Roles of CUP-5, the Caenorhabditis elegans orthologue of human TRPML1, in lysosome and gut granule biogenesis
© Campbell and Fares; licensee BioMed Central Ltd. 2010
Received: 4 February 2010
Accepted: 11 June 2010
Published: 11 June 2010
CUP-5 is a Transient Receptor Potential protein in C. elegans that is the orthologue of mammalian TRPML1. Loss of TRPML1 results in the lysosomal storage disorder Mucolipidosis type IV. Loss of CUP-5 results in embryonic lethality and the accumulation of enlarged yolk granules in developing intestinal cells. The embryonic lethality of cup-5 mutants is rescued by mutations in mrp-4, which is required for gut granule differentiation. Gut granules are intestine-specific lysosome-related organelles that accumulate birefringent material. This link between CUP-5 and gut granules led us to determine the roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells.
We show that CUP-5 protein localizes to lysosomes, but not to gut granules, in developing intestinal cells. Loss of CUP-5 results in defects in endo-lysosomal transport in developing intestinal cells of C. elegans embryos. This ultimately leads to the appearance of enlarged terminal vacuoles that show defective lysosomal degradation and that have lysosomal and endosomal markers. In contrast, gut granule biogenesis is normal in the absence of CUP-5. Furthermore, loss of CUP-5 does not result in inappropriate fusion or mixing of content between lysosomes and gut granules.
Using an in vivo model of MLIV, we show that there is a defect in lysosomal transport/biogenesis that is earlier than the presumed function of TRPML1 in terminal lysosomes. Our results indicate that CUP-5 is required for the biogenesis of lysosomes but not of gut granules. Thus, cellular phenotypes in Mucolipidosis type IV are likely not due to defects in lysosome-related organelle biogenesis, but due to progressive defects in lysosomal transport that lead to severe lysosomal dysfunction.
Lysosomes are the major degradative organelles of endocytosed, phagocytosed, and autophagocytosed material [1, 2]. Lysosomes also have specialized functions, for example fusing with the plasma membrane to initiate wound repair and mediating some cell death pathways [3–5]. Lysosome biogenesis is a dynamic process, in which late endosomes fuse with lysosomes resulting in a hybrid late endosome/lysosomal organelle . Late endosomes and lysosomes are reformed from these hybrid organelles, a process that requires the release of intra-organellar Ca2+ [6, 7].
Some tissues have additional organelles called lysosome-related organelles (LROs) that are acidic, contain some lysosomal proteins, and have cell type-specific functions . Although LROs derive from the endosomal system, they are different from bona fide lysosomes in composition, morphology, and function. Examples of LROs include organelles with storage or secretion functions, such as melanosomes in melanocytes, platelet-dense granules in platelets, and acrosomes in sperm cells [8–10]. In C. elegans embryos, gut granules are LROs found in intestinal cells at all stages of development [11–14]. These gut granules contain lipids, birefringent material that is autofluorescent under several wavelengths of light, and gut granule-specific proteins. The function and biogenesis of gut granules is not completely understood.
In C. elegans, CUP-5 is required for the biogenesis of lysosomes in scavenger cells called coelomocytes [15, 16]. CUP-5 is the sole orthologue of mammalian TRPML1 that is encoded by MCOLN1, mutations in which cause Mucolipidosis type IV (MLIV) in humans [15, 17]. Many MLIV-associated defects that are linked to lysosomal dysfunction have been described. Some examples include an enlargement of lysosomes that accumulate both lipid and water soluble material, a delay in the transport of endocytosed lactosylceramide (LacCer) from late endosomes/lysosomes to the Golgi Apparatus, a delay in the degradation in and/or transport of endocytosed lipids and proteins to lysosomes, and a delay in the degradation of autophagosome material . Similar to the MLIV phenotypes, worms with a cup-5 mutation have enlarged endo-lysosomal compartments in several cell types, including developing intestinal cells and coelomocytes [15, 19]. Pulse-chase studies in coelomocytes have shown that CUP-5 is required for the biogenesis of lysosomes, the earliest MLIV-associated defect in the endocytic pathway that has yet been described .
Loss of CUP-5 results in embryonic lethality . In these cup-5 mutant embryos, there is a significant enlargement of yolk granules and a defect in the degradation of endocytosed yolk proteins in developing intestinal cells . Loss of MRP-4 was shown to rescue both the yolk granule/yolk degradation defects and the embryonic lethality of cup-5 mutant . However, MRP-4 is an ATP-Binding Cassette (ABC) transporter that is required for gut granule differentiation in developing intestinal cells . Because of the role of MRP-4 in LRO/gut granule differentiation and the mrp-4 mutant rescue of cup-5 mutants, we hypothesized that CUP-5 may play a role in LRO biogenesis or differentiation, in addition to its role in lysosome biogenesis. We therefore initiated this study to decipher possible roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells.
Results and discussion
Discrete late endocytic organelles and gut granules in the developing intestine
Two late-stage endo-lysosomal organelles (yolk granules and lysosomes) and one LRO (gut granules) have been identified in developing intestinal cells of embryos [11, 22]. However, because there are no systematic studies that visualized all three organelles in developing intestinal cells, it is not clear whether they indeed represent discrete and separate compartments. We therefore first carried out colocalization studies in wild type embryos to define these organelles using established markers. We confined our analysis to specific stages of embryonic development, "comma" to "1.5-fold" stages, when the yolk granule defect is clearly observed in cup-5 mutants .
Yolk granules, lysosomes, and gut granules represent distinct compartments of developing intestinal cells. YP170::GFP did not colocalize with any of the gut granule markers at steady state (Fig. 1A, B; Additional file 1 Fig. S1B). This indicates that yolk granules are discrete and separate from gut granules. Similarly, YP170::GFP did not colocalize with LMP-1::TagRFP(S158T) at steady state (Fig. 1C). This indicates that yolk granules and lysosomes are distinct compartments; yolk is likely transported to lysosomes but is degraded in these organelles. Finally, LMP-1::GFP did not colocalize with PGP-2 or autofluorescence at steady state (Fig. 1D; Additional file 1 Fig. S1C). This indicates that lysosomes and gut granules are also discrete and separate organelles. Having defined the identities of these organelles by light microscopy studies, we then did localization and mutational analyses to decipher roles of CUP-5 in organelle biogenesis and transport in developing intestinal cells.
cup-5(zu223) lethality is primarily due to defects in the developing intestine
We also made a Pelt-2::mCherry::cup-5 expression construct. This mCherry::CUP-5 fusion protein only partially rescues cup-5(zu223) embryonic lethality (Fig. 2A), indicating that mCherry interferes with CUP-5 activity. However, mCherry::CUP-5 and GFP::CUP-5 show 100% colocalization (data not shown). In addition, GFP::CUP-5 and mCherry::CUP-5 show the same colocalization with LMP-1 in lysosomes (Fig. 2B, C). Thus mCherry::CUP-5 is a useful marker for the subcellular localization of CUP-5.
CUP-5 protein localizes to lysosomes
We had previously localized CUP-5 to lysosomes of coelomocytes . We wanted to determine whether CUP-5 localizes only to lysosomes of developing intestinal cells or whether it is also found on yolk granules and gut granules. We used our transgenic worms expressing either GFP::CUP-5 or mCherry::CUP-5 in intestinal cells for these localization studies.
mCherry::CUP-5 fully colocalizes with LMP-1::GFP (Fig. 2B). We see similar colocalization between GFP::CUP-5 and LMP-1::TagRFP(S158T) (Fig. 2C). Note that in addition to lysosomes, LMP-1 also localizes to, or close to, the plasma membrane of developing intestinal cells. Consistent with the lysosomal localization of CUP-5, there was no colocalization between mCherry::CUP-5 and YP170::GFP at steady state (Fig. 2D). We also did not detect any colocalization between GFP::CUP-5 and any gut granule markers (Fig. 2E; Additional file 2 Fig. S2).
Therefore, CUP-5 protein localizes to lysosomes at steady state in developing intestinal cells. We next assayed whether loss of CUP-5 causes content mixing between yolk granules, lysosomes, and gut granules.
Yolk Granule and lysosome content mixing in the cup-5(zu223) mutant
Alteration in size and content of yolk granules/lysosomes in the cup-5(zu223) mutant
We had previously shown an enlargement of the total population of yolk granules and an accumulation of YP170::GFP in C. elegans cup-5(zu223) mutants . We visualized markers for the different organelles in wild type and cup-5(zu223) embryos using identical microscopy parameters that allow us to compare sizes of organelles and intensities of staining of each marker. To determine the sizes of the compartments, we relied on endogenous markers, YP170::GFP for yolk granules, LMP-1::GFP for lysosomes, and autofluorescence for gut granules; this eliminates potential variability due to exogenous staining with dyes. Indeed, Nile Red staining of gut granules showed significant variability in staining quality between different experiments. We also used PGP-2 antibody immunostaining to confirm sizes of gut granules.
We next compared these two populations of YP70::GFP compartments in the cup-5 mutant to yolk granules in wild type. The YP170::GFP-only compartments in the cup-5(zu223) mutant were larger than YP170::GFP compartments in wild type (1.54 ± 0.38 μm2 compared with 0.92 ± 0.05 μm2, p-value = 1.92 × 10-7), although the intensities of GFP were similar in both cases (p-value = 0.14) (Fig. 4A). The compartments that contained both YP170::GFP and LMP-1::TagRFP(S158T) in the cup-5(zu223) mutant were much larger than YP170::GFP yolk granules in wild type (2.93 ± 0.61 μm2 compared with 0.92 ± 0.05 μm2, p-value = 1.80 × 10-22), and had a higher intensity of GFP than wild type (p-value = 2.10 × 10-9) (Fig. 4A). Thus in the absence of CUP-5, there is a defect in yolk granules/late endosomes prior to the lysosomal expansion described in MLIV cells: yolk granules/late endosomes progressively increase in size before they start accumulating lysosomal markers and showing defects in degradation.
Consistent with the colocalization of LMP-1 and YP170 in the absence of CUP-5, LMP-1::GFP puncta were also larger in cup-5(zu223) than in wild type (1.37 ± 0.40 μm2 compared with 0.71 ± 0.05 μm2, p-value = 6.19 × 10-4) (Fig. 4B). Furthermore, the intensity of LMP-1::GFP was also higher in cup-5(zu223) developing intestinal cells, indicating a defect in the turnover of LMP-1 (p-value = 5.02 × 10-7) (Fig. 4B).
In contrast to the yolk granule/lysosome defect, there was no apparent effect of the loss of CUP-5 on gut granule size. Gut granule sizes were similar in cup-5(zu223) and in wild type embryos based on UV fluorescence (1.17 ± 0.07 μm2 for mutant, 1.11 ± 0.06 μm2 for wild type, p-value = 0.25) and PGP-2 immunofluorescence (0.70 ± 0.04 μm2 for mutant, 0.76 ± 0.04 μm2 for wild type, p-value = 0.06) (Fig. 4C, D). However, the intensity per unit area of autofluorescent material was statistically lower per unit area in cup-5(u223) mutant embryos compared with wild type embryos (p-value = 2.3 × 10-16) (Fig. 4D), while the intensity per unit area of PGP-2 immunofluorescence was similar in both wild type and mutant embryos (p-value = 0.57) (Fig. 4C). Thus while the biogenesis of gut granules is normal, the loss of CUP-5 may have indirect effects on the composition of these granules; the severe yolk granule/lysosome defect in the cup-5 mutant may affect the trafficking of some gut granule proteins.
C. elegans strains
The following strains were used in this study: GS2886: unc-36(e251); bIs1(vit-2::gfp; pRF4); RT258: unc-119(ed3); pwIs50(lmp-1::gfp, unc-119(+); NP952: cup-5(zu223) unc-36(e251)/qC1; bIs1(vit-2::gfp; pRF4)[16, 20]. We created, using bombardment methods, strains with the following transgenes : cdIs184( Pelt-2::GFP:CUP-5; punc-119(+)-myo-2::GFP); cdIs190( Pelt-2::mCherry:CUP-5; punc-119(+)-myo-2::GFP); cdIs194(lmp-1::TagRFP(S158T); unc-119(+)-ttx-3::gfp). TagRFP(S158T) is a bright monomeric red fluorescent protein .
The LMP-1::TagRFP(S158T) plasmid was made by replacing the GFP with TagRFP(S158T) coding sequences in a plasmid that expresses an LMP-1::GFP fusion protein . The ~742 bp TagRFP(S158T) cDNA was PCR amplified (primers CACACAACCGGTAGAAAAAATGGTGTCTAAGGGCGAAGAGC and CACACAGAATTCCAAAGCTTGTGGGCTTTTATTAAGTTTGTGCCCCAGTTTG), restriction digested with Age I + Eco RI and ligated into the 2979 bp fragment of pHD43 to make plasmid pHD491. The 3532 bp Sph I + Age I fragment of pHD491 was then ligated to the 3946 bp Sph I + Age I fragment of pPD117.01-LMP-1 to make plasmid pHD499 used to make transgenic lines.
The GFP::CUP-5 and mCherry::CUP-5 plasmids were made by inserting the elt-2 promoter to existing GFP::CUP-5 and mCherry::CUP-5 plasmids (pJF168 and pHD260, respectively). The elt-2 promoter was PCR amplified (primers CACACACCGGGCTGCAGGAATTCACATA and CACACACGCGGATCCATTCTATAAT) from pJM260, and restriction digested with Pst I and Bst UI. Both GFP::CUP-5 and mCherry::CUP-5, pJF168 and pHD260, respectively, were restriction digested with Bmt I, incubated with T4 DNA Polymerase to create a blunt end, and then digested with Pst I. The promoter fragment (5057 bp) was then ligated to the pJF168 fragment (6074 bp) and pHD260 fragment (6227 bp) to make pHD423 and pHD429, used to make transgenic lines.
Nile Red is a lipophilic membrane-permeable dye that diffuses across membranes and fluoresces when it complexes with lipids [24, 26, 36]. For Nile Red staining, adult worms were placed on OP50 bacteria suspended in 60 ug/ml Nile Red, on NGM plates with 60 ng/ml Nile Red (Invitrogen, Carlsbad, CA). Worms were allowed to lay embryos overnight, and embryos were immediately imaged. For imaging of live worm embryos (Nile Red, UV imaging, and fluorescently tagged constructs), embryos were resuspended in M9 buffer and dropped onto 3% agar pads; coverslips were added and sealed with petroleum jelly.
For PGP-2 immunostaining, embryos were dropped onto slides coated with poly-l-lysine (Sigma, St. Louis, MO) and covered with a coverslip. Slides were quickly frozen on a metal plate cooled over dry ice, and coverslips were flicked off the slides. Slides then underwent one 15-minute 100% methanol wash at -20°. Slides were washed twice with PBST (PBS plus 0.1% Tween), and blocked with PBS, 1% nonfat milk, 1% BSA, 0.1% Tween for 2 hours at 4°. Anti-PGP-2 antibody was added at 1:500 dilution in the above blocking buffer and left overnight at 4°, followed by three 10-minute washes in PBST . Secondary anti-rabbit Cy3 antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was added at 1:200 dilution in blocking buffer for two hours at room temperature, followed by three 10-minute washes in PBST. Excessive moisture was wicked away from embryos, and mounting media (Invitrogen) was added prior to imaging.
Confocal images were taken on a Zeiss LSM510 (Carl Zeiss, Oberkochen, Germany) laser scanning confocal, using LSM imaging software. Epifluorescence images were taken on a Leica DMRXA compound microscope (Leica Microsystems, Wetzlar, Germany), using MetaMorph software (Molecular Devices, Sunnyvale, CA).
For colocalization analysis and quantification of organelle size and intensity, MetaMorph (Molecular Devices) was used, and Microsoft Excel was used for statistical analysis. For each embryo measured, two 7 × 7 μm boxes were drawn, and all compartments within each box were measured for area and intensity per unit area. For YP170::GFP and LMP-1::GFP experiments, one z-section for each of five embryos of each genotype was measured. For PGP-2 and UV experiments one z-section of five embryos of each genotype was measured. We used two-tailed t-test p-values to determine significance in all experiments. Figs. 1, 2, 3 and 4 and Additional files were assembled using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA), and Fig. 5 was assembled using Canvas (ACD Systems, Victoria, BC Canada).
Mucolipidosis type IV
We thank Greg Hermann for the anti-PGP-2 antibodies and Jim McGhee for the elt-2 promoter expression plasmid. This work was funded by a March of Dimes grant (#6-FY07-205) to H. Fares and an NIH/NCI T32 CA09213-30 Cancer Biology Training Grant to E.M. Campbell. The authors declare that they have no competing financial interests.
- Eskelinen EL, Saftig P: Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta. 2009, 1793 (4): 664-673. 10.1016/j.bbamcr.2008.07.014.View ArticlePubMed
- Luzio JP, Parkinson MD, Gray SR, Bright NA: The delivery of endocytosed cargo to lysosomes. Biochem Soc Trans. 2009, 37 (5): 1019-1021. 10.1042/BST0371019.View ArticlePubMed
- Gyrd-Hansen M, Farkas T, Fehrenbacher N, Bastholm L, Hoyer-Hansen M, Elling F, Wallach D, Flavell R, Kroemer G, Nylandsted J: Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9. Mol Cell Biol. 2006, 26 (21): 7880-7891. 10.1128/MCB.00716-06.PubMed CentralView ArticlePubMed
- Reddy A, Caler EV, Andrews NW: Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes. Cell. 2001, 106 (2): 157-169. 10.1016/S0092-8674(01)00421-4.View ArticlePubMed
- Yamashima T, Tonchev AB, Tsukada T, Saido TC, Imajoh-Ohmi S, Momoi T, Kominami E: Sustained calpain activation associated with lysosomal rupture executes necrosis of the postischemic CA1 neurons in primates. Hippocampus. 2003, 13 (7): 791-800. 10.1002/hipo.10127.View ArticlePubMed
- Luzio JP, Pryor PR, Bright NA: Lysosomes: fusion and function. Nat Rev Mol Cell Biol. 2007, 8 (8): 622-632. 10.1038/nrm2217.View ArticlePubMed
- Bright NA, Gratian MJ, Luzio JP: Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol. 2005, 15 (4): 360-365. 10.1016/j.cub.2005.01.049.View ArticlePubMed
- Huizing M, Helip-Wooley A, Westbroek W, Gunay-Aygun M, Gahl WA: Disorders of lysosome-related organelle biogenesis: clinical and molecular genetics. Annu Rev Genomics Hum Genet. 2008, 9: 359-386. 10.1146/annurev.genom.9.081307.164303.PubMed CentralView ArticlePubMed
- Dell'Angelica EC, Mullins C, Caplan S, Bonifacino JS: Lysosome-related organelles. FASEB J. 2000, 14 (10): 1265-1278. 10.1096/fj.14.10.1265.View ArticlePubMed
- Raposo G, Marks MS, Cutler DF: Lysosome-related organelles: driving post-Golgi compartments into specialisation. Curr Opin Cell Biol. 2007, 19 (4): 394-401. 10.1016/j.ceb.2007.05.001.PubMed CentralView ArticlePubMed
- Hermann GJ, Schroeder LK, Hieb CA, Kershner AM, Rabbitts BM, Fonarev P, Grant BD, Priess JR: Genetic analysis of lysosomal trafficking in Caenorhabditis elegans. Mol Biol Cell. 2005, 16 (7): 3273-3288. 10.1091/mbc.E05-01-0060.PubMed CentralView ArticlePubMed
- Clokey GV, Jacobson LA: The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech Ageing Dev. 1986, 35 (1): 79-94. 10.1016/0047-6374(86)90068-0.View ArticlePubMed
- Chitwood BG, Chitwood MB: Introduction to Nematology. 1974, Baltimore: University Park Press
- Rabbitts BM, Ciotti MK, Miller NE, Kramer M, Lawrenson AL, Levitte S, Kremer S, Kwan E, Weis AM, Hermann GJ: glo-3, a novel Caenorhabditis elegans gene, is required for lysosome-related organelle biogenesis. Genetics. 2008, 180 (2): 857-871. 10.1534/genetics.108.093534.PubMed CentralView ArticlePubMed
- Fares H, Greenwald I: Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog. Nat Genet. 2001, 28 (1): 64-68. 10.1038/88281.PubMed
- Treusch S, Knuth S, Slaugenhaupt SA, Goldin E, Grant BD, Fares H: Caenorhabditis elegans functional orthologue of human protein h-mucolipin-1 is required for lysosome biogenesis. Proc Natl Acad Sci USA. 2004, 101 (13): 4483-4488. 10.1073/pnas.0400709101.PubMed CentralView ArticlePubMed
- Bach G: Mucolipidosis type IV. Mol Genet Metab. 2001, 73 (3): 197-203. 10.1006/mgme.2001.3195.View ArticlePubMed
- Puertollano R, Kiselyov K: TRPMLs: in sickness and in health. Am J Physiol Renal Physiol. 2009, 296 (6): F1245-1254. 10.1152/ajprenal.90522.2008.PubMed CentralView ArticlePubMed
- Schaheen L, Patton G, Fares H: Suppression of the cup-5 Mucolipidosis type IV-related lysosomal dysfunction by the inactivation of an ABC transporter in C. elegans. Development. 2006, 133 (19): 3939-3948. 10.1242/dev.02575.View ArticlePubMed
- Schaheen L, Dang H, Fares H: Basis of lethality in C. elegans lacking CUP-5, the Mucolipidosis type IV orthologue. Dev Biol. 2006, 293 (2): 382-391. 10.1016/j.ydbio.2006.02.008.View ArticlePubMed
- Currie E, King B, Lawrenson AL, Schroeder LK, Kershner AM, Hermann GJ: Role of the Caenorhabditis elegans multidrug resistance gene, mrp-4, in gut granule differentiation. Genetics. 2007, 177 (3): 1569-1582. 10.1534/genetics.107.080689.PubMed CentralView ArticlePubMed
- Kostich M, Fire A, Fambrough DM: Identification and molecular-genetic characterization of a LAMP/CD68-like protein from Caenorhabditis elegans. J Cell Sci. 2000, 113 (Pt 14): 2595-2606.PubMed
- Bossinger O, Schierenberg E: Cell-cell communication in the embryo of Caenorhabditis elegans. Dev Biol. 1992, 151 (2): 401-409. 10.1016/0012-1606(92)90180-O.View ArticlePubMed
- Bossinger O, Schierenberg E: Early embryonic induction in C. elegans can be inhibited with polysulfated hydrocarbon dyes. Dev Biol. 1996, 176 (1): 17-21. 10.1006/dbio.1996.9998.View ArticlePubMed
- Grant B, Hirsh D: Receptor-mediated endocytosis in the Caenorhabditis elegans oocyte. Mol Biol Cell. 1999, 10 (12): 4311-4326.PubMed CentralView ArticlePubMed
- Schroeder LK, Kremer S, Kramer MJ, Currie E, Kwan E, Watts JL, Lawrenson AL, Hermann GJ: Function of the Caenorhabditis elegans ABC transporter PGP-2 in the biogenesis of a lysosome-related fat storage organelle. Mol Biol Cell. 2007, 18 (3): 995-1008. 10.1091/mbc.E06-08-0685.PubMed CentralView ArticlePubMed
- Fukushige T, Hawkins MG, McGhee JD: The GATA-factor elt-2 is essential for formation of the Caenorhabditis elegans intestine. Dev Biol. 1998, 198 (2): 286-302.PubMed
- Kiselyov K, Jennigs JJ, Rbaibi Y, Chu CT: Autophagy, mitochondria and cell death in lysosomal storage diseases. Autophagy. 2007, 3 (3): 259-262.PubMed CentralView ArticlePubMed
- LaPlante JM, Falardeau J, Sun M, Kanazirska M, Brown EM, Slaugenhaupt SA, Vassilev PM: Identification and characterization of the single channel function of human mucolipin-1 implicated in Mucolipidosis type IV, a disorder affecting the lysosomal pathway. FEBS Lett. 2002, 532 (1-2): 183-187. 10.1016/S0014-5793(02)03670-0.View ArticlePubMed
- LaPlante JM, Sun M, Falardeau J, Dai D, Brown EM, Slaugenhaupt SA, Vassilev PM: Lysosomal exocytosis is impaired in Mucolipidosis type IV. Mol Genet Metab. 2006, 89 (4): 339-348. 10.1016/j.ymgme.2006.05.016.View ArticlePubMed
- Vergarajauregui S, Connelly PS, Daniels MP, Puertollano R: Autophagic dysfunction in Mucolipidosis type IV patients. Hum Mol Genet. 2008, 17 (17): 2723-2737. 10.1093/hmg/ddn174.PubMed CentralView ArticlePubMed
- Vergarajauregui S, Puertollano R: Mucolipidosis type IV: the importance of functional lysosomes for efficient autophagy. Autophagy. 2008, 4 (6): 832-834.PubMed CentralView ArticlePubMed
- Praitis V, Casey E, Collar D, Austin J: Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics. 2001, 157 (3): 1217-1226.PubMed CentralPubMed
- Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY: Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods. 2008, 5 (6): 545-551. 10.1038/nmeth.1209.PubMed CentralView ArticlePubMed
- Schaheen B, Dang H, Fares H: Derlin-dependent accumulation of integral membrane proteins at cell surfaces. J Cell Sci. 2009, 122 (Pt 13): 2228-2239. 10.1242/jcs.048892.View ArticlePubMed
- Greenspan P, Mayer EP, Fowler SD: Nile red: a selective fluorescent stain for intracellular lipid droplets. J Cell Biol. 1985, 100 (3): 965-973. 10.1083/jcb.100.3.965.View ArticlePubMed
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