Limits...
Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J - Mol. Biol. Cell (2011)

Bottom Line: Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin.Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3.We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Switzerland.

ABSTRACT
Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Show MeSH

Related in: MedlinePlus

Cholesterol accumulation does not impair the release of hexosaminidase and the transport of Lamp1 to the plasma membrane. (A) BHK cells treated with 3 mg/ml U18666A for 16 h (“16h U18”) were stimulated or not with ionomycin as in Figure 1A, labeled with anti-Lamp1 antibodies before fixation, and stained with filipin to reveal cholesterol. Bars = 10 μm. (B and C) Cells were treated with U18666A before stimulation with ionomycin as in (A), and hexosaminidase release (B) was measured as in Figure 1A. Mean ± SEM of three experiments is shown. In parallel, cell surface biotinylation (C) was analyzed as in Figure 1D. (D) Control cells or cells treated with U18666A were fixed, permeabilized, and double-labeled with filipin and anti-Lamp1 antibodies. High magnification view of the boxed area shows Lamp1-positive structures in the cell periphery that are not labeled by filipin (arrowheads). (E) BHK cells were transfected with Lamp1-GFP, treated with U18666A, fixed, and labeled with filipin. Basal slice of Z-stacking is shown in comparison to an equatorial Z slice (right panel, only merge shown). Arrowheads point at peripheral Lamp1-GFP structures devoid of cholesterol.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113771&req=5

Figure 2: Cholesterol accumulation does not impair the release of hexosaminidase and the transport of Lamp1 to the plasma membrane. (A) BHK cells treated with 3 mg/ml U18666A for 16 h (“16h U18”) were stimulated or not with ionomycin as in Figure 1A, labeled with anti-Lamp1 antibodies before fixation, and stained with filipin to reveal cholesterol. Bars = 10 μm. (B and C) Cells were treated with U18666A before stimulation with ionomycin as in (A), and hexosaminidase release (B) was measured as in Figure 1A. Mean ± SEM of three experiments is shown. In parallel, cell surface biotinylation (C) was analyzed as in Figure 1D. (D) Control cells or cells treated with U18666A were fixed, permeabilized, and double-labeled with filipin and anti-Lamp1 antibodies. High magnification view of the boxed area shows Lamp1-positive structures in the cell periphery that are not labeled by filipin (arrowheads). (E) BHK cells were transfected with Lamp1-GFP, treated with U18666A, fixed, and labeled with filipin. Basal slice of Z-stacking is shown in comparison to an equatorial Z slice (right panel, only merge shown). Arrowheads point at peripheral Lamp1-GFP structures devoid of cholesterol.

Mentions: As expected, cholesterol was predominantly present in the plasma membrane of control cells and accumulated in perinuclear structures containing Lamp1 after drug treatment (Figure 2, A and D). Strikingly, however, U18666A had essentially no effect on hexosaminidase release (Figure 2B) and Lamp1 appearance at the plasma membrane measured by light microscopy (Figure 2A), or cell surface biotinylation (Figure 2C). Neither was the small increase in Tfn-R biotinylation observed after ionomycin treatment sensitive to U18666A treatment. Similarly, endocytosed antibodies against the late endocytic lipid lysobisphosphatidic acid (LBPA; Kobayashi et al., 1998), which also cause cholesterol accumulation in late endocytic compartments and phenocopy NPC (Kobayashi et al., 1999), failed to affect Lamp1 appearance at the plasma membrane in ionomycin-treated cells (Supplemental Figure S1A).


Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Laulagnier K, Schieber NL, Maritzen T, Haucke V, Parton RG, Gruenberg J - Mol. Biol. Cell (2011)

Cholesterol accumulation does not impair the release of hexosaminidase and the transport of Lamp1 to the plasma membrane. (A) BHK cells treated with 3 mg/ml U18666A for 16 h (“16h U18”) were stimulated or not with ionomycin as in Figure 1A, labeled with anti-Lamp1 antibodies before fixation, and stained with filipin to reveal cholesterol. Bars = 10 μm. (B and C) Cells were treated with U18666A before stimulation with ionomycin as in (A), and hexosaminidase release (B) was measured as in Figure 1A. Mean ± SEM of three experiments is shown. In parallel, cell surface biotinylation (C) was analyzed as in Figure 1D. (D) Control cells or cells treated with U18666A were fixed, permeabilized, and double-labeled with filipin and anti-Lamp1 antibodies. High magnification view of the boxed area shows Lamp1-positive structures in the cell periphery that are not labeled by filipin (arrowheads). (E) BHK cells were transfected with Lamp1-GFP, treated with U18666A, fixed, and labeled with filipin. Basal slice of Z-stacking is shown in comparison to an equatorial Z slice (right panel, only merge shown). Arrowheads point at peripheral Lamp1-GFP structures devoid of cholesterol.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113771&req=5

Figure 2: Cholesterol accumulation does not impair the release of hexosaminidase and the transport of Lamp1 to the plasma membrane. (A) BHK cells treated with 3 mg/ml U18666A for 16 h (“16h U18”) were stimulated or not with ionomycin as in Figure 1A, labeled with anti-Lamp1 antibodies before fixation, and stained with filipin to reveal cholesterol. Bars = 10 μm. (B and C) Cells were treated with U18666A before stimulation with ionomycin as in (A), and hexosaminidase release (B) was measured as in Figure 1A. Mean ± SEM of three experiments is shown. In parallel, cell surface biotinylation (C) was analyzed as in Figure 1D. (D) Control cells or cells treated with U18666A were fixed, permeabilized, and double-labeled with filipin and anti-Lamp1 antibodies. High magnification view of the boxed area shows Lamp1-positive structures in the cell periphery that are not labeled by filipin (arrowheads). (E) BHK cells were transfected with Lamp1-GFP, treated with U18666A, fixed, and labeled with filipin. Basal slice of Z-stacking is shown in comparison to an equatorial Z slice (right panel, only merge shown). Arrowheads point at peripheral Lamp1-GFP structures devoid of cholesterol.
Mentions: As expected, cholesterol was predominantly present in the plasma membrane of control cells and accumulated in perinuclear structures containing Lamp1 after drug treatment (Figure 2, A and D). Strikingly, however, U18666A had essentially no effect on hexosaminidase release (Figure 2B) and Lamp1 appearance at the plasma membrane measured by light microscopy (Figure 2A), or cell surface biotinylation (Figure 2C). Neither was the small increase in Tfn-R biotinylation observed after ionomycin treatment sensitive to U18666A treatment. Similarly, endocytosed antibodies against the late endocytic lipid lysobisphosphatidic acid (LBPA; Kobayashi et al., 1998), which also cause cholesterol accumulation in late endocytic compartments and phenocopy NPC (Kobayashi et al., 1999), failed to affect Lamp1 appearance at the plasma membrane in ionomycin-treated cells (Supplemental Figure S1A).

Bottom Line: Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin.Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3.We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Switzerland.

ABSTRACT
Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Show MeSH
Related in: MedlinePlus