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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.

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Related in: MedlinePlus

Ionomycin-induced release of hexosaminidase and transport of Lamp1 to the plasma membrane. (A and B) A431 cells (A) or BHK cells (B) were stimulated with 5 μM ionomycin (Iono) or not (NS) for the indicated time. Then hexosaminidase or LDH activity was measured in the medium and in total cell lysates. The enzyme activity released in the medium is expressed as a percentage of the total activity in lysates. (A) shows the mean ± SEM of five and four experiments for hexosaminidase and LDH, respectively, and (B) shows typical kinetics of ionomycin stimulation with or without 1 mM EGTA. No LDH release was observed before 10 min of ionomycin stimulation. (C) BHK cells were stimulated or not as in A and incubated on ice with anti-Lamp1 antibodies without permeabilization (left column, “cell surface Lamp1”), fixed with PFA and labeled with fluorescent secondary antibody. Pictures after Z-stacking show homogeneous Lamp1 distribution on the cell surface (“Basal”) and a clear peripheral labeling at the equatorial Z-position (“Equatorial”). Alternatively, cells were fixed, permeabilized, and labeled with anti-Lamp1 antibodies to reveal total Lamp1 (right column). After stimulation, we observed little change in the distribution of intracellular Lamp1, but peripheral labeling was sometimes observed (arrowhead) as shown in the high magnification view of the boxed area. Bars = 5 mm. (D–F) After stimulation as in A and B, cell surface proteins of BHK (D) or A431 (E and F) were biotinylated (“Biot”) on ice and lysed. In controls, the biotinylation agent was omitted (NoB). Lysates were incubated with streptavidin-coated beads, and bound biotinylated proteins (“strepta-bound”) were eluted, separated by SDS gel, and analyzed by Western blotting using the indicated antibodies. (D) 3% and (E) 2% from nonstimulated lysates were separated in parallel. (F) The experiment in E was quantified, and the ratio of stimulated versus nonstimulated biotinylated-Lamp1 signals is shown. The results were normalized to biot-TfnR signal and are expressed as “fold increase.”
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Figure 1: Ionomycin-induced release of hexosaminidase and transport of Lamp1 to the plasma membrane. (A and B) A431 cells (A) or BHK cells (B) were stimulated with 5 μM ionomycin (Iono) or not (NS) for the indicated time. Then hexosaminidase or LDH activity was measured in the medium and in total cell lysates. The enzyme activity released in the medium is expressed as a percentage of the total activity in lysates. (A) shows the mean ± SEM of five and four experiments for hexosaminidase and LDH, respectively, and (B) shows typical kinetics of ionomycin stimulation with or without 1 mM EGTA. No LDH release was observed before 10 min of ionomycin stimulation. (C) BHK cells were stimulated or not as in A and incubated on ice with anti-Lamp1 antibodies without permeabilization (left column, “cell surface Lamp1”), fixed with PFA and labeled with fluorescent secondary antibody. Pictures after Z-stacking show homogeneous Lamp1 distribution on the cell surface (“Basal”) and a clear peripheral labeling at the equatorial Z-position (“Equatorial”). Alternatively, cells were fixed, permeabilized, and labeled with anti-Lamp1 antibodies to reveal total Lamp1 (right column). After stimulation, we observed little change in the distribution of intracellular Lamp1, but peripheral labeling was sometimes observed (arrowhead) as shown in the high magnification view of the boxed area. Bars = 5 mm. (D–F) After stimulation as in A and B, cell surface proteins of BHK (D) or A431 (E and F) were biotinylated (“Biot”) on ice and lysed. In controls, the biotinylation agent was omitted (NoB). Lysates were incubated with streptavidin-coated beads, and bound biotinylated proteins (“strepta-bound”) were eluted, separated by SDS gel, and analyzed by Western blotting using the indicated antibodies. (D) 3% and (E) 2% from nonstimulated lysates were separated in parallel. (F) The experiment in E was quantified, and the ratio of stimulated versus nonstimulated biotinylated-Lamp1 signals is shown. The results were normalized to biot-TfnR signal and are expressed as “fold increase.”

Mentions: We used the calcium ionophore, ionomycin, to induce a transient rise in cytosolic calcium concentration. To avoid possible toxic effects of the drug, we optimized the conditions of the treatment (see Materials and Methods) so that cell survival (unpublished data) was not affected. Cell integrity was measured by the release of the abundant cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium (Figure 1A). Because LDH release occurred after prolonged incubation with ionomycin (Figure 1B), all studies were performed at earlier time points (≤10 min). After the ionomycin treatment, small but significant amounts of hexosaminidase, a lysosomal enzyme, were released into the extracellular medium, corresponding to ≈1% of the total cell-associated activity in A431 (Figure 1A) and BHK cells (Figure 1B). This release was abolished by ethylene glycol tetra­acetic acid (EGTA) in the medium (Figure 1B). Consistently, we found that ionomycin induced the appearance of Lamp1 at the plasma membrane, a process that could be unambiguously observed by immunofluorescence in nonpermeabilized BHK (Figure 1C, left panels). An analysis by time-lapse total internal reflection fluorescence (TIRF) microscopy confirmed that ionomycin addition caused Lamp1 to diffuse in rapid flashes away from punctae close to the plasma membrane, presumably corresponding to the fusion of individual Lamp1-positive vesicles with the plasma membrane (Supplemental Movie 1). Cell permeabilization revealed that, much like with hexosaminidase (Figure 1A), the majority of Lamp1 remained intracellular with only a minor proportion being transferred to the plasma membrane (Figure 1C, right panels). To quantify Lamp1 transport to the plasma membrane, the cell surface was biotinylated after ionomycin treatment (Parton et al., 1992; Gottardi et al., 1995). Lamp1 rapidly appeared at the plasma membrane and reached a plateau within 5–10 min with a 10-fold increase over nonstimulated controls (Figure 1, D and E, quantification in F), corresponding to 2–3% of total cellular Lamp1 (Figure 1D). Much like hexosaminidase release (Figure 1B), the process was abolished by calcium chelation with EGTA (Figure 1, E and F). By contrast, actin was not biotinylated, confirming that cells remained intact during the experiment (Figure 1, D and E). We also observed that biotinylation of cell surface transferrin receptor (Tfn-R) was slightly increased, perhaps suggesting that a small fraction of Tfn-R follows the same calcium-dependent exocytic route as Lamp1 or that a calcium rise also triggers exocytosis from some early/recycling endosome subpopulation. In any case, our observations, which confirm previous studies (Rodriguez et al., 1997; Andrews, 2000), support the notion that a calcium rise triggers the fusion of compartments containing hexosaminidase and Lamp1 with the plasma membrane.


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)

Ionomycin-induced release of hexosaminidase and transport of Lamp1 to the plasma membrane. (A and B) A431 cells (A) or BHK cells (B) were stimulated with 5 μM ionomycin (Iono) or not (NS) for the indicated time. Then hexosaminidase or LDH activity was measured in the medium and in total cell lysates. The enzyme activity released in the medium is expressed as a percentage of the total activity in lysates. (A) shows the mean ± SEM of five and four experiments for hexosaminidase and LDH, respectively, and (B) shows typical kinetics of ionomycin stimulation with or without 1 mM EGTA. No LDH release was observed before 10 min of ionomycin stimulation. (C) BHK cells were stimulated or not as in A and incubated on ice with anti-Lamp1 antibodies without permeabilization (left column, “cell surface Lamp1”), fixed with PFA and labeled with fluorescent secondary antibody. Pictures after Z-stacking show homogeneous Lamp1 distribution on the cell surface (“Basal”) and a clear peripheral labeling at the equatorial Z-position (“Equatorial”). Alternatively, cells were fixed, permeabilized, and labeled with anti-Lamp1 antibodies to reveal total Lamp1 (right column). After stimulation, we observed little change in the distribution of intracellular Lamp1, but peripheral labeling was sometimes observed (arrowhead) as shown in the high magnification view of the boxed area. Bars = 5 mm. (D–F) After stimulation as in A and B, cell surface proteins of BHK (D) or A431 (E and F) were biotinylated (“Biot”) on ice and lysed. In controls, the biotinylation agent was omitted (NoB). Lysates were incubated with streptavidin-coated beads, and bound biotinylated proteins (“strepta-bound”) were eluted, separated by SDS gel, and analyzed by Western blotting using the indicated antibodies. (D) 3% and (E) 2% from nonstimulated lysates were separated in parallel. (F) The experiment in E was quantified, and the ratio of stimulated versus nonstimulated biotinylated-Lamp1 signals is shown. The results were normalized to biot-TfnR signal and are expressed as “fold increase.”
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Related In: Results  -  Collection

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

Figure 1: Ionomycin-induced release of hexosaminidase and transport of Lamp1 to the plasma membrane. (A and B) A431 cells (A) or BHK cells (B) were stimulated with 5 μM ionomycin (Iono) or not (NS) for the indicated time. Then hexosaminidase or LDH activity was measured in the medium and in total cell lysates. The enzyme activity released in the medium is expressed as a percentage of the total activity in lysates. (A) shows the mean ± SEM of five and four experiments for hexosaminidase and LDH, respectively, and (B) shows typical kinetics of ionomycin stimulation with or without 1 mM EGTA. No LDH release was observed before 10 min of ionomycin stimulation. (C) BHK cells were stimulated or not as in A and incubated on ice with anti-Lamp1 antibodies without permeabilization (left column, “cell surface Lamp1”), fixed with PFA and labeled with fluorescent secondary antibody. Pictures after Z-stacking show homogeneous Lamp1 distribution on the cell surface (“Basal”) and a clear peripheral labeling at the equatorial Z-position (“Equatorial”). Alternatively, cells were fixed, permeabilized, and labeled with anti-Lamp1 antibodies to reveal total Lamp1 (right column). After stimulation, we observed little change in the distribution of intracellular Lamp1, but peripheral labeling was sometimes observed (arrowhead) as shown in the high magnification view of the boxed area. Bars = 5 mm. (D–F) After stimulation as in A and B, cell surface proteins of BHK (D) or A431 (E and F) were biotinylated (“Biot”) on ice and lysed. In controls, the biotinylation agent was omitted (NoB). Lysates were incubated with streptavidin-coated beads, and bound biotinylated proteins (“strepta-bound”) were eluted, separated by SDS gel, and analyzed by Western blotting using the indicated antibodies. (D) 3% and (E) 2% from nonstimulated lysates were separated in parallel. (F) The experiment in E was quantified, and the ratio of stimulated versus nonstimulated biotinylated-Lamp1 signals is shown. The results were normalized to biot-TfnR signal and are expressed as “fold increase.”
Mentions: We used the calcium ionophore, ionomycin, to induce a transient rise in cytosolic calcium concentration. To avoid possible toxic effects of the drug, we optimized the conditions of the treatment (see Materials and Methods) so that cell survival (unpublished data) was not affected. Cell integrity was measured by the release of the abundant cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium (Figure 1A). Because LDH release occurred after prolonged incubation with ionomycin (Figure 1B), all studies were performed at earlier time points (≤10 min). After the ionomycin treatment, small but significant amounts of hexosaminidase, a lysosomal enzyme, were released into the extracellular medium, corresponding to ≈1% of the total cell-associated activity in A431 (Figure 1A) and BHK cells (Figure 1B). This release was abolished by ethylene glycol tetra­acetic acid (EGTA) in the medium (Figure 1B). Consistently, we found that ionomycin induced the appearance of Lamp1 at the plasma membrane, a process that could be unambiguously observed by immunofluorescence in nonpermeabilized BHK (Figure 1C, left panels). An analysis by time-lapse total internal reflection fluorescence (TIRF) microscopy confirmed that ionomycin addition caused Lamp1 to diffuse in rapid flashes away from punctae close to the plasma membrane, presumably corresponding to the fusion of individual Lamp1-positive vesicles with the plasma membrane (Supplemental Movie 1). Cell permeabilization revealed that, much like with hexosaminidase (Figure 1A), the majority of Lamp1 remained intracellular with only a minor proportion being transferred to the plasma membrane (Figure 1C, right panels). To quantify Lamp1 transport to the plasma membrane, the cell surface was biotinylated after ionomycin treatment (Parton et al., 1992; Gottardi et al., 1995). Lamp1 rapidly appeared at the plasma membrane and reached a plateau within 5–10 min with a 10-fold increase over nonstimulated controls (Figure 1, D and E, quantification in F), corresponding to 2–3% of total cellular Lamp1 (Figure 1D). Much like hexosaminidase release (Figure 1B), the process was abolished by calcium chelation with EGTA (Figure 1, E and F). By contrast, actin was not biotinylated, confirming that cells remained intact during the experiment (Figure 1, D and E). We also observed that biotinylation of cell surface transferrin receptor (Tfn-R) was slightly increased, perhaps suggesting that a small fraction of Tfn-R follows the same calcium-dependent exocytic route as Lamp1 or that a calcium rise also triggers exocytosis from some early/recycling endosome subpopulation. In any case, our observations, which confirm previous studies (Rodriguez et al., 1997; Andrews, 2000), support the notion that a calcium rise triggers the fusion of compartments containing hexosaminidase and Lamp1 with the plasma membrane.

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