scholarly journals Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase.

1996 ◽  
Vol 134 (4) ◽  
pp. 935-947 ◽  
Author(s):  
H Radhakrishna ◽  
R D Klausner ◽  
J G Donaldson
Keyword(s):  
Plasma Membrane ◽  
Aluminum Fluoride ◽  
Wild Type ◽  
Lipoxygenase Pathway ◽  
Transfected Cells ◽  
Defective Mutant ◽  
Transferrin Uptake ◽  
Adp Ribosylation Factor ◽  
Stimulation Of ◽  
In Cells

To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.

Na+/H+ exchanger isoform 6 (NHE6/SLC9A6) is involved in clathrin-dependent endocytosis of transferrin

2011 ◽  
Vol 301 (6) ◽  
pp. C1431-C1444 ◽  
Author(s):  
Lou Xinhan ◽  
Masafumi Matsushita ◽  
Manami Numaza ◽  
Akira Taguchi ◽  
Keiji Mitsui ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Ph Regulation ◽  
Microscopic Analysis ◽  
Cholera Toxin B Subunit ◽  
Wild Type ◽  
B Subunit ◽  
Epidermal Growth ◽  
Transferrin Uptake ◽  
In Cells

In mammalian cells, nine conserved isoforms of the Na+/H+ exchanger (NHE) are known to be important for pH regulation of the cytoplasm and organellar lumens. NHE1–5 are localized to the plasma membrane, whereas NHE6–9 are localized to distinct organelles. NHE6 is localized predominantly in endosomal compartments but is also found in the plasma membrane. To investigate the role of NHE6 in endocytosis, we established NHE6-knockdown HeLa cells and analyzed the effect of this knockdown on endocytotic events. The expression level of NHE6 in knockdown cells was decreased to ∼15% of the level seen in control cells. Uptake of transferrin was also decreased. No effect was found on the endocytosis of epidermal growth factor or on the cholera toxin B subunit. Moreover, in the NHE6-knockdown cells, transferrin uptake was found to be affected in the early stages of endocytosis. Microscopic analysis revealed that, at 2 min after the onset of endocytosis, colocalization of NHE6, clathrin, and transferrin was observed, which suggests that NHE6 was localized to endocytotic, clathrin-coated vesicles. In addition, in knockdown cells, transferrin-positive endosomes were acidified, but no effect was found on cytoplasmic pH. In cells overexpressing wild-type NHE6, increased transferrin uptake was observed, but no such increase was seen in cells overexpressing mutant NHE6 deficient in ion transport. The luminal pH in transferrin-positive endosomes was alkalized in cells overexpressing wild-type NHE6 but normal in cells overexpressing mutant NHE6. These observations suggest that NHE6 regulates clathrin-dependent endocytosis of transferrin via pH regulation.

scholarly journals An Effector Domain Mutant of Arf6 Implicates Phospholipase D in Endosomal Membrane Recycling

2006 ◽  
Vol 17 (1) ◽  
pp. 327-335 ◽  
Author(s):  
Olivera A. Jovanovic ◽  
Fraser D. Brown ◽  
Julie G. Donaldson
Keyword(s):  
Phospholipase D ◽  
Aluminum Fluoride ◽  
Membrane Recycling ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Endosomal Membrane ◽  
Endosomal Membranes ◽  
Stimulation Of ◽  
In Cells

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.

scholarly journals ADP-Ribosylation Factor 6 Regulates a Novel Plasma Membrane Recycling Pathway

1997 ◽  
Vol 139 (1) ◽  
pp. 49-61 ◽  
Author(s):  
Harish Radhakrishna ◽  
Julie G. Donaldson
Keyword(s):  
Plasma Membrane ◽  
Bound State ◽  
Wild Type ◽  
Α Subunit ◽  
Adp Ribosylation ◽  
Defective Mutant ◽  
Membrane Compartment ◽  
Recycling Pathway ◽  
Mutant Forms ◽  
Adp Ribosylation Factor

ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor α subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

scholarly journals ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ

2003 ◽  
Vol 162 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Michael Krauss ◽  
Masahiro Kinuta ◽  
Markus R. Wenk ◽  
Pietro De Camilli ◽  
Kohji Takei ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Synaptic Membranes ◽  
Direct Stimulation ◽  
Vesicle Membranes ◽  
Phosphatidylinositol Phosphate ◽  
Presynaptic Plasma Membrane ◽  
Phosphatidylinositol 4 ◽  
Adp Ribosylation Factor ◽  
Stimulation Of

Clathrin-mediated endocytosis of synaptic vesicle membranes involves the recruitment of clathrin and AP-2 adaptor complexes to the presynaptic plasma membrane. Phosphoinositides have been implicated in nucleating coat assembly by directly binding to several endocytotic proteins including AP-2 and AP180. Here, we show that the stimulatory effect of ATP and GTPγS on clathrin coat recruitment is mediated at least in part by increased levels of PIP2. We also provide evidence for a role of ADP-ribosylation factor 6 (ARF6) via direct stimulation of a synaptically enriched phosphatidylinositol 4-phosphate 5-kinase type Iγ (PIPKIγ), in this effect. These data suggest a model according to which activation of PIPKIγ by ARF6-GTP facilitates clathrin-coated pit assembly at the synapse.

scholarly journals ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucyl-phenylalanine stimulation of human neutrophils

1997 ◽  
Vol 325 (3) ◽  
pp. 581-585 ◽  
Author(s):  
C. P. MORGAN ◽  
H. SENGELOV ◽  
J. WHATMORE ◽  
N. BORREGAARD ◽  
S. COCKCROFT
Keyword(s):  
Plasma Membrane ◽  
Phospholipase D ◽  
Small Gtpases ◽  
Human Neutrophils ◽  
Secretory Vesicles ◽  
Rho Proteins ◽  
Adp Ribosylation ◽  
Activated Neutrophils ◽  
Adp Ribosylation Factor ◽  
Stimulation Of

Phospholipase D (PLD) is responsible for the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Human neutrophils contain PLD activity which is regulated by the small GTPases, ADP-ribosylation factor (ARF) and Rho proteins. In this study we have examined the subcellular localization of the ARF-regulated PLD activity in non-activated neutrophils and cells ‘primed‘ with N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe). We report that PLD activity is localized at the secretory vesicles in control cells and is mobilized to the plasma membrane upon stimulation with fMetLeuPhe. We conclude that the ARF-regulated PLD activity is translocated to the plasma membrane by secretory vesicles upon stimulation of neutrophils with fMetLeuPhe in inflammatory/priming doses. We propose that this relocalization of PLD is important for the subsequent events occurring during neutrophil activation.

Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human proCRH gene: intracellular storage and regulated secretion

1991 ◽  
Vol 7 (2) ◽  
pp. 97-104 ◽  
Author(s):  
M. G. Castro ◽  
J. Brooke ◽  
A. Bullman ◽  
M. Hannah ◽  
B. P. Glynn ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Tumour Cell Line ◽  
Wild Type ◽  
Transfected Cells ◽  
Releasing Hormone ◽  
Intracellular Storage ◽  
Processed Products ◽  
Crh Receptors ◽  
Stimulation Of ◽  
Acth Release

ABSTRACT The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1–76), ACTH, β-lipotrophin and β-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. ArT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1–41). On stimulation with the secretagogue noradrenaline, CRH(1–41) was released into the medium, while the precursor was not. Whilst treatment of wild-type ArT-20 cells with exogenous CRH(1–41) (1 nm) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.

ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements

1999 ◽  
Vol 112 (6) ◽  
pp. 855-866 ◽  
Author(s):  
H. Radhakrishna ◽  
O. Al-Awar ◽  
Z. Khachikian ◽  
J.G. Donaldson
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Dominant Negative ◽  
Aluminum Fluoride ◽  
Cortical Actin ◽  
Primary Human Fibroblast ◽  
Recycling Endosome ◽  
Gtp Binding ◽  
Rho Family ◽  
In Cells

The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.

scholarly journals Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP

2001 ◽  
Vol 155 (7) ◽  
pp. 1307-1318 ◽  
Author(s):  
Erica M. TenBroek ◽  
Paul D. Lampe ◽  
Joell L. Solan ◽  
James K. Reynhout ◽  
Ross G. Johnson
Keyword(s):  
Plasma Membrane ◽  
Negative Charge ◽  
Molar Ratio ◽  
Wild Type ◽  
Positive Regulation ◽  
Major Site ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Cx 43 ◽  
Stimulation Of

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

scholarly journals A Human Dynamin-related Protein Controls the Distribution of Mitochondria

1998 ◽  
Vol 143 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Elena Smirnova ◽  
Dixie-Lee Shurland ◽  
Sergey N. Ryazantsev ◽  
Alexander M. van der Bliek
Keyword(s):  
Environmental Changes ◽  
Large Mass ◽  
Mitochondrial Morphology ◽  
Related Protein ◽  
Wild Type ◽  
Gtpase Domain ◽  
Transfected Cells ◽  
Organelle Morphology ◽  
Endocytic Pathways ◽  
In Cells

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

scholarly journals Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

1995 ◽  
Vol 128 (6) ◽  
pp. 1003-1017 ◽  
Author(s):  
P J Peters ◽  
V W Hsu ◽  
C E Ooi ◽  
D Finazzi ◽  
S B Teal ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Membrane System ◽  
Endocytic Pathway ◽  
Coat Proteins ◽  
Wild Type ◽  
Gtp Binding ◽  
Defective Mutant

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

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