Macropinocytosis in physiology, disease and therapy

09:05-09:30 Macropinocytosis and cellular growth control

Professor Joel Swanson, University of Michigan, USA

Abstract

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.

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09:30-09:45 Discussion

09:45-10:15 Constitutive vs inducible macropinocytosis in macrophages

Professor Sergio Grinstein, The Hospital for Sick Children, Canada

Abstract

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'.

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10:15-10:30 Discussion

10:30-11:00 Coffee

11:00-11:30 Macropinosome formation, maturation and membrane recycling

Professor Julie Donaldson, National Institutes of Health, USA

Abstract

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.

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11:30-11:45 Discussion

11:45-12:15 Learning how to form and process macropinosomes with Dictyostelium amoeba

Dr Jason King, University of Sheffield, UK

Abstract

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.

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12:15-12:30 Discussion

13:30-14:00 Unexpected roles for macropinocytosis in cell migration

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

Abstract

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.

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14:00-14:15 Discussion

14:15-14:45 Macropinocytosis: Ras and PIP3 driven endocytosis

Professor Rob Kay, MRC Laboratory for Molecular Biology, UK

Abstract

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.

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14:45-15:00 Discussion

15:00-15:30 Tea

15:30-16:00 Macropinosomes as launch-pads for inflammation in macrophages

Professor Jenny Stow, University of Queensland, Australia

Abstract

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.

16:00-16:15 Discussion

16:15-18:30 Poster session

09:00-09:30 How mammalian cells regulate the use of proteins as nutrients

Dr Wilhelm Palm, German Cancer Research Center, Germany

Abstract

The cells of mammalian organisms are surrounded by diverse nutrients, including low-molecular weight nutrients such as glucose and amino acids as well as various macromolecules. Cells can import free amino acids through cell surface transporters or recover amino acids through macropinocytosis and lysosomal degradation of extracellular proteins. While it was assumed that mammalian cells rely on uptake of free amino acids, the group demonstrates that macropinocytosis and lysosomal catabolism of extracellular proteins can support cell survival and proliferation in amino acid-deprived environments. Nutrient uptake is regulated by growth factors and their effectors, the Ras and PI3-kinase signalling pathways. Consistently, oncogenic mutations in Ras or PI3-kinase, which promote macropinocytosis, allow cancer cells to proliferate by exploiting extracellular proteins as nutrients. However, concerted inputs from growth factor signalling and protein catabolism also activate the nutrient-sensing kinase mTORC1, which blocks lysosomal degradation of ingested protein. Inhibiting mTORC1 results in increased catabolism of macropinocytosed proteins and enhances cell proliferation during nutrient-depleted conditions in vitro and within vascularly compromised tumours in vivo. This suggests that in response to Ras and PI3-kinase signalling, macropinocytosis dominates as a nutrient acquisition strategy when a decline in intracellular amino acid levels leads to inactivation of mTORC1. Together, these findings identify signalling pathways that regulate the usage of macropinocytosis of proteins as an amino acid source.

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09:30-09:45 Discussion

09:45-10:35 Short talks

10:35-11:00 Coffee

11:00-11:30 Macropinocytosis through the lens of Ebolavirus

Professor Robert Davey, Texas Biomedical Research Institute, USA

Abstract

Viruses offer advantages over conventional cell biology probes being discreet particles made of a few well defined proteins. They are easily labelled with fluorescent tags and are distinct by electron microscopy. Furthermore, once inside the cell, virus gene expression is readily detected by antibody staining or PCR. Like other viruses, Ebola virus is an obligate parasite, relying on host proteins for infection, from the point of cell entry, through virus replication and finally egress from the cell. The group is focused on identifying host factors that control early steps of Ebola virus entry into cells. Earlier, the group was the first to demonstrate that Ebola virus is internalized into cells by a macropinocytosis-like mechanism. More recently, the group has developed high throughput screening systems, based on virus infection, to identify small molecules and siRNA-suppressed host factors that inhibit virus infection by blocking macropinocytosis. Using this approach the group has identified a novel class of small molecules that appear to potently and specifically inhibit Ebola virus infection by interfering with macropinocytosis while not affecting other endocytic pathways. The group has also identified host proteins involved in the autophagy pathway that have prominent roles early in virus entry that are also important in early macropinosome formation. Professor Davey will discuss the latest understanding of Ebola virus uptake into cells and compare and contrast virus uptake to general macropinocytosis mechanism.

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11:30-11:45 Discussion

11:45-12:35 Short talks

13:30-14:00 Regulation of macropinocytosis in Ras-driven tumours

Dr Cosimo Commisso, NCI Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, USA

14:00-14:15 Discussion

14:15-14:45 Regulation and consequences of non-canonical autophagy resembling LC3-Associated Phagocytosis or LAP

Dr Michael Overholtzer, Memorial Sloan Kettering Cancer Center, USA

Abstract

Autophagy proteins were discovered by their regulation of macroautophagy but are now known to have parallel functions in related processes that also involve LC3 lipidation. Notably, LC3-Associated Phagocytosis or LAP is a parallel function of core autophagy proteins that involves lipidation of LC3 onto phagosomes and other endocytic membranes including macropinosomes. Dr Overholtzer's group has found that LAP is induced by changes to the osmotic environment of macropinosomes or endolysosomes. Here Dr Overholtzer will discuss new data uncovering regulation of LAP and the consequences of this process that differ from macroautophagy. The group finds that LAP induction leads to selective turnover of endolysosomal membrane and the ion channel TRPML1.

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14:45-15:00 Discussion

15:00-15:30 Tea

15:30-15:45 Strategies to inhibit the intracellular target Ras with potent antibody mimetic proteins

Dr Ralph Minter, Medimmune, UK

Abstract

Antibodies, and antibody-like mimetic proteins, are known to be excellent therapeutic molecules for addressing extracellular human targets. However, less is known about their potential as inhibitors of intracellular targets, which make up two-thirds of the expressed human genome. Mutations in the intracellular target Ras are strong oncogenic drivers of many cancers but the target Ras is still not addressed by any current therapies. Dr Minter describes an antibody mimetic, DARPin K27, which blocks Ras by inhibiting nucleotide exchange of GDP to GTP. Intracellular expression of K27 significantly reduces the amount of active Ras, inhibits downstream signalling, in particular the levels of phosphorylated ERK and AKT, and slows the growth in soft agar of HCT116 cells. K27 is the most potent non-covalent inhibitor of nucleotide exchange reported, showing consistent effects across wild type and oncogenic mutant forms of Ras. In addition the group has isolated a second DARPin K55, which is selective for the active, GTP form of Ras and inhibits signalling by directly blocking interactions with downstream effector molecules such as Raf. The isolation and characterisation of these antibody-like molecules enable us to explore novel strategies to (i) deliver functional macromolecules into cells and (ii) understand the biological implications of Ras inhibition by different mechanisms.

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15:45-16:00 Enhancing intracellular delivery of mRNA

Dr Arpan Desai, AstraZeneca, UK

Abstract

Modified mRNAs offer a potentially transformative way of treating disease by triggering the body’s natural processes for protein production. A major challenge for therapeutic application is productive delivery of modified mRNA to cell cytoplasm for protein translation. The use of lipid nanoparticle (LNP) based vehicles to aid delivery of nucleic acids is well documented, however key challenges remain with lack of efficacy observed in some target tissue types associated with poor uptake and endosomal entrapment. Several mechanisms for LNP uptake have been proposed, including macropinocytosis however a detailed understanding remains elusive. This work aims to unravel the key mechanisms controlling cellular uptake and intracellular trafficking of LNPs and mRNA cargo across different cell lines. Dr Desai's group first screened uptake of LNPs and productive delivery of mRNA cargo across a range of tumour cell types. Cellular uptake and cytoplasmic delivery was monitored using fluorescently labelled LNPs delivering mRNAs encoding a reporter protein (eGFP).  The group found delivery to be highly variable, with some tumour types completely refractile to LNP based delivery. The group then applied a phenotypic screening approach using selected libraries of small molecule compounds alongside RNA interference strategies with the objective to identify pathways that if perturbed would enhance LNP mediated delivery. This work has resulted in a greater understanding of the cellular mechanisms that control LNP based delivery and fate of mRNA cargo and will facilitate the design of next generation intracellular delivery technologies.

16:00-16:15 Discussion

16:15-17:00 Overview and future directions