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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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Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions (>50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10−5 and 7.76 × 10−6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.
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fig1: Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions (>50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10−5 and 7.76 × 10−6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.

Mentions: To visualize the effect of these treatments, cells were fixed and stained with filipin (Sobo et al., 2007) to detect cholesterol (Fig. 1 A and Fig. S1 B) or stained for CD63 to label LEs (Fig. 1 B and Fig. S1 B). Images were made at identical microscope settings and show that these cholesterol-manipulating treatments decreased (statin) or increased (U-18666A or siNPC1) intracellular cholesterol levels (Fig. 1, A and C; and Fig. S1 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)

Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions (>50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10−5 and 7.76 × 10−6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.
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Related In: Results  -  Collection

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fig1: Chemical and genetic cholesterol manipulations and LEs. (A) Modulation of intracellular cholesterol levels. Mel JuSo cells were cultured in normal medium (FCS), cholesterol-depleted medium supplemented with lovastatin (statin), or normal medium supplemented with U-18666A, as indicated. Fixed cells were stained with filipin to detect cholesterol. A color look up table (LUT) shows fluorescence intensities. n > 100 for each condition. (B) Effects of intracellular cholesterol manipulation (control [CTRL], U-18666A, or statin treatment) on LE positioning in RILP- or ORP1L-silenced Mel JuSo cells. Cells were stained for the LE marker CD63 (red), endogenous ORP1L (green), and actin (blue). The position of the CD63-positive vesicles relative to the nucleus (radial distribution) was determined for the various conditions (>50 cells per condition), binned, and plotted (right). The distributions were statistically different according to the Jonckheere-Terpstra test for control siRNA (P = 1.52 × 10−5 and 7.76 × 10−6 for CD63 and ORP1L, respectively) only. No difference was detected for siRILP (P = 0.20 and 0.54 for CD63 and ORP1L, respectively) and for siORP1L (P = 0.29 for CD63). (C) Dynein motor activity and clustering of LEs in NPC1-silenced cells. Mel JuSo cells were transfected with siRNA for NPC1 for 72 h and, after 48 h, transfected with an expression construct for p50dynamitin to disrupt the dynein–dynactin motor. Fixed cells were stained for CD63 and p50dynamitin to mark the overexpressing cells. n > 100. Bars: (A and C)10 µm; (B) 20 µm.
Mentions: To visualize the effect of these treatments, cells were fixed and stained with filipin (Sobo et al., 2007) to detect cholesterol (Fig. 1 A and Fig. S1 B) or stained for CD63 to label LEs (Fig. 1 B and Fig. S1 B). Images were made at identical microscope settings and show that these cholesterol-manipulating treatments decreased (statin) or increased (U-18666A or siNPC1) intracellular cholesterol levels (Fig. 1, A and C; and Fig. S1 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