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Joel A Swanson

University of Michigan, Microbiology & Immunology, Faculty Member

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Papers by Joel A Swanson

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity

Journal of Cell Biology, Feb 1, 1981

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The ... more Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry . Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes depleted of actin and myosin are incubated with rabbit skeletal muscle F-actin . Pre-extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity . Lipid vesicles, heat-denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity . Heat-denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin crosslinking activity of D. discoideum membranes . In the absence of MgATP, Triton X-100 extraction of isolated D. discoideum membranes results in a Triton-insoluble residue composed of actin, myosin, and associated membrane proteins . The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane . Interactions of cytoskeletal structures with eukaryotic cell membranes are thought to be important for maintenance of cellular shape, endo-and exocytosis, movement, and cell-cell and cell-substrate attachment (1,. Although many membrane-cytoskeleton interactions may be transient, many are stable enough to allow the isolation of membranes with part of the cytoskeleton still attached . Actin is one of the most frequently identified cytoskeletal proteins in such preparations (86) . Much is known about the interactions between actin and soluble cytoplasmic proteins (40, 79), but less is known about the interactions between actin and membranes. Amoebae of the cellular slime mold, Dictyostelium discoi-396

Differential Association of Phosphatidylinositol 3-Kinase, SHIP-1, and PTEN with Forming Phagosomes

Molecular Biology of the Cell, Jul 1, 2007

In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [... more In macrophages, enzymes that synthesize or hydrolyze phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P 3 ] regulate Fc␥ receptor-mediated phagocytosis. Inhibition of phosphatidylinositol 3-kinase (PI3K) or overexpression of the lipid phosphatases phosphatase and tensin homologue (PTEN) and Src homology 2 domain-containing inositol phosphatase (SHIP-1), which hydrolyze PI(3,4,5)P 3 to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], respectively, inhibit phagocytosis in macrophages. To examine how these enzymes regulate phagosome formation, the distributions of yellow fluorescent protein (YFP) chimeras of enzymes and pleckstrin homology (PH) domains specific for their substrates and products were analyzed quantitatively. PTEN-YFP did not localize to phagosomes, suggesting that PTEN regulates phagocytosis globally within the macrophage. SHIP1-YFP and p85-YFP were recruited to forming phagosomes. SHIP1-YFP sequestered to the leading edge and dissociated from phagocytic cups earlier than did p85-cyan fluorescent protein, indicating that SHIP-1 inhibitory activities are restricted to the early stages of phagocytosis. PH domain chimeras indicated that early during phagocytosis, PI(3,4,5)P 3 was slightly more abundant than PI(3,4)P 2 at the leading edge of the forming cup. These results support a model in which phagosomal PI3K generates PI(3,4,5)P 3 necessary for later stages of phagocytosis, PTEN determines whether those late stages can occur, and SHIP-1 regulates when and where they occur by transiently suppressing PI(3,4,5)P 3 -dependent activities necessary for completion of phagocytosis.

The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy

Nature Communications, Aug 10, 2021

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular ... more Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the threedimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3′phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3′-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

Alveolar macrophage‐derived extracellular vesicles inhibit endosomal fusion of influenza virus

The EMBO Journal, 2020

Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned... more Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.

Macropinocytosis, mTORC1 and cellular growth control

Cellular and molecular life sciences : CMLS, 2018

The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extr... more The growth and proliferation of metazoan cells are driven by cellular nutrient status and by extracellular growth factors. Growth factor receptors on cell surfaces initiate biochemical signals that increase anabolic metabolism and macropinocytosis, an actin-dependent endocytic process in which relatively large volumes of extracellular solutes and nutrients are internalized and delivered efficiently into lysosomes. Macropinocytosis is prominent in many kinds of cancer cells, and supports the growth of cells transformed by oncogenic K-Ras. Growth factor receptor signaling and the overall metabolic status of the cell are coordinated in the cytoplasm by the mechanistic target-of-rapamycin complex-1 (mTORC1), which positively regulates protein synthesis and negatively regulates molecular salvage pathways such as autophagy. mTORC1 is activated by two distinct Ras-related small GTPases, Rag and Rheb, which associate with lysosomal membranes inside the cell. Rag recruits mTORC1 to the lysos...

Reverse Engineering the Intracellular Self-Assembly of a Functional Mechanopharmaceutical Device

Scientific reports, Jan 13, 2018

Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructi... more Weakly basic, poorly soluble chemical agents could be exploited as building blocks for constructing sophisticated molecular devices inside the cells of living organisms. Here, using experimental and computational approaches, we probed the relationship between the biological mechanisms mediating lysosomal ion homeostasis and the self-assembly of a weakly basic small molecule building block (clofazimine) into a functional, mechanopharmaceutical device (intracellular Crystal-Like Drug Inclusions - "CLDIs") in macrophage lysosomes. Physicochemical considerations indicate that the intralysosomal stabilization of the self-assembled mechanopharmaceutical device depends on the pH of the weakly basic building block and its affinity for chloride, both of which are consistent with the pH and chloride content of a physiological lysosomal microenvironment. Most importantly, in vitro and in silico studies revealed that high expression levels of the vacuolar ATPase (V-ATPase), irrespecti...

Growth factor signaling to mTORC1 by amino acid–laden macropinosomes

Journal of Cell Biology, 2015

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors ... more The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid–dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow–derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficientl...

Signaling for Phagocytosis

Phagocyte-Pathogen Interactions

Phagocytes ingest a variety of particles, cells, and microbes by use of actin-rich, contractile e... more Phagocytes ingest a variety of particles, cells, and microbes by use of actin-rich, contractile extensions of plasma membrane that close into intracellular membranous organelles called phagosomes. Signaling for phagocytosis begins when cell surface receptors engage particle-bound molecular ligands. The distinct component activities localize to subregions of forming phagosomes and may be coordinated by membrane-associated proteins and phospholipids that diffuse in the plane of the membrane bilayer. Different receptors use distinct mechanisms for regulating the actin cytoskeleton and the movements of membrane, producing a variety of morphologies for forming phagosomes. Some elements of phagocytic signaling are both constructive and interpretive. This chapter emphasizes the constructive signals. The cytosolic signaling proteins bind to the receptors or associated proteins, and a complex aggregate assembles around the receptor by diffusion and trapping. Secondary signals include the enzymatic modification of phospholipids and other membrane-associated proteins that in turn organize the cytoplasmic movements of phagocytosis. The underlying mechanism of phagocytosis appears to depend less on localized extension of the plasma membrane and more on controlled invagination of the cell surface by actin-myosin contractile activities. The morphogenesis of phagosomes suggests that actinmyosin-like contractility is essential to phagocytosis. The chapter reviews the signals for various different receptor-mediated phagocytic processes. The patterns of GTPase activities and the corresponding patterns of movement for actin, myosin, and membranes may be coordinated by patterns of membrane lipids in the inner leaflet of the inner membrane of the phagocytic cup that appear and disappear through the course of phagocytosis.

Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca++]i

The Journal of cell biology, 1986

The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the... more The calcium ion has been implicated as a cytosolic signal or regulator in phagocytosis. Using the Ca++-sensitive photoprotein aequorin we have measured intracellular free Ca++ ion concentration ([Ca++]i) in thioglycolate-elicited mouse peritoneal macrophages during phagocytosis and IgG-induced spreading. Macrophages plated on glass were loaded with aequorin and [Ca++]i was then measured from cell populations, both as previously described (McNeil, P. L., and D. L. Taylor, 1985, Cell Calcium, 6:83-92). Aequorin indicated a resting [Ca++]i in adherent macrophages of 84 nM and was responsive to changes in [Ca++]i induced by the addition of Mg-ATP (0.1 mM) or serum to medium. However, during the 15 min required for phagocytosis of seven or eight IgG-coated erythrocytes per macrophage loaded with aequorin, we measured no change in [Ca++]i. Similarly, the ligation of Fc-receptors that occurs when macrophages spread on immune complex-coated coverslips did not change macrophage [Ca++]i. In c...

Listeria monocytogenes exploits cystic fibrosis transmembrane conductance regulator (CFTR) to escape the phagosome

Proceedings of the National Academy of Sciences, 2011

Virulence of the intracellular pathogen Listeria monocytogenes ( Listeria ) requires escape from ... more Virulence of the intracellular pathogen Listeria monocytogenes ( Listeria ) requires escape from the phagosome into the host cytosol, where the bacteria replicate. Phagosomal escape is a multistep process characterized by perforation, which is dependent on the pore-forming toxin listeriolysin O (LLO), followed by rupture. The contribution of host factors to Listeria phagosomal escape is incompletely defined. Here we show that the cystic fibrosis transmembrane conductance regulator (CFTR) facilitates Listeria cytosolic entry. CFTR inhibition or mutation suppressed Listeria vacuolar escape in culture, and inhibition of CFTR in wild-type mice before oral inoculation of Listeria markedly decreased systemic infection. We provide evidence that high chloride concentrations may facilitate Listeria vacuolar escape by enhancing LLO oligomerization and lytic activity. We propose that CFTR transiently increases phagosomal chloride concentration after infection, potentiating LLO pore formation a...

Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages

The Journal of experimental medicine, 1989

Incubation of murine bone marrow-derived macrophages (BMM) in medium containing recombinant macro... more Incubation of murine bone marrow-derived macrophages (BMM) in medium containing recombinant macrophage colony-stimulating factor (rM-CSF) stimulated influx, efflux, and the net accumulation of the fluid-phase pinocytic marker, lucifer yellow (LY). Stimulation was dose dependent, occurred within 5 min of addition of the growth factor, and was sustained. Previous experiments had shown that BMM treated with PMA were stimulated to accumulate LY, but compared with rM-CSF-treated cells, the onset of stimulation in PMA-treated macrophages was slower. In further comparisons of rM-CSF- and PMA-stimulated LY accumulation, it was found that rM-CSF-stimulated pinocytosis could be abolished by pretreatment with 0.5 mg/ml trypsin, whereas neither unstimulated nor PMA-stimulated LY accumulation was affected by trypsin pretreatment. These findings indicate that the rM-CSF response was initiated at the cell surface, while the PMA response occurred via intracellular (or trypsin-resistant) receptors. ...

Salmonella stimulate macrophage macropinocytosis and persist within spacious phagosomes

The Journal of experimental medicine, 1994

Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrop... more Light microscopic studies of phagocytosis showed that Salmonella typhimurium entered mouse macrophages enclosed in spacious phagosomes (SP). Viewed by time-lapse video microscopy, bone marrow-derived macrophages exposed to S. typhimurium displayed generalized plasma membrane ruffling and macropinocytosis. Phagosomes containing Salmonella were morphologically indistinguishable from macropinosomes. SP formation was observed after several methods of bacterial opsonization, although bacteria opsonized with specific IgG appeared initially in small phagosomes that later enlarged. In contrast to macropinosomes induced by growth factors, which shrink completely within 15 min, SP persisted in the cytoplasm, enlarging often by fusion with macropinosomes or other SP. A Salmonella strain containing a constitutive mutation in the phoP virulence regulatory locus (PhoPc) induced significantly fewer SP. Similar to Yersinia enterocolitica, PhoPc bacteria entered macrophages in close-fitting phagosom...

Cellular and Molecular Aspects of Amoeboid Movement

Cold Spring Harbor Symposia on Quantitative Biology, 1982

... 8 M. SDS-poly-acrylamide gels of these two fractions reveal that they contain prominent polyp... more

The breadth of macropinocytosis research

Philosophical Transactions of the Royal Society B, Dec 17, 2018

Dynamics of cytoskeletal proteins during Fc receptor-mediated phagocytosis in macrophages

Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskele-... more Particle ingestion by phagocytosis results from sequential rearrangements of the actin cytoskele-ton and overlying membrane. To assemble a chronology of molecular events during phagosome formation and to examine the contributions of phosphoinositide 3-kinase (PI 3-kinase) to these dynamics, a method was developed for synchronizing Fc receptor-mediated phagocytosis by murine macrophages. Erythrocytes opsonized with complement component C3bi were bound to macrophages at 37°C, a condition that does not favor particle phagocytosis. Addition of soluble anti-erythrocyte IgG resulted in rapid opsonization of the bound erythrocytes, followed by their immediate internalization via phagocytosis. Cellular content of F-actin, as measured by binding of rhodamine-phalloidin, increased transiently during phagocytosis, and this increase was not diminished by inhibitors of PI 3-kinase. Immunofluorescence localization of myosins in macro-phages fixed at various times during phagocytosis indicated tha...

Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium

Infection and Immunity, 1996

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive af... more The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained access...

A contractile activity that closes phagosomes in macrophages

Journal of Cell Science, 1999

Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the... more Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, o...

Dorsal ruffles enhance activation of Akt by growth factors

Journal of Cell Science, 2018

In fibroblasts, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulate... more In fibroblasts, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) stimulate the formation of actin-rich, circular dorsal ruffles (CDR) and phosphatidylinositol 3-kinase (PI3K)-dependent phosphorylation of Akt. To test the hypothesis that CDR increase synthesis of phospho-Akt (pAkt), we analyzed the contributions of CDR to Akt phosphorylation in response to PDGF and EGF. CDR appeared within several minutes of growth factor addition, coincident with a peak of pAkt. Microtubule depolymerization with nocodazole blocked CDR formation and inhibited phosphorylation of Akt in response to EGF but not PDGF. Quantitative immunofluorescence showed increased concentrations of Akt, pAkt and PIP3, the phosphoinositide product of PI3K that activates Akt, concentrated in CDR and ruffles. EGF stimulated lower maximal levels of pAkt than did PDGF, which suggested that Akt phosphorylation requires amplification in CDR only when PI3K activities are low. Accordingly, stimulation wit...

Macropinosomes as units of signal transduction

Philosophical Transactions of the Royal Society B: Biological Sciences, 2019

Macropinosome formation occurs as a localized sequence of biochemical activities and associated m... more Macropinosome formation occurs as a localized sequence of biochemical activities and associated morphological changes, which may be considered a form of signal transduction leading to the construction of an organelle. Macropinocytosis may also convey information about the availability of extracellular nutrients to intracellular regulators of metabolism. Consistent with this idea, activation of the metabolic regulator mechanistic target of rapamycin complex-1 (mTORC1) in response to acute stimulation by growth factors and extracellular amino acids requires internalization of amino acids by macropinocytosis. This suggests that macropinocytosis is necessary for mTORC1-dependent growth of metazoan cells, both as a route for delivery of amino acids to sensors associated with lysosomes and as a platform for growth factor-dependent signalling to mTORC1 via phosphatidylinositol 3-kinase (PI3K) and the Akt pathway. Because the biochemical signals required for the construction of macropinosom...

Intracellular delivery of macromolecules