Macropinocytosis in physiology, disease and therapy

Macropinocytosis has long been implicated as a mechanism for growing cells to assimilate extracellular nutrients. Studies of murine macrophages and embryonic fibroblasts showed that activation of the growth-associated metabolic regulator mechanistic target of rapamycin complex-1 (mTORC1) in response to growth factors and extracellular amino acids requires internalization of amino acids by macropinocytosis. This led the group to propose that macropinocytosis is necessary for mTORC1-dependent growth of metazoan cells, as both a route for nutrient delivery into lysosomes and a platform for growth factor-dependent signalling to mTORC1 via PI 3-kinase (PI3K) and Akt. A functional actin cytoskeleton is required for macropinocytic cup formation and activation of mTORC1 by growth factors and amino acids. Although activation of Akt by some growth factors occurs independent of the actin cytoskeleton, maximal activation of Akt by stimuli that elicit weak Akt responses requires that cells be capable of forming macropinocytic cups or circular dorsal ruffles. This indicates that growth factors and chemokines which trigger low maximal levels of Akt activity require actin-based macropinocytic cup formation for localized amplification of PI3K-dependent responses. In this way, ruffles and cups could organize signalling by extracellular ligands into stochastic, structure-dependent cascades of chemical reactions that stimulate cell growth or orient cell migration.

Professor Joel Swanson, University of Michigan, USA

Professor Joel Swanson, University of Michigan, USA

Joel Swanson, Professor of Microbiology and Immunology, trained at Rutgers University (BA, Biology), Ohio State University (M.S., Botany), and Princeton University (PhD, Biology), followed by post-doctoral studies at Rockefeller University and Columbia College of Physicians and Surgeons. He served on the faculty at Harvard Medical School and the University of Michigan Medical School. His laboratory has a longstanding interest of heterophagy, which is the cellular ingestion of extracellular particles and solutes by macropinocytosis, phagocytosis and endocytosis, and the subsequent degradation of ingested materials in lysosomes. His approach emphasizes molecular and microscopic studies of the spatial organization of cytoplasm in cells. Recent work has demonstrated roles for macropinocytosis in the regulation of cell growth in macrophages and other cell types.

Like other cells, macrophages respond to growth promoters by ruffling their membrane, which in turn promotes macropinocytosis. However, the surveillance role of macrophages requires ongoing sampling of their environment, which is performed by constitutive macropinocytosis. The latter occurs continuously, even in the presence of growth promoters, generating smaller macropinosomes. Constitutive macropinocytosis requires extracellular calcium and is mediated by calcium-sensing receptors. Both inducible and constitutive macropinocytosis depend on actin and on phosphatidylinositol 3,4,5-trisphosphate (PIP3), but only the inducible form is sensitive to amiloride analogues. Constitutive macropinocytosis is active in anti-inflammatory macrophages, but negligible in pro-inflammatory macrophages, such as those polarized by treatment with GM-CSF, LPS and/or IFNγ. Inflammatory macrophages have reduced levels of PIP3, which accounts at least in part for their inability to perform constitutive macropinocytosis.

Oxidized LDL (oxLDL), the source of the vascular plaque responsible for heart attacks and strokes, is taken up by macrophages via scavenger receptors. Unlike conventional receptor-mediated endocytosis, oxLDL uptake requires actin polymerization. The evidence indicates that constitutive macropinocytosis is largely responsible for oxLDL internalization by non-inflammatory macrophages, in a scavenger receptor-dependent manner. The group has termed this process 'receptor-assisted macropinocytosis'.

Professor Sergio Grinstein, The Hospital for Sick Children, Canada

Professor Sergio Grinstein, The Hospital for Sick Children, Canada

Dr Sergio Grinstein completed his PhD in 1976 at the Centro de Investigacion y Estudios Avanzados, in Mexico City.  He then spent two years as a post-doctoral fellow at the Hospital For Sick Children in Toronto, followed by a year in the Department of Biochemistry at the Federal Institute of Technology in Zurich.  He is currently working at the Hospital For Sick Children in Toronto and has been Professor of Biochemistry at the University of Toronto since 1988. 

Dr Grinstein is interested in the cell physiology and biophysics of innate immunity, particularly phagocytosis and host-pathogen interactions.

Macropinocytosis is an unusual form of endocytosis that has fascinated cell biologists for decades. Recent advances in live cell imaging have allowed the study of this process in more detail. Professor Donaldson's lab has focused on how plasma membrane is shaped to form the macropinosome, how the macropinosome is brought into the cell interior and how membrane is sorted out from the maturing macropinosome. They find a requirement for microtubules and dynein for macropinocytosis that is driven by Ras. In addition, Arf6 and its effector, the JIP3 microtubule motor scaffold protein are also involved in macropinosome formation and transport through the lamellar region. Once past the lamellar actin/myosin arc, actin is shed from the macropinosome and it begins to undergo cargo sorting for membrane recycling back to the cell surface. Cargo entering into the macropinosome are mostly clathrin-independent cargo proteins and during sorting, select proteins (CD98 and CD147) leave the macropinosome for recycling. Retromer components are recruited onto the macropinosome during this sorting out of cargo. Similar sorting of cargo is observed in other cells but the large size of the macropinosome enables us to track cargo sorting in space and time.

Professor Julie Donaldson, National Institutes of Health, USA

Professor Julie Donaldson, National Institutes of Health, USA

Julie Donaldson received her PhD from the University of Maryland in 1988 and did her postdoctoral research training at NIH before starting her own lab in 1995 at the National Heart, Lung and Blood Institute at the NIH. She is currently a Senior Investigator in the Cell Biology and Physiology Center. Her research program focuses on understanding the mechanism and physiological function of clathrin-independent endocytosis (CIE) and the subsequent routing and fate of endocytosed membrane proteins. CIE is the entry mechanism for many cell surface proteins including cell adhesion molecules, ion channels, nutrient transporters and microbial pathogens. Macropinocytosis is another form of CIE that provides a unique platform for following membrane dynamics.

Macropinocytosis requires the co-ordinated regulation of both the cytoskeleton to form the cup-shaped protrusions, as well as the vesicular trafficking machinery to process them after internalisation. The group is trying to understand both these processes using Dictyostelium amoeba, which use macropinocytosis for feeding.  

Macropinocytic cups are able to self-assemble stochastically, without any external physical scaffolds or localised signals. This requires cells to generate a ring of protrusion, driven by localised actin polymerisation, that encircles a static membrane domain that defines the cup interior. Dr King will discuss a new mechanism by which the Ras and Rac small GTPases differentially regulated across the protrusive rim and cup interior, in order to spatially modulate the cytoskeleton and generate protrusions that efficiently engulf fluid. 

After engulfment cells must rapidly recycle any surface components before they are degraded, whilst orchestrating a complex series of trafficking steps that ensure the efficient processing of the internalised material. Dr King will therefore also discuss how the group is using the amoeba model to dissect these processes to provide a mechanistic understanding of macropinosome maturation.

Dr Jason King, University of Sheffield, UK

Dr Jason King, University of Sheffield, UK

Jason King is currently a Royal Society University Research Fellow at the University of Sheffield, having moved there in 2013 to start his independent group. Their main interests are understanding how cells both form, and regulate the maturation of macropinosomes and phagosomes. Prior to this Dr King worked as a postdoc in the laboratory of Robert Insall at the Beatson Institute for Cancer Research in Glasgow, UK, studying the cytoskeleton and cell migration, before developing interests in autophagy and trafficking. This followed on from his PhD work in the lab of Adrian Harwood at Cardiff University, studying how lithium treatment (still widely used to tread bipolar disorder) affects cellular signalling and in particular phosphinositide signalling. The group’s current work on macropinocytosis brings all these interests together in order to determine how both the cytoskeleton and vesicular trafficking are coordinated to facilitate fluid uptake and processing.

The migration of immune cells is guided by specific chemical signals, such as chemokine gradients. Nevertheless, their trajectories can also be diverted by physical cues and obstacles imposed by the cellular environment, such as topography, rigidity, adhesion, or hydraulic resistance. On the example of hydraulic resistance, it was shown that neutrophil preferentially follow paths of least resistance, a phenomenon referred to as barotaxis. The group here combined quantitative imaging and physical modelling to show that barotaxis results from a force imbalance at the scale of the cell, which is amplified by the acto-myosin intrinsic polarization capacity. Strikingly, macropinocytosis specifically confers to immature dendritic cells a unique capacity to overcome this physical bias, which enhances their space exploration capacity in vivo and promotes their tissue-patrolling function. Conversely, mature dendritic cells, which down-regulate macropinocytosis, are sensitive to hydraulic resistance. Theoretical modelling predicts that being barotactic helps them avoid dead-ends while migrating to lymph nodes to initiate the adaptive immune response. The group concludes that the physical properties of the microenvironment of moving cells can introduce biases in their migratory behaviours but that specific active mechanisms such as macropinocytosis have emerged to diminish the influence of these biases, allowing motile cells to reach their final destination and efficiently fulfill their functions.

Professor Ana-Maria Lennon-Duménil, Institut Curie, France

Professor Ana-Maria Lennon-Duménil, Institut Curie, France

Ana-Maria Lennon-Duménil is a cell biologist, team-leader in the ‘Immunity and Cancer’ Department at the Curie Institute in Paris since 2004.  Her career has focused on combining cell biology with other disciplines such as genetics (PhD), biochemistry and organic chemistry (post-doctoral stage), and biophysics (group-leader) to address key questions concerning immune cells. She has successfully coordinated for eight years a consortium named ‘Dendritic Cell Migration in Confined Environments’ that includes Matthieu Piel (Institut Curie) and Raphaël Voituriez (UPMC), and generated more than 20 publications, some in very prestigious journals such as Cell, Science, Nature Cell Biol., Nature Cell Physics, Nature Comm. Her work as an independent investigator has opened a yet unexplored research line on the coordination of cell function and cell migration.

Macropinocytosis probably originated as a feeding mechanism in unicellular organisms and is used in this way by amoebae today. Dictyostelium amoebae form several large macropinosomes per minute in rich medium and can take up a significant fraction of their entire volume of fluid per hour, with macropinocytosis accounting for more than 90% of this. Macropinocytic cups are organized around intense patches of activated Ras and PIP3 in the plasma membrane, whose size is regulated by the RasGAP, NF1. These patches recruit the SCAR/WAVE complex to their periphery, thus initiating a hollow ring of actin polymerization to form the walls of the macropinocytic cup. PIP3 is essential for efficient macropinocytosis in Dictyostelium, as in mammals, and we find that the downstream protein kinase, PKB, which is recruited to the plasma membrane by PIP3, is similarly important. The group has identified a number of targets for PKB, among which regulators of small G-proteins are prominent. Professor Kay will also describe how active-Ras/PIP3 patches are unexpectedly sensitive to disruption of cytoskeletal organization.

Professor Rob Kay, MRC Laboratory for Molecular Biology, UK

Professor Rob Kay, MRC Laboratory for Molecular Biology, UK

Rob Kay’s group is interested in finding fundamental mechanisms of macropinocytosis using Dictyostelium amoebae and mammalian cells as models. Rob is a group leader at the MRC Laboratory of Molecular Biology in Cambridge. He studied Biochemistry at UCL and was an EMBO fellow in Freiburg before working at the Mill Hill laboratory of the Imperial Cancer Research Fund with John Cairns and Julian Gross. He is a member of EMBO and former Howard Hughes International Fellow.

Enhanced ruffling and macropinocytosis on pathogen-activated macrophages help to drive innate immune and inflammatory responses. These membrane domains function in tissue surveillance, antigen capture, receptor activation and as signaling domains for pro-or anti-inflammatory transcriptional programs. With the high temporal and spatial resolution of lattice light sheet microscopy for live cell imaging the group has been able to resolve new features of dorsal ruffles and redefine their dynamic closure and transition to macropinosomes. These features depict how different sized ruffles and macropinosomes can be made. Moreover, the group has defined roles for sequential Rab GTPases in the formation of LPS-induced, enlarged ruffles and macropinosomes. Toll-like receptor (TLR) signalling also occurs on early macropinosomes where the group has shown a TLR4/coreceptor/GEF/Rab complex recruits PI3K for Akt/mTORC1 signalling. This signalling pathway biases cytokine outputs and is a key mechanism for driving an M2-like macrophage program for resolution of TLR-induced inflammation. The highly dynamic, rapidly transitioning domains on early macropinosomes support TLR signalling and protein sorting that determines the outcome of inflammation.

Professor Jenny Stow, University of Queensland, Australia

Professor Jenny Stow, University of Queensland, Australia

Professor Jennifer Stow heads the Protein Trafficking and Inflammation research group at The University of Queensland’s Institute for Molecular Bioscience (IMB) in Brisbane, Australia. Her tenure at IMB has included appointments as IMB Deputy Director (Research), Head of the Division of Molecular Cell Biology, NHMRC Principal Research Fellow and Wellcome Trust Senior Medical Research Fellow. Earlier appointments include as Assistant Professor at Massachusetts General Hospital/Harvard Medical School and postdoctoral fellow in the department of Cell Biology, Yale University School of Medicine, following award of her PhD from Monash University, Australia.  The Stow group use multiscale microscopy and live cell imaging, coupled with biochemistry, to define key macrophage molecules, receptor signalling and trafficking pathways that regulate inflammation.