Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

Abstract

It has been shown that the treatment with 3beta-[2-(diethylamino)ethoxy] androst-5-en-17-one (U18666A) causes the accumulation of cholesterol and the cation-independent mannose 6-phosphate receptor (CIMPR) in late endosomal/lysosomal compartments in BHK cells. The present study reports on a study of the effect of U18666A on CIMPR distribution in more detail in HeLa cells. When cells were treated with U18666A for 20 h, the intense perinuclear signal for CIMPR corresponding to the trans-Golgi network (TGN) disappeared and lamp1-negative punctate signals, scattered in the perinuclear region were detected. CIMPR then began to accumulate in lamp1-positive compartments 48 h after addition of the drug. Double immunofluorescence microscopy showed that U18666A-induced mannose 6-phosphate receptor-containing compartments (U-MPRCs), which were formed in the early phase of the redistribution, contained no marker for the TGN, late endosomes or lysosomes. Approximately half of the structures contained transferrin that had been internalized for 20 min, and cathepsin D, the majority of which appeared to be its precursor form. Immunoelectron-microscopic analysis revealed that U-MPRCs are composed of multivesicular bodies, irregularly shaped structures, and vesicular structures adjacent to the multivesicular bodies. These results suggest that U18666A treatment primarily suppresses the CIMPR transport pathways to late endosomes and from transferrin-containing endosomes, both of which may be dependent on cholesterol function.

The luminal domain participates in the endosomal trafficking of the cation-independent mannose 6-phosphate receptor

“…In HeLa cells, U18666A causes an accumulation of CIMPR in Tfn-positive aberrant endosomal structures together with one of its ligands, cathepsin D [41]. Interestingly, the present study revealed that this accumulation was dependent on M6P-ligand binding, because G-CIMPRfull R/A did not show this alteration.…”

“…Although it has been reported that this drug also induces the redistribution of CIMPR from the TGN to late endosomal compartments in BHK cells [53], we recently reported that, in HeLa cells, CIMPR is redistributed into aberrant Tfn-positive endosomal structures after a 20 h treatment with U18666A [41]. As shown in Fig.…”

“…Immunofluorescence microscopy and double labeling with cholesterol using filipin were carried out as described previously [41].…”

The lipid composition of autophagic vacuoles regulates expression of multilamellar bodies

“…MLBs were also induced in the Mv1Lu type II alveolar cell line by treatment with U18666A, a compound that promotes lysosomal cholesterol accumulation, independently of Mgat5 transgene expression. U18666A treatment has been shown to induce MLB formation in CHO cells (Lusa et al, 2001) but not in HeLa cells (Tomiyama et al, 2004), MDCK or mammary carcinoma cells (data not shown). Lysosomal cholesterol accumulation is therefore not sufficient to induce MLB formation and other cell-type specific factors are required.…”

Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments

“…The endosomal trafficking defects observed in NPCD cells extend to proteins such as IGF2/MPR, NPC1, and annexin II, all of which utilize the endosomal recycling pathway (42,74). Electron microscopy studies have shown that within the LEs of NPCD cells these proteins are trapped in the cholesterol-enriched membrane-bound vesicular structures (47).…”