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Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.

Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W, Neefjes J - J. Cell Biol. (2009)

Bottom Line: Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L.At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors.Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands.

ABSTRACT
Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

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Cholesterol-dependent MCSs between LEs and the ER allowing in trans VAP interactions with the Rab7–RILP complex. (A) Electron micrographs of multivesicular bodies in Mel JuSo cells expressing HA-VAP and different variants of GFP-ORP1L, as indicated. Sections were stained with anti-HA antibodies detected with 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 C. (B) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes was determined in Mel JuSo cells expressing the ORP1L variants as shown in A. Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. (C) Electron micrographs of multivesicular bodies in HA-VAP– and GFP-ORP1L–expressing MelJuSo cells exposed to different cholesterol-manipulating conditions, as indicated. Sections were stained with anti-HA antibodies marked by 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 F. (D) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes from MelJuSo cells expressing ORP1L under various conditions of cholesterol manipulation (as shown in C). Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. Bars, 200 nm.
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fig8: Cholesterol-dependent MCSs between LEs and the ER allowing in trans VAP interactions with the Rab7–RILP complex. (A) Electron micrographs of multivesicular bodies in Mel JuSo cells expressing HA-VAP and different variants of GFP-ORP1L, as indicated. Sections were stained with anti-HA antibodies detected with 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 C. (B) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes was determined in Mel JuSo cells expressing the ORP1L variants as shown in A. Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. (C) Electron micrographs of multivesicular bodies in HA-VAP– and GFP-ORP1L–expressing MelJuSo cells exposed to different cholesterol-manipulating conditions, as indicated. Sections were stained with anti-HA antibodies marked by 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 F. (D) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes from MelJuSo cells expressing ORP1L under various conditions of cholesterol manipulation (as shown in C). Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. Bars, 200 nm.

Mentions: The localization of VAP and ORP1L was subsequently visualized by EM in more detail. EM has a significantly higher spatial resolution than CLSM, which has a maximal resolution of ∼240 nm. MelJuSo cells were transfected with GFP-ΔORD, -ORP1L, or -ΔORDPHDPHD together with HA-tagged VAP-A. After expression of the ORP1L variants was observed (by GFP fluorescence), cells were processed for immuno-EM and stained with HA antibodies to detect VAP-A (Fig. 8 A). The frequency of ER contacts with LEs and the surface of the LEs covered by ER structures was quantified (Fig. 8 B). Note that HA-VAP was never detected at LE membranes, excluding fusion of the LE and ER under these conditions. The ER, as labeled by HA-tagged VAP-A, had to be within 40 nm of the LE-limiting membrane to be considered a contact. Most LEs (>95%) interacted, often over large surface areas, with the ER when ΔORD was expressed. Both the number of ER–LE interactions and the surface covered by the ER decreased when ORP1L was expressed. Additionally, contact sites were almost absent when the high cholesterol conformation of ORP1L (ΔORDPHDPHD) was expressed, suggesting that the cholesterol-dependent conformational state of ORP1L determines ER contact site formation with LEs.


Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.

Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W, Neefjes J - J. Cell Biol. (2009)

Cholesterol-dependent MCSs between LEs and the ER allowing in trans VAP interactions with the Rab7–RILP complex. (A) Electron micrographs of multivesicular bodies in Mel JuSo cells expressing HA-VAP and different variants of GFP-ORP1L, as indicated. Sections were stained with anti-HA antibodies detected with 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 C. (B) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes was determined in Mel JuSo cells expressing the ORP1L variants as shown in A. Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. (C) Electron micrographs of multivesicular bodies in HA-VAP– and GFP-ORP1L–expressing MelJuSo cells exposed to different cholesterol-manipulating conditions, as indicated. Sections were stained with anti-HA antibodies marked by 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 F. (D) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes from MelJuSo cells expressing ORP1L under various conditions of cholesterol manipulation (as shown in C). Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. Bars, 200 nm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: Cholesterol-dependent MCSs between LEs and the ER allowing in trans VAP interactions with the Rab7–RILP complex. (A) Electron micrographs of multivesicular bodies in Mel JuSo cells expressing HA-VAP and different variants of GFP-ORP1L, as indicated. Sections were stained with anti-HA antibodies detected with 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 C. (B) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes was determined in Mel JuSo cells expressing the ORP1L variants as shown in A. Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. (C) Electron micrographs of multivesicular bodies in HA-VAP– and GFP-ORP1L–expressing MelJuSo cells exposed to different cholesterol-manipulating conditions, as indicated. Sections were stained with anti-HA antibodies marked by 15-nm gold particles. The gold particles are highlighted by red dots, and the ER membrane (containing HA-VAP) is indicated by blue lines. The original images are shown in Fig. S5 F. (D) The percentage of multivesicular bodies contacting ER structures and the contact area between the ER and LE membranes from MelJuSo cells expressing ORP1L under various conditions of cholesterol manipulation (as shown in C). Over 50 multivesicular bodies were considered per condition. The data on contact area from two independent quantifications were binned as indicated, and error bars show SEM. Bars, 200 nm.
Mentions: The localization of VAP and ORP1L was subsequently visualized by EM in more detail. EM has a significantly higher spatial resolution than CLSM, which has a maximal resolution of ∼240 nm. MelJuSo cells were transfected with GFP-ΔORD, -ORP1L, or -ΔORDPHDPHD together with HA-tagged VAP-A. After expression of the ORP1L variants was observed (by GFP fluorescence), cells were processed for immuno-EM and stained with HA antibodies to detect VAP-A (Fig. 8 A). The frequency of ER contacts with LEs and the surface of the LEs covered by ER structures was quantified (Fig. 8 B). Note that HA-VAP was never detected at LE membranes, excluding fusion of the LE and ER under these conditions. The ER, as labeled by HA-tagged VAP-A, had to be within 40 nm of the LE-limiting membrane to be considered a contact. Most LEs (>95%) interacted, often over large surface areas, with the ER when ΔORD was expressed. Both the number of ER–LE interactions and the surface covered by the ER decreased when ORP1L was expressed. Additionally, contact sites were almost absent when the high cholesterol conformation of ORP1L (ΔORDPHDPHD) was expressed, suggesting that the cholesterol-dependent conformational state of ORP1L determines ER contact site formation with LEs.

Bottom Line: Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L.At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors.Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands.

ABSTRACT
Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

Show MeSH
Related in: MedlinePlus