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Encapsulated cargo internalized by fusogenic liposomes partially overlaps the endoplasmic reticulum.

Mustata RC, Grigorescu A, Petrescu SM - J. Cell. Mol. Med. (2009)

Bottom Line: Brefeldin A, which prevents Golgi-dependent retrograde trafficking, does not disrupt the cargo delivery to the ER.This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the reported SV40 caveolae pathways.Exploiting a cellular route linking the cell surface to the ER, fusogenic liposomes may become efficient drug delivery vehicles for ER stress and diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Splaiul Independentei, Bucharest, Romania.

ABSTRACT
Few endocytosed ligands, including bacterial toxins and simian virus 40 (SV40) have been shown to reach the endoplasmic reticulum (ER) in mammalian cells. Using calcein and fluorescently labelled lactoferrin encapsulated in fusogenic liposomes we found that the cargo uses a microtubule-based pathway with ER delivery. Endocytic uptake of the lipid vesicles was cholesterol dependent in all cell lines tested, including the caveolin-1-deficient human hepatoma 7 cell line. The ligand was transported in non-caveosome organelles requiring acidic pH for maturation, but able to escape the lysosomal route. These organelles were not recycling endosomes either, as shown by the lack of co-localization with recycling transferrin. Co-localization with the ER-tracker, orange fluorescent protein with KDEL signal retention and cholera toxin in live microscopy revealed an ER distribution of the fluorescent ligand. Brefeldin A, which prevents Golgi-dependent retrograde trafficking, does not disrupt the cargo delivery to the ER. This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the reported SV40 caveolae pathways. Exploiting a cellular route linking the cell surface to the ER, fusogenic liposomes may become efficient drug delivery vehicles for ER stress and diseases.

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Internalization of vesicles-containing calcein results in calcein accumulation within the ER. MDBK cells were incubated with liposome-loaded calcein and RhPE liposomes for 30 min. at 37°C, washed, and kept in culture for the indicated times. (A) 30 min. before visualization cells were incubated with the ER marker, ER-tracker (blue). Lipid marker RhPE and calcein were visualized in red and green channel, respectively. (B) Cells were treated for 30 min. at 37°C with ∼0.2 mM PC: Chol liposomes-included calcein, washed and chased 2 hrs in fresh medium. (C) Cells were treated with free calcein for 30 min. at 37°C, washed and incubated for 2.5 hrs in fresh medium. (D) MDBK cells transduced with ER-OFP (red) were incubated with liposome-included calcein for 30 min. at 37°C and imaged after 5 hrs chase. Scale bar 10 μm.
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fig01: Internalization of vesicles-containing calcein results in calcein accumulation within the ER. MDBK cells were incubated with liposome-loaded calcein and RhPE liposomes for 30 min. at 37°C, washed, and kept in culture for the indicated times. (A) 30 min. before visualization cells were incubated with the ER marker, ER-tracker (blue). Lipid marker RhPE and calcein were visualized in red and green channel, respectively. (B) Cells were treated for 30 min. at 37°C with ∼0.2 mM PC: Chol liposomes-included calcein, washed and chased 2 hrs in fresh medium. (C) Cells were treated with free calcein for 30 min. at 37°C, washed and incubated for 2.5 hrs in fresh medium. (D) MDBK cells transduced with ER-OFP (red) were incubated with liposome-included calcein for 30 min. at 37°C and imaged after 5 hrs chase. Scale bar 10 μm.

Mentions: To follow the intracellular trafficking of internalized vesicles, we used liposomes loaded with the membrane-impermeant calcein molecule [16] and liposomes labelled with the lipophilic marker RhPE. MDBK cells were pulsed with equal amounts of both liposomes for 30 min. and chased for 5 hrs at 37°C. RhPE liposomes were slowly internalized in vesicular structures found mainly near the plasma membrane (Fig. 1A). Starting 2 hrs from internalization, most of the liposomal lipids and calcein accumulated around the nuclei, showing a partial overlapping with the ER-tracker, a cell-permeant dye, highly selective for the ER [17]. Although the ER-tracker stained both perinuclear and peripheral ER, confirming previous reports [18], calcein and RhPE fluorescence were confined to the perinuclear area. Further confirmation for the ER localization of calcein came from its overlapping with another ER marker, the orange fluorescent protein KDEL (Fig. 1D). This was a surprising finding, because these types of liposomes that destabilize at acidic pH have been reported to deliver their content mainly to the cytoplasm [19, 20]. In contrast, free calcein and pH-insensitive liposomes made of PC: Chol were inefficiently internalized (Fig. 1B and C). Moreover, the calcein cargo did not overlap with the Golgi, lysosomes or mitochondria markers tested (Fig. S1). Similarly, liposome-treated HeLa cells showed a prominent overlapping of calcein with the two ER markers suggesting that this maybe a more common intracellular traffic route (Fig. 3).


Encapsulated cargo internalized by fusogenic liposomes partially overlaps the endoplasmic reticulum.

Mustata RC, Grigorescu A, Petrescu SM - J. Cell. Mol. Med. (2009)

Internalization of vesicles-containing calcein results in calcein accumulation within the ER. MDBK cells were incubated with liposome-loaded calcein and RhPE liposomes for 30 min. at 37°C, washed, and kept in culture for the indicated times. (A) 30 min. before visualization cells were incubated with the ER marker, ER-tracker (blue). Lipid marker RhPE and calcein were visualized in red and green channel, respectively. (B) Cells were treated for 30 min. at 37°C with ∼0.2 mM PC: Chol liposomes-included calcein, washed and chased 2 hrs in fresh medium. (C) Cells were treated with free calcein for 30 min. at 37°C, washed and incubated for 2.5 hrs in fresh medium. (D) MDBK cells transduced with ER-OFP (red) were incubated with liposome-included calcein for 30 min. at 37°C and imaged after 5 hrs chase. Scale bar 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4516470&req=5

fig01: Internalization of vesicles-containing calcein results in calcein accumulation within the ER. MDBK cells were incubated with liposome-loaded calcein and RhPE liposomes for 30 min. at 37°C, washed, and kept in culture for the indicated times. (A) 30 min. before visualization cells were incubated with the ER marker, ER-tracker (blue). Lipid marker RhPE and calcein were visualized in red and green channel, respectively. (B) Cells were treated for 30 min. at 37°C with ∼0.2 mM PC: Chol liposomes-included calcein, washed and chased 2 hrs in fresh medium. (C) Cells were treated with free calcein for 30 min. at 37°C, washed and incubated for 2.5 hrs in fresh medium. (D) MDBK cells transduced with ER-OFP (red) were incubated with liposome-included calcein for 30 min. at 37°C and imaged after 5 hrs chase. Scale bar 10 μm.
Mentions: To follow the intracellular trafficking of internalized vesicles, we used liposomes loaded with the membrane-impermeant calcein molecule [16] and liposomes labelled with the lipophilic marker RhPE. MDBK cells were pulsed with equal amounts of both liposomes for 30 min. and chased for 5 hrs at 37°C. RhPE liposomes were slowly internalized in vesicular structures found mainly near the plasma membrane (Fig. 1A). Starting 2 hrs from internalization, most of the liposomal lipids and calcein accumulated around the nuclei, showing a partial overlapping with the ER-tracker, a cell-permeant dye, highly selective for the ER [17]. Although the ER-tracker stained both perinuclear and peripheral ER, confirming previous reports [18], calcein and RhPE fluorescence were confined to the perinuclear area. Further confirmation for the ER localization of calcein came from its overlapping with another ER marker, the orange fluorescent protein KDEL (Fig. 1D). This was a surprising finding, because these types of liposomes that destabilize at acidic pH have been reported to deliver their content mainly to the cytoplasm [19, 20]. In contrast, free calcein and pH-insensitive liposomes made of PC: Chol were inefficiently internalized (Fig. 1B and C). Moreover, the calcein cargo did not overlap with the Golgi, lysosomes or mitochondria markers tested (Fig. S1). Similarly, liposome-treated HeLa cells showed a prominent overlapping of calcein with the two ER markers suggesting that this maybe a more common intracellular traffic route (Fig. 3).

Bottom Line: Brefeldin A, which prevents Golgi-dependent retrograde trafficking, does not disrupt the cargo delivery to the ER.This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the reported SV40 caveolae pathways.Exploiting a cellular route linking the cell surface to the ER, fusogenic liposomes may become efficient drug delivery vehicles for ER stress and diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Cell Biology, Institute of Biochemistry of Romanian Academy, Splaiul Independentei, Bucharest, Romania.

ABSTRACT
Few endocytosed ligands, including bacterial toxins and simian virus 40 (SV40) have been shown to reach the endoplasmic reticulum (ER) in mammalian cells. Using calcein and fluorescently labelled lactoferrin encapsulated in fusogenic liposomes we found that the cargo uses a microtubule-based pathway with ER delivery. Endocytic uptake of the lipid vesicles was cholesterol dependent in all cell lines tested, including the caveolin-1-deficient human hepatoma 7 cell line. The ligand was transported in non-caveosome organelles requiring acidic pH for maturation, but able to escape the lysosomal route. These organelles were not recycling endosomes either, as shown by the lack of co-localization with recycling transferrin. Co-localization with the ER-tracker, orange fluorescent protein with KDEL signal retention and cholera toxin in live microscopy revealed an ER distribution of the fluorescent ligand. Brefeldin A, which prevents Golgi-dependent retrograde trafficking, does not disrupt the cargo delivery to the ER. This new endocytic pathway making use of acidic endosome-like organelles is an alternative to the reported SV40 caveolae pathways. Exploiting a cellular route linking the cell surface to the ER, fusogenic liposomes may become efficient drug delivery vehicles for ER stress and diseases.

Show MeSH
Related in: MedlinePlus