore demonstrates an evolutionarily conserved non-proteolytic role for presenilin, and γ-secretase component orthologs, in maintaining Dictyostelium lysosomal trafficking and autophagy. Abbreviations: Atg8: autophagy protein 8a; Aph-1: gamma-secretase subunit Aph-1; crtA: calreticulin; ER: endoplasmic reticulum; GFP: green fluorescent protein; GSK3B: glycogen synthase kinase 3 beta; Ncstn: nicastrin; PSEN1: presenilin 1; psenA and psenB: Dictyostelium presenilin A and B; TRITC; tetramethylrhodamine isothiocyanate.

PIKfyve/Fab1 is required for efficient V-ATPase and hydrolase delivery to phagosomes, phagosomal killing, and restriction of Legionella infection.

By engulfing potentially harmful microbes, professional phagocytes are continually at risk from intracellular pathogens. To avoid becoming infected, the host must kill pathogens in the phagosome before they can escape or establish a survival niche. Here, we analyse the role of the phosphoinositide (PI) 5-kinase PIKfyve in phagosome maturation and killing, using the amoeba and model phagocyte Dictyostelium discoideum. PIKfyve plays important but poorly understood roles in vesicular trafficking by catalysing formation of the lipids phosphatidylinositol (3,5)-bisphosphate (PI(3,5)2) and phosphatidylinositol-5-phosphate (PI(5)P). Here we show that its activity is essential during early phagosome maturation in Dictyostelium. Disruption of PIKfyve inhibited delivery of both the vacuolar V-ATPase and proteases, dramatically reducing the ability of cells to acidify newly formed phagosomes and digest their contents. Consequently, PIKfyve- cells were unable to generate an effective antimicrobial environment and efficiently kill captured bacteria. Moreover, we demonstrate that cells lacking PIKfyve are more susceptible to infection by the intracellular pathogen Legionella pneumophila. We conclude that PIKfyve-catalysed phosphoinositide production plays a crucial and general role in ensuring early phagosomal maturation, protecting host cells from diverse pathogenic microbes.