Neurodegeneration in Niemann-Pick Type C disease and Huntington's disease: impact of defects in membrane trafficking.

Although neurodegenerative diseases exhibit distinct pathologies, such as affected neuronal cell population, age of onset, and pathological symptoms, overlapping characteristics can be observed at the cellular level. In particular, several neurodegenerative diseases display defects in intracellular vesicular trafficking. Here we discuss the range of cellular phenotypes observed in two rare neurodegenerative diseases, Niemann-Pick Type C and Huntington's Disease, both of which involve vesicular trafficking defects that may contribute to neuronal cell death. In NPC, the primary defect is cholesterol and glycosphingolipid accumulation, but NPC mutant cells display widespread trafficking alterations. In HD, protein aggregates are a hallmark feature, but HD cells also exhibit changes in vesicular traffic, including axonal transport and early endosomal trafficking, that likely impact neuronal cell viability. Here we discuss current therapies that seek to address cellular defects in NPC and HD and describe areas of investigation that may lead to new therapeutic treatment.

Efficient uptake of Yersinia pseudotuberculosis via integrin receptors involves a Rac1-Arp 2/3 pathway that bypasses N-WASP function.

Efficient uptake of Yersinia pseudotuberculosis into cultured mammalian cells is the result of high-affinity binding of invasin to beta1 chain integrins. We demonstrate here that uptake requires Rac1 and Arp 2/3 function. Bacterial uptake was stimulated by GTPgammaS, but was inhibited in mammalian cells transfected with the interfering Rac1-N17 derivative. Rac1 was found to be activated in response to integrin engagement by invasin, whereas Rac1 and Arp 2/3 were found to be intensely localized around phagosomes bearing bacteria, indicating a specific role for Rac1 signalling from the nascent phagosome to downstream effectors. To determine whether the Arp 2/3 complex was a component of this proposed pathway, cells overproducing various derivatives of Scar1/WAVE1, an Arp 2/3-binding protein, were analysed. Sequestration of Arp 2/3 away from the phagocytic cup as a result of Scar1/WAVE1 overproduction dramatically inhibited uptake. To determine whether signalling from Rac1 to Arp 2/3 occurred via N-WASP, uptake was analysed in a cell line lacking expression of WASP and N-WASP. Uptake was unaffected by the absence of these proteins, indicating that beta1 integrin signalling from Rac1 to Arp 2/3 can occur in the absence of N-WASP function.

An essential role for ARF6-regulated membrane traffic in adherens junction turnover and epithelial cell migration.

We describe a novel role for the ARF6 GTPase in the regulation of adherens junction (AJ) turnover in MDCK epithelial cells. Expression of a GTPase-defective ARF6 mutant, ARF6(Q67L), led to a loss of AJs and ruffling of the lateral plasma membrane via mechanisms that were mutually exclusive. ARF6-GTP-induced AJ disassembly did not require actin remodeling, but was dependent on the internalization of E-cadherin into the cytoplasm via vesicle transport. ARF6 activation was accompanied by increased migratory potential, and treatment of cells with hepatocyte growth factor (HGF) induced the activation of endogenous ARF6. The effect of ARF6(Q67L) on AJs was specific since ARF6 activation did not perturb tight junction assembly or cell polarity. In contrast, dominant-negative ARF6, ARF6(T27N), localized to AJs and its expression blocked cell migration and HGF-induced internalization of cadherin-based junctional components into the cytoplasm. Finally, we show that ARF6 exerts its role downstream of v-Src activation during the disassembly of AJs. These findings document an essential role for ARF6- regulated membrane traffic in AJ disassembly and epithelial cell migration.

ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA.

In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Galphaq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.

Actin assembly at membranes controlled by ARF6.

The small GTPase, ADP-ribosylation factor-6 (ARF6), has been implicated in regulating membrane traffic and remodeling cortical F-actin. Using real-time video analysis of actin assembly in living cells, we investigated the function and mechanism of ARF6 in control of actin assembly. Expression of an activated form of ARF6 that mimicks the GTP-bound form of the GTPase induced actin assembly resulting in the movement of vesicle-like particles, some of which contain markers for pinosomes. Activated ARF6 also stimulated actin assembly at foci on the ventral surface of the cell and stimulated fluid phase pinocytosis. Particle motility induced by ARF6 involved Arp2/3 complex, tyrosine kinase activity, phospholipase D (PLD) and D3-phosphoinositides, but not phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). We conclude that ARF6 regulates actin assembly for pinosome motility and at foci on the ventral cell surface.

EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization.

We have identified a human cDNA encoding a novel protein, exchange factor for ARF6 (EFA6), which contains Sec7 and pleckstrin homology domains. EFA6 promotes efficient guanine nucleotide exchange on ARF6 and is distinct from the ARNO family of ARF1 exchange factors. The protein localizes to a dense matrix on the cytoplasmic face of plasma membrane invaginations, induced on its expression. We show that EFA6 regulates endosomal membrane recycling and promotes the redistribution of transferrin receptors to the cell surface. Furthermore, expression of EFA6 induces actin-based membrane ruffles that are inhibited by co-expression of dominant-inhibitory mutant forms of ARF6 or Rac1. Our results demonstrate that by catalyzing nucleotide exchange on ARF6 at the plasma membrane and by regulating Rac1 activation, EFA6 coordinates endocytosis with cytoskeletal rearrangements.

Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes.

Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte-ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70(S6 kinase) but appeared to involve a phospholipid kinase.

Subcellular distribution and differential expression of endogenous ADP-ribosylation factor 6 in mammalian cells.

ADP-ribosylation factor (ARF) 6 has been shown to play a role in vesicular transport; however, the expression and subcellular localization of the endogenous protein have not been clearly delineated. In this study, an ARF6-specific monoclonal antibody was raised and used to examine the subcellular distribution and expression of ARF6 in various tissues and during the differentiation of several well characterized cell types. We found that ARF6 localizes in both the cytosol and membranes of all tissues and cells tested. Moreover, ARF6 in 3T3-L1 adipocytes is principally localized on the plasma membrane, but substantial amounts are detected in the cytosolic and intracellular membrane fractions. We observed an increased expression of ARF6 during the differentiation of B lymphocytes to plasmocytes. However, the expression of ARF6 decreased during adipogenesis and monocyte differentiation. In contrast, the expression of other ARFs, detected by the monoclonal antibody 1D9, did not significantly change during differentiation of the aforementioned cell types. Taken together, our results indicate that ARF6 is a broadly expressed, differentially regulated GTPase that is present in cytoplasm and on both cell-surface and intracellular membranes and whose functions may include tissue-specific effects on vesicular trafficking during cellular differentiation.

ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

A role for POR1, a Rac1-interacting protein, in ARF6-mediated cytoskeletal rearrangements.

The ARF6 GTPase, the least conserved member of the ADP ribosylation factor (ARF) family, associates with the plasma membrane and intracellular endosome vesicles. Mutants of ARF6 defective in GTP binding and hydrolysis have a marked effect on endocytic trafficking and the gross morphology of the peripheral membrane system. Here we report that expression of the GTPase-defective mutant of ARF6, ARF6(Q67L), remodels the actin cytoskeleton by inducing actin polymerization at the cell periphery. This cytoskeletal rearrangement was inhibited by co-expression of ARF6(Q67L) with deletion mutants of POR1, a Rac1-interacting protein involved in membrane ruffling, but not with the dominant-negative mutant of Rac1, Rac1(S17N). A synergistic effect between POR1 and ARF6 for the induction of actin polymerization was detected. Furthermore, we observed that ARF6 interacts directly with POR1 and that this interaction was GTP dependent. These findings indicate that ARF6 and Rac1 function on distinct signaling pathways to mediate cytoskeletal reorganization, and suggest a role for POR1 as an important regulatory element in orchestrating cytoskeletal rearrangements at the cell periphery induced by ARF6 and Rac1.

Uncoupling of membrane ruffling and pinocytosis during Ras signal transduction.

Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, our study demonstrates that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant, via a Sindbis virus vector, resulted in a dramatic stimulation of membrane ruffling without affecting the uptake of horseradish peroxidase. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulated both membrane ruffling and horseradish peroxidase uptake. The Ha-Ras(G12V)-stimulated pinocytosis but not membrane ruffling was abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not membrane ruffling. Our data indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling.

Myristoylation is required for the intracellular localization and endocytic function of ARF6.

ADP-ribosylation factors (ARFs) are modified by a myristate group that is covalently linked to the second glycine residue at the amino terminus. With the recent evidence that ARF6 localizes to the cell periphery and plays a regulatory role in endocytic traffic, we have investigated the role of myristoylation on the membrane association, biological activity, and subcellular distribution of ARF6 in intact cells. A Gly2Ala mutation produced a nonmyristoylated protein that failed to associate with membranes, was cytosolic, and had no effect on endocytic transport. To determine if a different form of lipid modification could restore membrane association and biological activity, a nonmyristoylated ARF6 derivative was constructed that contained a prenyl group at the carboxy terminus. Prenylated ARF6 bound efficiently to membranes, but had no effect on receptor-mediated endocytosis and was mislocalized to distinct intracellular structures. Thus, although prenylation can replace myristoylation for membrane association, the latter appears to be critical for the proper targeting and biological activity of ARF6.

Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic.

Trimeric G proteins have emerged as important regulators of membrane trafficking. To explore a role for G beta gamma in endosome fusion, we have taken advantage of beta-adrenergic receptor kinase (beta ARK), an enzyme translocated to membranes by interaction with G beta gamma. The COOH terminus of beta ARK (beta ARKct) has a G beta gamma-binding domain which blocks some G beta gamma-mediated processes. We found that beta ARKct and peptide G, a peptide derived from beta ARKct, inhibit in vitro endosome fusion. Interestingly, peptide G and ARF share sequence similarity. Peptide G and beta ARKct reversed ARF-mediated inhibition of endosome fusion and blocked ARF binding to membranes. Using an ARF fusion protein, we show that both G beta gamma and G alpha s interact with the small GTPase ARF, an interaction that is regulated by nucleotide binding. We conclude that G proteins may participate in the regulation of vesicular trafficking by directly interacting with ARF, a cytosolic factor required for transport.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

A regulatory role for ARF6 in receptor-mediated endocytosis.

Adenosine diphosphate-ribosylation factor 6 (ARF6), ARF6 mutants, and ARF1 were transiently expressed in Chinese hamster ovary cells, and the effects on receptor-mediated endocytosis were assessed. Overexpressed ARF6 localized to the cell periphery and led to a redistribution of transferrin receptors to the cell surface and a decrease in the rate of uptake of transferrin. Similar results were obtained when a mutant defective in guanosine triphosphate hydrolysis was expressed. Expression of a dominant negative mutant, ARF6(T27N), resulted in an intracellular distribution of transferrin receptors and an inhibition of transferrin recycling to the cell surface. In contrast, overexpression of ARF1 had little or no effect on these parameters of endocytosis.

Mammalian glycosyltransferases prefer glycosyl phosphoryl dolichols rather than glycosyl phosphoryl polyprenols as substrates for oligosaccharyl synthesis.

We have studied the effectiveness of polyprenyl-P-mannose and polyprenol-P-glucose as donor substrates for the dolichyl-P-mannose:Man5(GlcNAc)2-PP-dolichol mannosyltransferase and the dolichyl-P-glucose:Man9(GlcNAc)2-PP-dolichol glucosyltransferase, respectively. The polyprenol moiety differs from dolichol only in the unsaturation of the terminal isoprene unit of the molecule. Based on the kinetics of the reactions, we have found that both glycosyltransferases have higher apparent Kms and lower apparent Vmaxs using polyprenyl-P-monosaccharides as substrates rather than the dolichyl-P-monosaccharides. The products formed with the polyprenyl-P-sugars were the same as those formed by the dolichol-linked sugars, indicating that the polyprenol substrates could be utilized by the glycosyltransferases in vitro. The results also indicate that the dolichyl-P-sugars and the polyprenyl-P-sugars compete for the same binding site on the enzyme. These findings are significant in terms of understanding the glycosylation phenotypes of Chinese hamster ovary cell mutants of the Lec9 complementation group, which lack the ability to convert polyprenol into dolichol.

Partial purification and properties of a glucosyltransferase that synthesizes Glc1Man9(GlcNAc)2-pyrophosphoryldolichol.

The glucosyltransferase that transfers the first glucose residue from dolichyl-P-glucose to Man9-(GlcNAc)2-PP-dolichol has been solubilized from porcine aorta and purified 720-fold. The purification strategy involved ammonium sulfate precipitation followed by ion-exchange, gel filtration, and hydroxylapatite column chromatographies. Analysis of the products produced by enzyme fractions at different stages of purification indicate that three different glucosyltransferases are involved in the conversion of Man9(GlcNAc)2-PP-dolichol to Glc3Man9(GlcNAc)2PP-dolichol. the first glucosyltransferase appears to be specific for dolichyl-P-glucose as the donor substrate. Man9(GlcNAc)2-PP-dolichol, Man7(GlcNAc)2-PP-dolichol, and Man5(GlcNAc)2-PP-dolichol (with two different oligosaccharide structures) were tested for their ability to accept glucose from dolichyl-P-glucose. Studies on the comparative rates of transfer of glucose to these different acceptor substrates demonstrated that Man9(GlcNAc)2-PP-dolichol accepts glucose at a higher initial rate and to a greater extent than does Man7(GlcNAc)2-PP-dolichol and the biosynthetic Man5(GlcNAc)2-PP-dolichol. The other Man5(GlcNAc)2-PP-dolichol (i.e. Man alpha 1,6[Man alpha 1,3]-Man alpha 1,6[Man alpha 1,3]Man beta 1,4GlcNAc beta 1, GlcNAc) was not an acceptor, indicating that the Man alpha 1,2-Man alpha 1,2Man alpha 1,3Man arm is necessary. Man9(Glc-NAc)2 and Man9(GlcNAc)2-protein were not acceptors, indicating that both the lipid and the oligosaccharide portion of Man9(GlcNAc)2-PP-dolichol are required for enzyme activity. The partially purified enzyme has a pH optimum of 6.5 and exhibits a requirement for divalent metal ions.