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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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ORP1L controls cholesterol-dependent LE positioning. (A) Cholesterol-dependent ORP1L vesicle positioning. Mel JuSo cells expressing mRFP-ORP1L were cultured under control conditions (FCS) or conditions causing decreased (statin) or increased (U-18666A) cholesterol levels. Actin was stained with falloidin (green) to mark the cell boundaries before analyses by CLSM. n > 100. (B) ORP1L is dominant in vesicle repositioning as the result of cholesterol manipulations. Mel JuSo cells expressing mRFP-ΔORDPHDPHD or mRFP-ΔORD were treated and imaged as described in A. n > 100. Bars, 10 µm.
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fig3: ORP1L controls cholesterol-dependent LE positioning. (A) Cholesterol-dependent ORP1L vesicle positioning. Mel JuSo cells expressing mRFP-ORP1L were cultured under control conditions (FCS) or conditions causing decreased (statin) or increased (U-18666A) cholesterol levels. Actin was stained with falloidin (green) to mark the cell boundaries before analyses by CLSM. n > 100. (B) ORP1L is dominant in vesicle repositioning as the result of cholesterol manipulations. Mel JuSo cells expressing mRFP-ΔORDPHDPHD or mRFP-ΔORD were treated and imaged as described in A. n > 100. Bars, 10 µm.

Mentions: To test whether LE positioning requires cholesterol sensing by ORP1L, MelJuSo cells were transfected with mRFP-ORP1L before manipulating cholesterol levels. Cholesterol reduction by statin and lipid-free medium scattered the mRFP-ORP1L–positive vesicles, whereas U-18666A treatment positioned LEs around the MTOC (Fig. 3 A and Fig. S3). Removal of the cholesterol-binding ORD or its replacement by tandem PH domains rendered positioning of LEs insensitive to cholesterol levels. ΔORD induced scattering of LEs under all cholesterol-manipulated conditions, whereas ΔORDPHDPHD always induced dense clustering (Fig. 3 B and Fig. S3). Microscopy analyses revealed that the expression of the ORP1L constructs was increased by six- to 15-fold upon ectopic expression (unpublished data). We conclude that functional ORP1L is required for translating LE cholesterol content into LE positioning.


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)

ORP1L controls cholesterol-dependent LE positioning. (A) Cholesterol-dependent ORP1L vesicle positioning. Mel JuSo cells expressing mRFP-ORP1L were cultured under control conditions (FCS) or conditions causing decreased (statin) or increased (U-18666A) cholesterol levels. Actin was stained with falloidin (green) to mark the cell boundaries before analyses by CLSM. n > 100. (B) ORP1L is dominant in vesicle repositioning as the result of cholesterol manipulations. Mel JuSo cells expressing mRFP-ΔORDPHDPHD or mRFP-ΔORD were treated and imaged as described in A. n > 100. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712958&req=5

fig3: ORP1L controls cholesterol-dependent LE positioning. (A) Cholesterol-dependent ORP1L vesicle positioning. Mel JuSo cells expressing mRFP-ORP1L were cultured under control conditions (FCS) or conditions causing decreased (statin) or increased (U-18666A) cholesterol levels. Actin was stained with falloidin (green) to mark the cell boundaries before analyses by CLSM. n > 100. (B) ORP1L is dominant in vesicle repositioning as the result of cholesterol manipulations. Mel JuSo cells expressing mRFP-ΔORDPHDPHD or mRFP-ΔORD were treated and imaged as described in A. n > 100. Bars, 10 µm.
Mentions: To test whether LE positioning requires cholesterol sensing by ORP1L, MelJuSo cells were transfected with mRFP-ORP1L before manipulating cholesterol levels. Cholesterol reduction by statin and lipid-free medium scattered the mRFP-ORP1L–positive vesicles, whereas U-18666A treatment positioned LEs around the MTOC (Fig. 3 A and Fig. S3). Removal of the cholesterol-binding ORD or its replacement by tandem PH domains rendered positioning of LEs insensitive to cholesterol levels. ΔORD induced scattering of LEs under all cholesterol-manipulated conditions, whereas ΔORDPHDPHD always induced dense clustering (Fig. 3 B and Fig. S3). Microscopy analyses revealed that the expression of the ORP1L constructs was increased by six- to 15-fold upon ectopic expression (unpublished data). We conclude that functional ORP1L is required for translating LE cholesterol content into LE positioning.

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