A role for phosphoinositide 3-kinase in the completion of macropinocytosis and phagocytosis by macrophages

Phosphoinositide 3-kinase (PI 3-kinase) has been implicated in growth factor signal transduction and vesicular membrane traffic. It is thought to mediate the earliest steps leading from ligation of cell surface receptors to increased cell surface ruffling. We show here that inhibitors of PI 3-kinase inhibit endocytosis in macrophages, not by interfering with the initiation of the process but rather by preventing its completion. Consistent with earlier studies, the inhibitors wortmannin and LY294002 inhibited fluid-phase pinocytosis and Fc receptor-mediated phagocytosis, but they had little effect on the receptor-mediated endocytosis of diI-labeled, acetylated, low density lipoprotein. Large solute probes of endocytosis reported greater inhibition by wortmannin than smaller probes did, indicating that macropinocytosis was affected more than micropinocytosis. Since macropinocytosis and phagocytosis are actin-mediated processes, we expected that their inhibition by wortmannin resulted from deficient signaling from macrophage colony-stimulating factor (M-CSF) receptors or Fc receptors to the actin cytoskeleton. However, video microscopy showed cell surface ruffling in wortmannin-treated cells, and increased ruffling after addition of M-CSF or phorbol myristate acetate. Quantitative measurements of video data reported slightly diminished ruffling in wortmannin-treated cells. Remarkably, the ruffles that formed in wortmannin-treated macrophages all receded into the cytoplasm without closing into macropinosomes. Similarly, wortmannin and LY294002 did not inhibit the extension of actin-rich pseudopodia along IgG- opsonized sheep erythrocytes, but instead prevented them from closing into phagosomes. These findings indicate that PI 3-kinase is not necessary for receptor-mediated stimulation of pseudopod extension, but rather functions in the closure of macropinosomes and phagosomes into intracellular organelles.     

Regulation by intracellular Ca2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes [T. Kadowaki et al. (1986) J. Biol. Chem. 261, 16,141-16,147] and stimulate the fluid-phase endocytosis and exocytosis [Y. Miyata et al. (1988) Exp. Cell Res. 178, 73-83] in human epidermoid carcinoma KB cells. An increase in intracellular Ca2+ concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca2+ or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca2+ and cAMP. 125I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca2+- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.     

Morphologic changes in human carcinoma cells (A-431) stimulated by epidermal growth factor: effect of cholesterol and low-density lipoproteins on the ruffling response

Stimulation of A-431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk-phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography, we have investigated the effects of exogenous sterols thought to alter membrane fluidity. Light and scanning electron microscopy revealed a time- and concentration-dependent inhibition of ruffling (greater than 90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low-density lipoproteins (LDL). EGF-stimulated bulk-phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol-enriched medium (175 micrograms/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF-stimulated rounding. Morphometry of electron micrographs from cholesterol-treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF-stimulated ruffling and bulk-phase pinocytosis are related phenomena, whereas EGF-stimulated cell rounding is an independent process.     

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood‐brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin‐mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin‐mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.      

The role of complement receptor 3 (CR3) in Neisseria gonorrhoeae infection of human cervical epithelia

Neisseria gonorrhoeae is an important sexually transmitted pathogen and a major cofactor in HIV-1 infection. This organism uses different mechanisms to infect male and female genital tract epithelia. Receptor-mediated endocytosis of N. gonorrhoeae is the principle mechanism of entry into male urethral epithelial cells. Infection in men leads to a pronounced inflammatory response. In contrast, N. gonorrhoeae infection in women induces ruffling of the cervical epithelia, allowing a macropinocytic mechanism of entry. Infection in women is frequently asymptomatic, suggesting suppression of the inflammatory response. N. gonorrhoeae -induced membrane ruffling and inflammation suppression are consistent with the ability of this bacterium to enter cervical epithelial cells, in vitro and in vivo , by interaction with complement receptor 3 (CR3), a receptor that does not trigger an inflammatory response. This receptor is present on cervical epithelial cells but not on male urogenital tract epithelia. N. gonorrhoeae engagement of CR3 initiates a unique mechanism of bacterial-induced membrane ruffling and internalization. These studies explain why the pathology of N. gonorrhoeae infection differs between males and females. Additionally, the observation that this receptor is present on cervical epithelia may provide insight into the pathogenesis of other sexually transmitted pathogens     

Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Endocytosis is thought to play an essential role in the catabolism of HA due to the intracellular compartmentalization of the HA degrading hyaluronidase enzymes. Previous investigations have shown that keratinocytes, chondrocytes and breast tumor cell lines endocytose HA via the cell surface glycoprotein, CD44. However, other cell types endocytose HA using a CD44-independent mechanism that remains to be defined. The purpose of this study was to investigate HA endocytosis in B16-F10 melanoma cells. We found that B16-F10 melanoma cells expressed CD44 on their surfaces. Unexpectedly, CD44 did not play a role in the endocytosis of HA. Electron microscopy studies revealed that B16-F10 melanoma cells exhibited membrane ruffling, a characteristic feature of macropinocytosis, only after incubating the cells with the HA co-polymer. Moreover, B16-F10 melanoma cells endocytosed HA via macropinocytosis as assessed by drug inhibition studies and the co-localization of fluorescently labeled HA with fluorescent tracers under confocal microscopy. Based on these results, we conclude that induced macropinocytosis may provide a previously unrecognized avenue for HA endocytosis in some cell types.     

Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling

There is increasing interest in endocytosis that occurs independently of clathrin coats and the fates of membrane proteins internalized by this mechanism. The appearance of clathrin-independent endocytic and membrane recycling pathways seems to vary with different cell types and cargo molecules. In this review we focus on studies that have been performed using HeLa and COS cells as model systems for understanding this membrane trafficking system. These endosomal membranes contain signaling molecules including H-Ras, Rac1, Arf6 and Rab proteins, and a lipid environment rich in cholesterol and PIP(2) providing a unique platform for cell signaling. Furthermore, activation of some of these signaling molecules (H-Ras, Rac and Arf6) can switch the constitutive form of clathrin-independent endocytosis into a stimulated one, associated with PM ruffling and macropinocytosis.     

Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.     

Origin of ruffles: linkage to other protrusions, filopodia and lamellae

Although growth factor-initiated cascades in cells are networked with mechanisms such as “inside-out signaling”, it is not known how these pathways are integrated. Earlier studies reported that ruffling was enhanced and filopodia reduced in transformed cells. Since dissecting relationships among features was impossible if subjective recognition was relied upon, features in two epithelial cell lines were recognized by latent factor analysis. Factor-based classification revealed four protrusion classes, but none of them corresponded to ruffles. Loss of filopodia, defined by factor 4 (F4) values, accounted for the greatest change in features of oncogenically transformed cells. Factor 5 (F5, lamella) was unchanged during transformation of an airway epithelium cell line. The tumor promoter, phorbol 12-myristate 13-acetate (PMA), increased ruffling but decreased filopodia. F4 retained this relationship to ruffling in untreated cells and at multiple times after treatment. F5 values decreased but were positively correlated with measures of ruffling. Because factors are created as mutually orthogonal variables, this suggested that ruffles were not flagged in factor analysis because they originate from other features. Actin filament capping with sub-micromolar cytochalasin D (Cyto D) suppressed ruffling without affecting F4 or F5. Cyto D increased factor 7 (F7) values, thus showing specificity for this feature. However, cytochalasin treatment of PMA-treated cells that had developed stress fibers increased F4 and decreased F5. The results suggest that PMA changes the state of the cytoskeleton, causing protrusions to show novel responses to Cyto D compared to untreated cells. Results suggest that the factors identify physiologically distinct features.     

Effects of endocytosis inhibitory drugs on rubella virus entry into VeroE6 cells

It has been suggested that infectious entry of rubella virus (RV) is conducted by receptor mediated endocytosis. To explore the cellular entry mechanism of RV, inhibitory effects of drugs affecting various endocytic pathways on RV entry into VeroE6 cells were analyzed. Results showed that RV infectious entry into VeroE6 cells is mediated by clathrin-dependent endocytosis and not by caveolae-mediated endocytosis. Moreover, chemical inhibition of macropinocytosis such as treatments of amiloride, actin and microtubule-disrupting drug significantly reduced RV infection. Considering that macropinocytosis is inducible endocytosis by cellular stimulations, clathrin-mediated endocytosis is likely to be a major route of RV infectious entry.     

B cell antigen receptor-induced Rac1 activation and Rac1-dependent spreading are impaired in transitional immature B cells due to levels of membrane cholesterol

The BCR-triggered responses of mature and transitional immature B cells differ at both the biochemical and functional level. In this study, we show that in mature B cells, BCR signaling triggers Vav phosphorylation and Rac1 activation. Furthermore, we demonstrate that although downstream actin-dependent BCR capping is independent of Rac1 activation, actin-dependent membrane ruffling and cell spreading are Rac1-dependent processes. In contrast, BCR-induced Vav phosphorylation and Rac1 activation is impaired in transitional immature B cells, resulting in defects in actin polymerization-dependent spreading and membrane ruffling while Rac1-independent BCR capping remains intact. Because transitional immature murine B cells maintain lower steady-state levels of plasma membrane cholesterol, we augmented their levels to that of mature B cells and found that BCR-induced Rac1 activation and Rac1-dependent membrane ruffling and cell spreading were restored. These studies provide a direct link between B cell cholesterol levels and downstream cellular signaling processes.     

An agent or agents produced by virus-transformed cells cause unregulated ruffling in untransformed cells

KNRK cells (a normal rat kidney [NRK] cell line transformed by Kirsten murine sarcoma virus) in sparse culture exhibit a highly ruffled morphology, but the cause of this ruffling is unknown. In this study, we have demonstrated that the continuous, excess ruffling on KNRK cells is caused by one or more soluble agents secreted by the KNRK cells themselves. To do this study, an assay for ruffling responses in live cell cultures was defined, and its reproducibility was demonstrated. This assay permitted observation of the kinetics of ruffling responses (percentage of cells ruffled as a function of time after stimulation). This method was used to compare the kinetics of ruffling induced by insulin, epidermal growth factor, fibroblast growth factor, glucose, and KNRK cell conditioned medium (CM). Ruffling was elicited on NRK cells by each of the polypeptide mitogens and nutrients, but, in each case, this ruffling subsided spontaneously within an hour. CM from KNRK cells also caused ruffling movements on untransformed NRK cells, but this ruffling continued for at least 20 h. This response was largely blocked by premixing the KNRK cell CM with rabbit IgG against rat transforming growth factor, type alpha, (TGF-alpha). KNRK cells made quiescent (ruffle free) by a pH shift (from 7.4 to 8.4) responded to insulin, glucose, and KNRK cell CM with kinetics similar to those observed for each of these factors in NRK cells. The unusual feature for the ruffle-inducing agent(s) produced by KNRK cells was that this activity was not subject, in either NRK or KNRK cells, to the cellular off-regulation that limits the responses to insulin or glucose. Thus, the continuous ruffling of KNRK cells is caused by their own unregulated ruffle-inducing agent or agents, which appear to include TGF-alpha. This work also demonstrates that kinetic analysis of cellular responses to exogenous factors can provide new insights into the regulatory mechanisms involved in the normal limitation of these responses.     

Cell-mediated co-action of transforming growth factors: incubation of type beta with normal rat kidney cells produces a soluble activity that prolongs the ruffling response to type alpha

Intense, continuous ruffling is a characteristic of many transformed cells, but untransformed cells ruffle intensely only briefly after exposure to growth factors. We reported previously that cells of a normal rat kidney (NRK) cell line transformed by Kirsten murine sarcoma virus secrete their own ruffle-inducing agent(s) that cause sustained ruffling in either themselves or untransformed NRK cells. In the present study, we examined the roles of the transforming growth factors TGF-alpha and TGF-beta in the induction and maintenance of ruffling in untransformed NRK cells and observed the following: TGF-alpha caused a transient epidermal growth factor (EGF)-like response, which could be blocked by prior exposure of cells to EGF or by antiserum directed against the COOH-terminus of TGF-alpha. TGF-beta caused no ruffling and did not itself prolong TGF-alpha ruffling. A new, buffer-soluble (transferable) mediator activity produced by incubation of TGF-beta with NRK cells for 6-h extended the duration of maximal TGF-alpha-induced ruffling by several-fold. This study demonstrates that TGF-alpha alone causes an EGF-like, transient ruffling response, but neither TGF-alpha or TGF-beta alone, nor the two together, cause transformation-associated sustained ruffling. Rather, TGF-alpha acts in concert with a new, TGF-beta-dependent activity. This new activity appears to inhibit normal cellular off-regulation of TGF-alpha-induced ruffling. Inhibition of the cellular off-regulation of a growth factor response could play a key role in the unregulated growth associated with malignancy.     

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells [published erratum appears in J Cell Biol 1990 Mar;110(3):859]

Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF- stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.     

Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways.     

Endocytosis: A Positive or a Negative Influence on Wnt Signalling?

Endocytosis, with subsequent targeting to lysosomes for degradation, is traditionally seen as a way for cells to terminate signalling. However, in a few instances, endocytosis has been demonstrated to contribute positively to signalling. Here we review recent work on the role of endocytosis in Wnt signalling. Biochemical evidence suggests that the branch of Wnt signalling that controls planar cell polarity (PCP) does require endocytosis, although how endocytosis of Frizzled receptors is translated into PCP in vivo remains unknown. With respect to the main signalling branch (called the canonical or beta-catenin pathway), the literature is divided as to whether endocytosis is required. Results of in vivo experiments are inconclusive because of the toxic side-effects of blocking endocytosis. Some results with cultured cells suggest the need for endocytosis in canonical signalling; however, it remains unclear whether the ligand-receptor complex must enter the cell by clathrin-mediated or caveolae-mediated endocytosis in order to signal. Means of specifically altering Wnt trafficking as well as of tracking the internalization route in different cell types are needed.     

Clathrin-dependent and independent endocytosis of glucose transporter 4 (GLUT4) in myoblasts: regulation by mitochondrial uncoupling

In myocytes and adipocytes, insulin increases glucose transporter 4 (GLUT4) exocytosis by promoting GLUT4 vesicle docking/fusion with the membrane. Less is known about the mechanism and regulation of GLUT4 endocytosis, particularly in myocytes. Here, we show that GLUT4 internalization in L6 myoblasts was inhibited in part by hypertonicity or clathrin heavy chain knockdown and in part by cholesterol depletion. Both strategies had additive effects, abolishing GLUT4 endocytosis. GLUT4 internalization was abrogated by expressing dominant-negative dynamin-2 but unaffected by inhibiting caveolar-dependent endocytosis through syntaxin-6 knockdown or caveolin mutants (which reduced lactosylceramide endocytosis). Insulin did not affect GLUT4 internalization rate or sensitivity to clathrin or cholesterol depletion. In contrast, the mitochondrial uncoupler dinitrophenol (DNP), which like insulin increases surface GLUT4, reduced GLUT4 (but not transferrin) internalization, an effect additive to that of depleting clathrin but not cholesterol. Trout GLUT4 (a natural variant of GLUT4 bearing different endocytic motifs) exogenously expressed in mammalian L6 cells internalized only through the cholesterol-dependent route that also included the non-clathrin-dependent cargo interleukin-2 receptor β, and DNP reduced internalization of both proteins. These results suggest that in muscle cells, GLUT4 internalizes simultaneously through clathrin-mediated endocytosis and a caveolae-independent but cholesterol- and dynamin-dependent route. Manipulating GLUT4 endocytosis to maintain surface GLUT4 may bypass insulin resistance.     

Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.     

Protein kinase C-dependent and -independent pathways in the growth factor-induced cytoskeletal reorganization

Human epidermoid carcinoma KB cells exhibit rapid induction of membrane ruffling in response to epidermal growth factor (EGF), insulin, and insulin-like growth factor-I (IGF-I) (Kadowaki, T., Koyasu, S., Nishida, E., Sakai, H., Takaku, F., Yahara, I., and Kasuga, M. (1986) J. Biol. Chem. 261, 16141-16147). We have analyzed the role of protein kinase C (PKC) in this response. Treatment of KB cells with 4 beta-phorbol 12,13-dibutyrate (PDBu) (100 ng/ml) for 30 min caused translocation of PKC to the membrane. This treatment completely inhibited the induction of membrane ruffling by EGF, insulin, and IGF-I. Prolonged treatment with PDBu (200 ng/ml for 15 h) induced complete depletion of the PKC activity in the cells. Under these conditions, EGF binding to cells and autophosphorylation of the EGF receptor occurred normally, while EGF could not induce membrane ruffling. In contrast, insulin- or IGF-I-induced membrane ruffling occurred normally in the PKC-depleted cells. Moreover, H-7 (PKC inhibitor) inhibited only EGF-induced membrane ruffling in a dose-dependent manner. We further found that EGF, but not insulin/IGF-I, caused transient translocation of PKC to the membrane. All these results suggest that PKC is required for the membrane ruffling induced by EGF but not for that induced by insulin/IGF-I. Therefore, there are PKC-dependent and independent pathways in the growth factor-induced membrane ruffling. Furthermore, we propose dual roles of PKC in the EGF signaling, a signal transmitting role and a negative feedback role.     

Endocytosis and exocytosis in hyphal growth

Two ancient processes, endocytosis and exocytosis, are employed by eukaryotic cells to shape their plasma membrane and interact with their environment. Filamentous fungi have adapted them to roles compatible with their unique ecological niche and morphology. These organisms are optimal systems in which to address questions such as how endocytosis is localized, how endocytosis and exocytosis interact, and how large molecules traverse eukaryotic cell walls. In the tips of filamentous (hyphal) cells, a ring of endocytosis encircles an apical crescent of exocytosis, suggesting that this area is able to support an endocytic recycling route, although both processes can occur in subapical regions as well. Endocytosis and exocytosis underlie growth, but also facilitate disease progression and secretion of industrially relevant compounds in these organisms. Here we highlight recent work on endocytosis and exocytosis in filamentous fungi.