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Lysosomal trafficking functions of mucolipin-1 in murine macrophages.

Thompson EG, Schaheen L, Dang H, Fares H - BMC Cell Biol. (2007)

Bottom Line: MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH.Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane.Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, Life Sciences South Room 531, University of Arizona, Tucson, AZ 85721, USA. ericgt@email.arizona.edu

ABSTRACT

Background: Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes.

Results: We show that mucolipin-1 localizes to lysosomal compartments in RAW264.7 mouse macrophages that show subcompartmental accumulations of endocytosed molecules. Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane.

Conclusion: Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

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Related in: MedlinePlus

Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones. A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.
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Figure 4: Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones. A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.

Mentions: Previous results had shown that CUP-5 in C. elegans is required for the transport of endocytosed BSA from late endosomes to lysosomes [35]. To determine whether ML1 is also required for lysosomal transport, we loaded the terminal compartments of RAW264.7, LS9 or LS10 cells with BSA-AlexaFluor 594 by incubating cells with the fluorescent marker the first day for 4 hours followed by a 24-hour incubation in the absence of the marker. Approximately 5% of LS9 and LS10 cells showed a significantly enlarged terminal compartment that contained the fluorescent endocytosed marker (Fig. 4A). In the absence of ML1-specific antibodies, we cannot determine whether these represent cells with the most reduction in ML1 levels or whether loss of ML1 in all cells would give a similarly low penetrant phenotype. The complete loss of ML1 in DT40 B lymphocytes has been reported not to affect the sizes of the terminal compartments [33].


Lysosomal trafficking functions of mucolipin-1 in murine macrophages.

Thompson EG, Schaheen L, Dang H, Fares H - BMC Cell Biol. (2007)

Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones. A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254603&req=5

Figure 4: Trafficking and endocytosis defect of proteins in MCOLN1 RNAi clones. A) Confocal images of RAW264.7, LS9, and LS10 cells whose terminal compartments were pre-loaded with BSA-AlexaFluor 594 (BSA-AF 594, red). BSA-AlexaFluor 488 (BSA-AF 488, green) was added for 10 minutes to the cells and the cells were chased for the indicated times before fixation. Bar is 5 μm. B) Western blots of HEL that was endocytosed for 5 minutes and chased for the indicated times. C) Quantitation of the HEL that remains in cells relative to the 0 time point. Bars represent standard deviations from two independent assays.
Mentions: Previous results had shown that CUP-5 in C. elegans is required for the transport of endocytosed BSA from late endosomes to lysosomes [35]. To determine whether ML1 is also required for lysosomal transport, we loaded the terminal compartments of RAW264.7, LS9 or LS10 cells with BSA-AlexaFluor 594 by incubating cells with the fluorescent marker the first day for 4 hours followed by a 24-hour incubation in the absence of the marker. Approximately 5% of LS9 and LS10 cells showed a significantly enlarged terminal compartment that contained the fluorescent endocytosed marker (Fig. 4A). In the absence of ML1-specific antibodies, we cannot determine whether these represent cells with the most reduction in ML1 levels or whether loss of ML1 in all cells would give a similarly low penetrant phenotype. The complete loss of ML1 in DT40 B lymphocytes has been reported not to affect the sizes of the terminal compartments [33].

Bottom Line: MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH.Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane.Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular and Cellular Biology, Life Sciences South Room 531, University of Arizona, Tucson, AZ 85721, USA. ericgt@email.arizona.edu

ABSTRACT

Background: Mucolipidosis Type IV is currently characterized as a lysosomal storage disorder with defects that include corneal clouding, achlorhydria and psychomotor retardation. MCOLN1, the gene responsible for this disease, encodes the protein mucolipin-1 that belongs to the "Transient Receptor Potential" family of proteins and has been shown to function as a non-selective cation channel whose activity is modulated by pH. Two cell biological defects that have been described in MLIV fibroblasts are a hyperacidification of lysosomes and a delay in the exit of lipids from lysosomes.

Results: We show that mucolipin-1 localizes to lysosomal compartments in RAW264.7 mouse macrophages that show subcompartmental accumulations of endocytosed molecules. Using stable RNAi clones, we show that mucolipin-1 is required for the exit of lipids from these compartments, for the transport of endocytosed molecules to terminal lysosomes, and for the transport of the Major Histocompatibility Complex II to the plasma membrane.

Conclusion: Mucolipin-1 functions in the efficient exit of molecules, destined for various cellular organelles, from lysosomal compartments.

Show MeSH
Related in: MedlinePlus