Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ialpha impairs localized actin remodeling and suppresses phagocytosis

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP(2) from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIalpha was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIalpha in particle engulfment, we generated a double mutant (D309N/R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KIalpha D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fcgamma receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP(2) and of F-actin in the phagosomal cup. These data suggest that PIPKIalpha activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIalpha appears to contribute to the transient changes in PIP(2) levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins.     

The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.     

Differential role of actin,clathrin, and dynamin in Fc gamma receptor-mediated endocytosis and phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.     

Role of ubiquitin and proteasomes in phagosome maturation

Phagosomes undergo multiple rounds of fusion with compartments of the endocytic pathway during the course of maturation. Despite the insertion of vast amounts of additional membrane, the phagosomal surface area remains approximately constant, implying active ongoing fission. To investigate the mechanisms underlying phagosomal fission we monitored the fate of Fcgamma receptors (FcgammaR), which are known to be cleared from the phagosome during maturation. FcgammaR, which show a continuous distribution throughout the membrane of nascent phagosomes were found at later times to cluster into punctate, vesicular structures, before disappearing. In situ photoactivation of receptors tagged with a light-sensitive fluorescent protein revealed that some of these vesicles detach, whereas others remain associated with the phagosome. By fusing FcgammaR to pH-sensitive fluorescent proteins, we observed that the cytoplasmic domain of the receptors enters an acidic compartment, indicative of inward budding and formation of multivesicular structures. The topology of the receptor was confirmed by flow cytometry of purified phagosomes. Phagosomal proteins are ubiquitylated, and ubiquitylation was found to be required for formation of acidic multivesicular structures. Remarkably, proteasomal function is also involved in the vesiculation process. Preventing the generation of multivesicular structures did not impair the acquisition of late endosomal and lysosomal markers, indicating that phagosomal fusion and fission are controlled separately.     

Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis.

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis. The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3'PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.     

Binding of heparan sulphate and heparin to control and virus-transformed cells

A cloned embryonic mouse cell line contained specific cell-surface receptors for heparin and both the number and affinity appeared to be unchanged in a simian-virus-40-transformed subclone. In competitive binding assays heparan sulphate from the control clone was bound preferentially compared to that from the transformed subclone, indicating that the altered sulphation of heparan sulphate from transformed cells results in a lowered affinity for cell-surface receptors. Evidence was obtained suggesting that endogenous proteoglycans were not held at the cell surface by binding to these receptors alone. However the possibility that proteoglycans embedded in the plasma membrane may interact with the receptor has not been ruled out.     

Ironing out the wrinkles of neutrophil phagocytosis

Though phagocytosis of microbes by professional phagocytes such as neutrophils is crucial for the survival of the host, it is still unclear how the apparent 'stretching' of the plasma membrane is achieved. Microscopically, pseudopod extension, particulate engulfment and phagosome closure all require seemingly large expansions of the cell surface area. Although actual membrane stretching can be ruled out on the basis of physical properties of lipid bilayers, the addition of new membrane from within the cell, either by exocytosis or phagosomal fusion with endoplasmic reticulum membrane, might provide an explanation. However, these events do not seem to have major roles during phagocytosis by neutrophils. Instead, neutrophils might use a more primitive mechanism, that is, the unfolding of surface membrane wrinkles, to provide the additional membrane for phagocytosis. Here, we briefly discuss why membrane unwrinkling provides a feasible hypothesis for membrane expansion during neutrophil phagocytosis, and suggest a potential molecular mechanism for neutrophil control over membrane surface wrinkles, and the potential signalling route.     

Regulation of G protein‐coupled receptor signaling by plasma membrane organization and endocytosis

The trafficking of G protein coupled‐receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine‐tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor‐receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin‐coated pit dynamics, and through the scaffolding adaptor protein β‐arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.      

Fc gamma R-mediated phagocytosis stimulates localized pinocytosis in human neutrophils

Engulfment of IgG-coated particles by neutrophils and macrophages is an essential component of the innate immune response. This process, known as phagocytosis, is triggered by clustering of FcgammaR at sites where leukocytes make contact with the opsonized particles. We found that phagocytosis is accompanied by a burst of fluid phase pinocytosis, which is largely restricted to the immediate vicinity of the phagosomal cup. FcgammaR-induced pinocytosis preceded and appeared to be independent of phagosomal sealing. Accordingly, fluid phase uptake was accentuated by actin depolymerization, which precludes phagocytosis. Stimulation of pinocytosis required phosphatidylinositol 3-kinase activity and was eliminated when changes in the cytosolic free Ca(2+) concentration were prevented. Because stimulation of FcgammaR also induces secretion, which is similarly calcium and phosphatidylinositol 3-kinase dependent, we studied the possible relationship between these events. Neutrophil fragments devoid of secretory granules (cytoplasts) were prepared by sedimentation through Ficoll gradients. Cytoplasts could perform FcgammaR-mediated phagocytosis, which was not accompanied by activation of pinocytosis. This observation suggests that granule exocytosis is required for stimulation of pinocytosis. Analysis of the cytosolic Ca(2+) dependence of secretion and pinocytosis suggests that primary (lysosomal) granule exocytosis is the main determinant of pinocytosis during FcgammaR stimulation. Importantly, primary granules are secreted in a polarized fashion near forming phagosomes. Focal pinocytosis during particle engulfment may contribute to Ag processing and presentation and/or to retrieval of components of the secretory machinery. Alternatively, it may represent an early event in the remodeling of the phagosomal membrane, leading to phagosomal maturation.     

Caveolin-1 regulates transforming growth factor (TGF)-beta/SMAD signaling through an interaction with the TGF-beta type I receptor

Transforming growth factor-beta (TGF-beta) signaling proceeds from the cell membrane to the nucleus through the cooperation of the type I and II serine/threonine kinase receptors and their downstream SMAD effectors. Although various regulatory proteins affecting TGF-beta-mediated events have been described, relatively little is known about receptor interactions at the level of the plasma membrane. Caveolae are cholesterol-rich membrane microdomains that, along with their marker protein caveolin-1 (Cav-1), have been implicated in the compartmentalization and regulation of certain signaling events. Here, we demonstrate that specific components of the TGF-beta cascade are associated with caveolin-1 in caveolae and that Cav-1 interacts with the Type I TGF-beta receptor. Additionally, Cav-1 is able to suppress TGF-beta-mediated phosphorylation of Smad-2 and subsequent downstream events. We localize the Type I TGF-beta receptor interaction to the scaffolding domain of Cav-1 and show that it occurs in a physiologically relevant time frame, acting to rapidly dampen signaling initiated by the TGF-beta receptor complex.     

Mechanisms of nutritive endocytosis. III. A freeze-fracture study of phagocytosis by digestive cells of Chlorohydra

Freeze-fracture was used to compare the ultrastructure of plasma with phagosomal membranes of digestive cells of green hydra. Changes in both the pattern and density of intramembrane particles (IMP) were evident during the transition from plasma to phagosomal membrane. Small particle-free regions and associated aggregates of three to eight IMP were observed in presumptive adherent and enveloping plasma membranes, as well as in fully formed phagosomal membranes. Larger particle-free regions were observed as rims around the tips of enveloping membranes which had nearly completed enclosure of particles. The density of IMP in newly formed phagosomal membranes was 1.5-fold greater than that in the parent plasma membrane from which they derived, and was greater also than in older phagosomal membranes.     

Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.     

Involvement of heterotrimeric G proteins in phagocytosis and recycling from the phagosomal compartment.

Phagocytosis is a receptor-mediated process by which specialized cell types engulf large extracellular particles. Phagosome maturation involves a series of intracellular membrane fusion and budding events resulting in the delivery of particles to compartments enriched in lysosomal hydrolases where they are digested. Substantial amounts of plasma membrane and many phagosomal proteins, such as receptors, rapidly recycle to the plasma membrane following phagosome formation. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the molecular machinery involved is largely unknown. To assess the participation of GTPases in phagocytosis and recycling from phagosomes we used aluminum fluoride (AIF(-)(4)), which activates the GDP-bound form of stimulatory and inhibitory trimeric G proteins. AlF(-)(4) inhibited both the uptake to and the recycling from the phagosomal compartment. Cholera toxin, which activates Galphas, and pertussis toxin, which uncouples Gi and Go from receptors, were effective inhibitors of phagocytosis. However, both toxins stimulated recycling from phagosomes. These results suggest that more than one GTP-binding protein participates either directly or indirectly not only in phagocytosis, but also in maturation and recycling from phagosomes, and thereby assign a role for heterotrimeric G proteins in controlling traffic through the phagocytic pathway.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Signaling-dependent immobilization of acylated proteins in the inner monolayer of the plasma membrane

Phospholipids play a critical role in the recruitment and activation of several adaptors and effectors during phagocytosis. Changes in lipid metabolism during phagocytosis are restricted to the phagocytic cup, the area of the plasmalemma lining the target particle. It is unclear how specific lipids and lipid-associated molecules are prevented from diffusing away from the cup during the course of phagocytosis, a process that often requires several minutes. We studied the mobility of lipid-associated proteins at the phagocytic cup by measuring fluorescence recovery after photobleaching. Lipid-anchored (diacylated) fluorescent proteins were freely mobile in the unstimulated membrane, but their mobility was severely restricted at sites of phagocytosis. Only probes anchored to the inner monolayer displayed reduced mobility, whereas those attached to the outer monolayer were unaffected. The immobilization persisted after depletion of plasmalemmal cholesterol, ruling out a role of conventional "rafts." Corralling of the probes by the actin cytoskeleton was similarly discounted. Instead, the change in mobility required activation of tyrosine kinases. We suggest that signaling-dependent recruitment of adaptors and effectors with lipid binding domains generates an annulus of lipids with restricted mobility.     

G proteins control diverse pathways of transmembrane signaling

Hormones, neurotransmitters, and autacoids interact with specific receptors and thereby trigger a series of molecular events that ultimately produce their biological effects. These receptors, localized in the plasma membrane, carry binding sites for ligands as diverse as peptides (e.g., glucagon, neuropeptides), lipids (e.g., prostaglandins), nucleosides and nucleotides (e.g., adenosine), and amines (e.g., catecholamines, serotonin). These receptors do not interest directly with their respective downstream effector (i.e., an ion channel and/or an enzyme that synthesizes a second messenger); rather, they control one or several target systems via the activation of an intermediary guanine nucleotide-binding regulatory protein or G protein. G proteins serve as signal transducers, linking extracellularly oriented receptors to membrane-bound effectors. Traffic in these pathways is regulated by a GTP (on)-GDP (off) switch, which is regulated by the receptor. The combination of classical biochemistry and recombinant DNA technology has resulted in the discovery of many members of the G protein family. These approaches, complemented in particular by electrophysiological experiments, have also identified several effectors that are regulated by G proteins. We can safely assume that current lists of G proteins and the functions that they control are incomplete.     

Synthesis, insertion into the plasma membrane, and turnover of alpha- bungarotoxin receptors in chick sympathetic neurons

alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.     

Agonist-dependent immobilization of chimeric bombesin/GRP receptors: dependence on c-Src activity and dissociation from internalization.

G-protein-coupled receptors (GPCRs) are membrane proteins that exhibit a decreased mobile fraction compared to a freely mobile plasma membrane protein. Recently, interest has focused on proteins other than heterotrimeric G-proteins that interact with GPCRs as scaffolding structures that affect receptor signal transduction. In order to investigate the physical state of receptors before and after agonist, we used fluorescence recovery after photobleaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to the intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) expressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells at 37 degrees C, addition of GRP (100 nM) caused a rapid decrease in GFP-GRP receptor mobile fraction from 0.8 +/- 0.1 to 0.49 +/- 0.05, which was independent of endocytosis. Concurrently, the remaining mobile GFP-GRPreceptors showed an increase in the diffusion rate with the half-time of fluorescent recovery, tau(1/2) = 46 +/- 7 s for untreated cells, decreasing to tau(1/2) = 30 +/- 6 s for cells treated with GRP. Prior treatment with the Src-specific inhibitor PP-2 (10 microM) blocked GFP-GRP receptor immobilization while treatment with the inactive analog PP-3 (10 microM) did not affect receptor immobilization. These data suggest that agonist-bound GPCR have increased plasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity.     

Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.     

Phosphoinositides in phagolysosome and autophagosome biogenesis

Interconversions of phosphoinositides play a pivotal role during phagocytosis and at the subsequent stages of phagosomal maturation into the phagolysosome. Several model systems have been used to study the role of phosphoinositides in phagosomal membrane remodelling. These include phagosomes formed by inanimate objects such as latex beads, or pathogenic bacteria, e.g. Mycobacterium tuberculosis. The latter category provides naturally occurring tools to dissect membrane trafficking processes governing phagolysosome biogenesis. M. tuberculosis persists in infected macrophages by blocking Rab conversion and affecting Rab effectors. One of the major Rab effectors involved in this process is the type III phosphatidylinositol 3-kinase hVPS34. The lipid kinase hVPS34 and its enzymatic product PtdIns3P are critical for the default pathway of phagosomal maturation into phagolysosomes. Mycobacteria block PtdIns3P production and thus arrest phagosomal maturation. PtdIns3P is also critical for the process of autophagy, recently recognized as an effector of innate immunity defenses. Induction of autophagy by pharmacological, physiological, or immunological means, overcomes mycobacterial phagosome maturation block in a PtdIns3P generation dependent manner and eliminates intracellular M. tuberculosis. PtdIns3P and PtdIns3P-dependent processes represent an important cellular nexus where fundamental trafficking processes, disease causing host-pathogen interactions, and innate and adaptive immunity defense mechanisms meet.