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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 ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for >20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10−6; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.
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fig7: Cholesterol-dependent ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for >20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10−6; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.

Mentions: If the integral ER protein VAP controls motor binding to LEs after recruitment by ORP1L, VAP should somehow contact these vesicles. To visualize this, MelJuSo cells were transfected with the various ORD variants of mRFP-ORP1L and stained for endogenous VAP (Fig. 7 A). Endogenous VAP accumulated on ΔORD- and, although to a lesser extent, on ORP1L-containing vesicles but was excluded from vesicles labeled by ΔORDPHDPHD, indicating that VAP recruitment is dependent on the conformation of ORP1L (Fig. 7 A). These results were confirmed in photoactivation experiments with MelJuSo cells expressing photoactivatable (PA)-GFP–tagged VAP-A and mRFP-ORP1L variants (Fig. S5 A and Videos 6–8) that also showed colocalization of VAP-A and ORP1L on moving vesicles. Accumulation of the ER protein VAP on LEs by ΔORD expression also coincided with the removal of p150Glued from RILP (Fig. S5 B). We then tested whether the ER–LE interactions could be observed by manipulating cholesterol levels in MelJuSo cells in which ORP1L and VAP were not ectopically expressed. TAP1-GFP labeled the ER (Reits et al., 2000). Decreasing cholesterol levels sufficed to induce the arrival of ER markers on LEs, as analyzed by CLSM and quantified by pixel analysis (Fig. 7 B).


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 ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for >20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10−6; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig7: Cholesterol-dependent ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for >20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10−6; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.
Mentions: If the integral ER protein VAP controls motor binding to LEs after recruitment by ORP1L, VAP should somehow contact these vesicles. To visualize this, MelJuSo cells were transfected with the various ORD variants of mRFP-ORP1L and stained for endogenous VAP (Fig. 7 A). Endogenous VAP accumulated on ΔORD- and, although to a lesser extent, on ORP1L-containing vesicles but was excluded from vesicles labeled by ΔORDPHDPHD, indicating that VAP recruitment is dependent on the conformation of ORP1L (Fig. 7 A). These results were confirmed in photoactivation experiments with MelJuSo cells expressing photoactivatable (PA)-GFP–tagged VAP-A and mRFP-ORP1L variants (Fig. S5 A and Videos 6–8) that also showed colocalization of VAP-A and ORP1L on moving vesicles. Accumulation of the ER protein VAP on LEs by ΔORD expression also coincided with the removal of p150Glued from RILP (Fig. S5 B). We then tested whether the ER–LE interactions could be observed by manipulating cholesterol levels in MelJuSo cells in which ORP1L and VAP were not ectopically expressed. TAP1-GFP labeled the ER (Reits et al., 2000). Decreasing cholesterol levels sufficed to induce the arrival of ER markers on LEs, as analyzed by CLSM and quantified by pixel analysis (Fig. 7 B).

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