Limits...
Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic.

Brown FD, Rozelle AL, Yin HL, Balla T, Donaldson JG - J. Cell Biol. (2001)

Bottom Line: PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment.Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L.These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institute of Health, Bethesda, MD 20892, USA.

ABSTRACT
ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

Show MeSH

Related in: MedlinePlus

Live cell dynamics of PIP2-labeled membranes after Arf6 activation. Cos cells were transfected with PH-GFP alone (A), EFA6 (B), or Arf6 Q67L (C) or PIP 5-kinase (D) and then imaged 18 h after transfection for ∼40 min. For serum add-back (E), PH-GFP–expressing cells were serum starved overnight. Serum (20%) was added immediately before imaging. Images of the subject cells taken at 0 and 40 min are shown. See also QuickTime videos 1–5 of each condition available at http://www.jcb.org/content/vol154/issue5. Each video shows the time points between the still images, are played at equivalent frame rates, and were recorded over the same approximate length of time. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196179&req=5

fig7: Live cell dynamics of PIP2-labeled membranes after Arf6 activation. Cos cells were transfected with PH-GFP alone (A), EFA6 (B), or Arf6 Q67L (C) or PIP 5-kinase (D) and then imaged 18 h after transfection for ∼40 min. For serum add-back (E), PH-GFP–expressing cells were serum starved overnight. Serum (20%) was added immediately before imaging. Images of the subject cells taken at 0 and 40 min are shown. See also QuickTime videos 1–5 of each condition available at http://www.jcb.org/content/vol154/issue5. Each video shows the time points between the still images, are played at equivalent frame rates, and were recorded over the same approximate length of time. Bars, 10 μm.

Mentions: As an alternative to AlF stimulation, we expressed EFA6 shown to be a specific GEF for Arf6 in in vitro assays and in cells (Franco et al., 1999). The majority of cells expressing EFA6 exhibited protrusions around the cell periphery where EFA6 and PIP2 colocalized (Fig. 2) . This phenotype could be observed in 57% of cells at 20 h and increased to 76% at 44 h after transfection (Table I). In about 5% of the cells, vacuolar endocytic structures that were positive for EFA6 and PH-GFP were observed in the central portion of the cells and also associated with the protrusions (Fig. 2 and Table I). However, this is an underestimate, since imaging of live cells expressing EFA6 revealed that all cells forming protrusions were undergoing active endocytosis and recycling (see Fig. 7) . These endosomal membranes also contained endogenous molecules that traffic through the Arf6 endosome such as major histocompatibility complex class I (MHC I; see Fig. 6) . EFA6-induced protrusion formation was inhibited in cells coexpressing the dominant negative mutant of Arf6, T27N (unpublished data), indicating that endogenous Arf6 mediates the effects of EFA6 as described previously (Franco et al., 1999). CD treatment also inhibited protrusions and resulted in localization of EFA6 on the tubular endosomal membranes (Fig. 2, bottom) as observed for Arf6 in Fig. 1.


Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic.

Brown FD, Rozelle AL, Yin HL, Balla T, Donaldson JG - J. Cell Biol. (2001)

Live cell dynamics of PIP2-labeled membranes after Arf6 activation. Cos cells were transfected with PH-GFP alone (A), EFA6 (B), or Arf6 Q67L (C) or PIP 5-kinase (D) and then imaged 18 h after transfection for ∼40 min. For serum add-back (E), PH-GFP–expressing cells were serum starved overnight. Serum (20%) was added immediately before imaging. Images of the subject cells taken at 0 and 40 min are shown. See also QuickTime videos 1–5 of each condition available at http://www.jcb.org/content/vol154/issue5. Each video shows the time points between the still images, are played at equivalent frame rates, and were recorded over the same approximate length of time. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196179&req=5

fig7: Live cell dynamics of PIP2-labeled membranes after Arf6 activation. Cos cells were transfected with PH-GFP alone (A), EFA6 (B), or Arf6 Q67L (C) or PIP 5-kinase (D) and then imaged 18 h after transfection for ∼40 min. For serum add-back (E), PH-GFP–expressing cells were serum starved overnight. Serum (20%) was added immediately before imaging. Images of the subject cells taken at 0 and 40 min are shown. See also QuickTime videos 1–5 of each condition available at http://www.jcb.org/content/vol154/issue5. Each video shows the time points between the still images, are played at equivalent frame rates, and were recorded over the same approximate length of time. Bars, 10 μm.
Mentions: As an alternative to AlF stimulation, we expressed EFA6 shown to be a specific GEF for Arf6 in in vitro assays and in cells (Franco et al., 1999). The majority of cells expressing EFA6 exhibited protrusions around the cell periphery where EFA6 and PIP2 colocalized (Fig. 2) . This phenotype could be observed in 57% of cells at 20 h and increased to 76% at 44 h after transfection (Table I). In about 5% of the cells, vacuolar endocytic structures that were positive for EFA6 and PH-GFP were observed in the central portion of the cells and also associated with the protrusions (Fig. 2 and Table I). However, this is an underestimate, since imaging of live cells expressing EFA6 revealed that all cells forming protrusions were undergoing active endocytosis and recycling (see Fig. 7) . These endosomal membranes also contained endogenous molecules that traffic through the Arf6 endosome such as major histocompatibility complex class I (MHC I; see Fig. 6) . EFA6-induced protrusion formation was inhibited in cells coexpressing the dominant negative mutant of Arf6, T27N (unpublished data), indicating that endogenous Arf6 mediates the effects of EFA6 as described previously (Franco et al., 1999). CD treatment also inhibited protrusions and resulted in localization of EFA6 on the tubular endosomal membranes (Fig. 2, bottom) as observed for Arf6 in Fig. 1.

Bottom Line: PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment.Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L.These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institute of Health, Bethesda, MD 20892, USA.

ABSTRACT
ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

Show MeSH
Related in: MedlinePlus