Macropinocytosis and cellular growth control
Professor Joel Swanson, University of Michigan, USA
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.
Constitutive vs inducible macropinocytosis in macrophages
Professor Sergio Grinstein, The Hospital for Sick Children, Canada
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'.
Macropinosome formation, maturation and membrane recycling
Professor Julie Donaldson, National Institutes of Health, USA
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.
Learning how to form and process macropinosomes with Dictyostelium amoeba
Dr Jason King, University of Sheffield, UK
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.