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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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LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (ED). The color LUT visualizes the differences in the ED panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments (>10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in >10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10−6). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E. n > 50 for each condition. Bars, 10 µm.
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fig4: LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (ED). The color LUT visualizes the differences in the ED panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments (>10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in >10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10−6). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E. n > 50 for each condition. Bars, 10 µm.

Mentions: To determine whether LE cholesterol content affects the conformation of ORP1L, an mRFP-ORP1L-GFP fusion protein was expressed to monitor intramolecular fluorescence resonance energy transfer (FRET). Energy transfer from a donor fluorophore to a suitable acceptor (Förster, 1948) is dependent on distance and orientation between the two fluorophores (Förster, 1948; Calleja et al., 2003) and was used in this study to reveal conformational changes within mRFP-ORP1L-GFP (Fig. 4 A).


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)

LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (ED). The color LUT visualizes the differences in the ED panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments (>10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in >10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10−6). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E. n > 50 for each condition. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2712958&req=5

fig4: LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (ED). The color LUT visualizes the differences in the ED panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments (>10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in >10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10−6). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E. n > 50 for each condition. Bars, 10 µm.
Mentions: To determine whether LE cholesterol content affects the conformation of ORP1L, an mRFP-ORP1L-GFP fusion protein was expressed to monitor intramolecular fluorescence resonance energy transfer (FRET). Energy transfer from a donor fluorophore to a suitable acceptor (Förster, 1948) is dependent on distance and orientation between the two fluorophores (Förster, 1948; Calleja et al., 2003) and was used in this study to reveal conformational changes within mRFP-ORP1L-GFP (Fig. 4 A).

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