We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome.

Macrophages and dendritic cells continuously survey their environment in search of foreign particles and soluble antigens. Such surveillance involves the ongoing extension of actin-rich protrusions and the consequent formation of phagosomes and macropinosomes. The signals inducing this constitutive cytoskeletal remodeling have not been defined. We report that, unlike nonphagocytic cells, macrophages and immature dendritic cells have elevated levels of phosphatidic acid (PA) in their plasma membrane. The plasmalemmal PA is synthesized by phosphorylation of diacylglycerol, which is in turn generated by a G protein-stimulated phospholipase C. Inhibition of diacylglycerol kinase activity results in the detachment of T-cell lymphoma invasion and metastasis-inducing protein 1 (TIAM1)-a Rac guanine exchange factor-from the plasma membrane, thereby depressing Rac activity and abolishing the constitutive ruffling and macropinocytosis that characterize macrophages and immature dendritic cells. Accumulation of PA and binding of TIAM1 to the membrane require the activity of phosphatidylinositol-4,5-bisphosphate 3-kinase. Thus a distinctive, constitutive pathway of PA biosynthesis promotes the actin remodeling required for immune surveillance.

Macropinocytosis mediates the uptake of antigens and of nutrients that dictate the regulation of cell growth by mechanistic target of rapamycin complex 1 (mTORC1). Because these functions differ in proinflammatory and anti-inflammatory macrophages, we compared the macropinocytic ability of two extreme polarization states. We found that anti-inflammatory macrophages perform vigorous macropinocytosis constitutively, while proinflammatory cells are virtually inactive. The total cellular content of Rho-family GTPases was higher in anti-inflammatory cells, but this disparity failed to account for the differential macropinocytic activity. Instead, reduced activity of Rac/RhoG was responsible for the deficient macropinocytosis of proinflammatory macrophages, as suggested by the stimulatory effects of heterologously expressed guanine nucleotide-exchange factors or of constitutively active (but not wild-type) forms of these GTPases. Similarly, differences in the activation state of phosphatidylinositol 3-kinase (PtdIns3K) correlated with the macropinocytic activity of pro- and anti-inflammatory macrophages. Differences in PtdIns3K and Rho-GTPase activity were attributable to the activity of calcium-sensing receptors (CaSRs), which appear to be functional only in anti-inflammatory cells. However, agonists of PtdIns3K, including cytokines, chemokines, and LPS, induced macropinocytosis in proinflammatory cells. Our findings revealed a striking difference in the macropinocytic ability of pro- and anti-inflammatory macrophages that correlates with their antigen-presenting and metabolic activity.

Anionic phospholipids can confer a net negative charge on biological membranes. This surface charge generates an electric field that serves to recruit extrinsic cationic proteins, can alter the disposition of transmembrane proteins and causes the local accumulation of soluble counterions, altering the local pH and the concentration of physiologically important ions such as calcium. Because the phospholipid compositions of the different organellar membranes vary, their surface charges are similarly expected to diverge. Yet, despite the important functional implications, remarkably little is known about the electrostatic properties of the individual organellar membranes. We therefore designed and implemented approaches to estimate the surface charges of the cytosolic membranes of various organelles in situ in intact cells. Our data indicate that the inner leaflet of the plasma membrane is most negative, with a surface potential of approximately -35 mV, followed by the Golgi complex > lysosomes > mitochondria ≈ peroxisomes > endoplasmic reticulum, in decreasing order.

Macrophages respond to changes in environmental stimuli by assuming distinct functional phenotypes, a phenomenon referred to as macrophage polarization. We generated classically (M1) and alternatively (M2) polarized macrophages--two extremes of the polarization spectrum--to compare the properties of their phagosomes. Specifically, we analyzed the regulation of the luminal pH after particle engulfment. The phagosomes of M1 macrophages had a similar buffering power and proton (equivalent) leakage permeability but significantly reduced proton-pumping activity compared with M2 phagosomes. As a result, only the latter underwent a rapid and profound acidification. By contrast, M1 phagosomes displayed alkaline pH oscillations, which were caused by proton consumption upon dismutation of superoxide, followed by activation of a voltage- and Zn(2+)-sensitive permeation pathway, likely HV1 channels. The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes. The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify. Thus, in M1 cells, elimination of pathogens through deployment of the microbicidal NADPH oxidase is given priority at the expense of delayed acidification. By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.

CX3CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX3CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX3CL1. We observed that the majority of cell surface CX3CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX3CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX3CL1 confinement and increased CX3CL1-ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand.

Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference-based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation-deactivation cycle of Ack.

To account for the many functions of phosphatidylinositol 4,5-bisphosphate (PIP(2)), several investigators have proposed that there are separate pools of PIP(2) in the plasma membrane. Recent experiments show the surface concentration of PIP(2) is indeed enhanced in regions where phagocytosis, exocytosis, and cell division occurs. Kinases that produce PIP(2) are also concentrated in these regions. However, how is the PIP(2) produced by these kinases prevented from diffusing rapidly away? First, proteins could act as "fences" around the perimeter of these regions. Second, some factor could markedly decrease the diffusion coefficient, D, of PIP(2) within these regions. We used fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) to investigate these two possibilities in the forming phagosomes of macrophages injected with fluorescent PIP(2). FCS measurements show that PIP(2) diffuses rapidly (D ~ 1 μm(2)/s) in both the forming phagosomes and unengaged plasma membrane. FRAP measurements show that the fluorescence from PIP(2) does not recover (>100 s) after photobleaching the entire forming phagosome but recovers rapidly (~10 s) in a comparable area of membrane outside the cup. These results (and similar data for a plasma membrane-anchored green fluorescent protein) support the hypothesis that a fence impedes the diffusion of PIP(2) into and out of forming phagosomes.

Much has been learned about the role of exofacial phosphatidylserine (PS) in apoptosis and blood clotting using annexin V. However, because annexins are impermeant and unable to bind PS at low calcium concentration, they are unsuitable for intracellular use. Thus little is known about the topology and dynamics of PS in the endomembranes of normal cells. We used two new probes-green fluorescent protein (GFP)-LactC2, a genetically encoded fluorescent PS biosensor, and 1-palmitoyl-2-(dipyrrometheneboron difluoride)undecanoyl-sn-glycero-3-phospho-L-serine (TopFluor-PS), a synthetic fluorescent PS analogue-to examine PS distribution and dynamics inside live cells. The mobility of PS was assessed by a combination of advanced optical methods, including single-particle tracking and fluorescence correlation spectroscopy. Our results reveal the existence of a sizable fraction of PS with limited mobility, with cortical actin contributing to the confinement of PS in the plasma membrane. We were also able to measure the dynamics of PS in endomembrane organelles. By targeting GFP-LactC2 to the secretory pathway, we detected the presence of PS in the luminal leaflet of the endoplasmic reticulum. Our data provide new insights into properties of PS inside cells and suggest mechanisms to account for the subcellular distribution and function of this phospholipid.

Mutations of the inositol 5-phosphatase OCRL cause Lowe syndrome (LS), characterized by congenital cataract, low IQ, and defective kidney proximal tubule resorption. A key subset of LS mutants abolishes OCRL's interactions with endocytic adaptors containing F&H peptide motifs. Converging unbiased methods examining human peptides and the unicellular phagocytic organism Dictyostelium discoideum reveal that, like OCRL, the Dictyostelium OCRL orthologue Dd5P4 binds two proteins closely related to the F&H proteins APPL1 and Ses1/2 (also referred to as IPIP27A/B). In addition, a novel conserved F&H interactor was identified, GxcU (in Dictyostelium) and the Cdc42-GEF FGD1-related F-actin binding protein (Frabin) (in human cells). Examining these proteins in D. discoideum, we find that, like OCRL, Dd5P4 acts at well-conserved and physically distinct endocytic stations. Dd5P4 functions in coordination with F&H proteins to control membrane deformation at multiple stages of endocytosis and suppresses GxcU-mediated activity during fluid-phase micropinocytosis. We also reveal that OCRL/Dd5P4 acts at the contractile vacuole, an exocytic osmoregulatory organelle. We propose F&H peptide-containing proteins may be key modifiers of LS phenotypes.

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer's disease-related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC-derived neurons for the investigation of neuronal cell biology and pathology.

T-cell immunoglobulin mucin protein 4 (TIM4), a phosphatidylserine (PtdSer)-binding receptor, mediates the phagocytosis of apoptotic cells. How TIM4 exerts its function is unclear, and conflicting data have emerged. To define the mode of action of TIM4, we used two distinct but complementary approaches: 1) we compared bone marrow-derived macrophages from wild-type and TIM4(-/-) mice, and 2) we heterologously expressed TIM4 in epithelioid AD293 cells, which rendered them competent for engulfment of PtdSer-bearing targets. Using these systems, we demonstrate that rather than serving merely as a tether, as proposed earlier by others, TIM4 is an active participant in the phagocytic process. Furthermore, we find that TIM4 operates independently of lactadherin, which had been proposed to act as a bridging molecule. Of interest, TIM4-driven phagocytosis depends on the activation of integrins and involves stimulation of Src-family kinases and focal adhesion kinase, as well as the localized accumulation of phosphatidylinositol 3,4,5-trisphosphate. These mediators promote recruitment of the nucleotide-exchange factor Vav3, which in turn activates small Rho-family GTPases. Gene silencing or ablation experiments demonstrated that RhoA, Rac1, and Rac2 act synergistically to drive the remodeling of actin that underlies phagocytosis. Single-particle detection experiments demonstrated that TIM4 and β1 integrins associate upon receptor clustering. These findings support a model in which TIM4 engages integrins as coreceptors to evoke the signal transduction needed to internalize PtdSer-bearing targets such as apoptotic cells.

Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P(2), which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P(2) and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P(2) available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.