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First published online 14 March 2006
doi: 10.1242/jcs.02958

Journal of Cell Science 119, 1494-1503 (2006)
Published by The Company of Biologists 2006
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An N-terminally acetylated Arf-like GTPase is localised to lysosomes and affects their motility

Irmgard Hofmann and Sean Munro*

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK


Figure 1
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  		Fig. 1. Arl8a and Arl8b localise to lysosomes. (A-D) Confocal micrographs of COS cells transfected with plasmids expressing either Arl8a-GFP or Arl8b-GFP from a CMV promoter. After fixation and permeabilization, the cells were labelled with antibodies against the indicated endogenous proteins CD63 (lysosomes), MPR (cation-dependent mannose 6-phosphate receptor, late endosomes) and EEA1 (early endosomes). (E) Confocal micrographs of NRK cells expressing Arl8b-GFP. Lysosomes were labelled with endocytosed dextran-tetramethylrhodamine using a four hour pulse at 1 mg/ml, followed by a 20-hour chase. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. Arl8 targeting is evolutionarily conserved but brefeldin A resistant. (A) Confocal micrographs of COS cells expressing the Drosophila Arl8 homologue (CG7891) with GFP at the C-terminus (Dm Arl8-GFP), and after fixation and permeabilisation labelled with antibody against the lysosomal protein CD63. (B) Confocal micrographs of COS cells expressing Arl8b-GFP and either untreated, or incubated in 5 µg/ml brefeldin A for 15 minutes, before fixation and labelling with the indicated antibodies to residents of the lysosome (LAMP2) and the Golgi (giantin). Bars, 10 µm.

 

Figure 3
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  		Fig. 3. The N-terminus of Arl8b is acetylated. (A) Alignment of human Arl8a and Arl8b with their relatives from Xenopus tropicalis, Drosophila melanogaster, Caenorhabditis elegans, Schistosoma japonicum, Arabidopsis thaliana, Neurospora crassa and Dictyostelium discoideum. Sequences were aligned with CLUSTAL W, and shaded in cases where more than half of the residues are related (grey) or identical (black). Hydrophobic amino acids are shown in red, arrows indicate the mutants analysed with combined point mutations in hydrophobic residues (red), or in individual conserved residues (black). (B) Alignment of the N-termini of human Arl8b, Arl1, Arf1 and ARFRP1. The second residue is shown in red. (C) MALDI mass spectrum of tryptic peptides from Arl8b-HA immunoprecipitated from transfected COS cells. Peptides with masses corresponding to expected digestion products of Arl8b are indicated with the sequence for the N-terminal peptide, or with residue numbers (o, oxidised). (D) Anti-HA immunoprecipitates from COS cells expressing LAT-HA or Arl8b-HA and labelled with [3H]palmitate. After gel electrophoresis, the precipitates were either probed with anti-HA antibodies or 3H-detected with fluorography.

 

Figure 4
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  		Fig. 4. The N-terminus of Arl8b is required for its lysosomal localisation. (A) Mutant forms of Arl8b, with the corresponding N-terminal peptides obtained after trypsin digestions of HA-tagged forms as in Fig. 3C. The sequence of the N-terminal peptides identified by MALDI were determined by tandem mass spectrometry (MS/MS). The mass of the peptide stated is for the protonated form, with that from Arl8b(L2F) being the form with an oxidised methionine that was more abundant than the native version and hence used for MS/MS sequencing. (B-D) Confocal micrographs of COS cells transfected with plasmids expressing the indicated forms of Arl8b fused to GFP and then, after fixation and permeabilisation, labelled with an anti-CD63 antibody to label lysosomes. (E-F) Confocal micrographs of COS cells transfected with plasmids expressing GFP (-G) fused to the indicated chimeras of Arf1 and Arl8b, and labelled with the indicated antibodies. Replacement of the first 18 residues of Arl8b with the first 15 of Arf1 does not prevent localisation to lysosomes (labelled with CD63), whereas the first 19 residues of Arl8b do not relocalise Arf1 (17-181) from the Golgi (labelled with golgin-245). Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Arl8b expression results in a more peripheral localization of lysosomes. (A) Confocal micrographs of COS cells expressing Arl8b-GFP or Arl8b(Q75L)-GFP and labelled with the antibodies to the indicated lysosomal membrane proteins. (B) As in (A), except that cells were trypsinised and plated on glass slides 90 minutes prior to fixation. (C) As in (A), except that cells were treated with 20 µM nocodazole for 4 hours before fixation, and then labelled for tubulin to confirm microtubule depolymerisation, or for the lysosomal antigen CD63. In such cells the lysosomes are scattered throughout the cytosol irrespective of the presence of Arl8b-GFP. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Arl8b stimulates lysosomal transport. (A) Still images of spinning-disc movies of lysosomes in processes of an untransfected NRK cell (con.), or of the same expressing Arl8b-GFP (Arl8b). Lysosomes were labelled by incubating cells for four hours with 1 mg/ml Alexa-Fluor-568-dextran, followed by a 20-hour chase. In both cases the cell body is at the top, and lysosomes moving toward, or away from, the cell body are indicated with open or closed triangles respectively. Bars, 5 µm. (B) Table summarizing the analysis of movies of NRK cells transfected with Arl8b-GFP, Arl8b(Q75L)-GFP or of untransfected cells. Lysosomes were labelled as in (A) and cell extensions of five cells each were imaged for three minutes at two frames per second. Individual lysosomes were tracked relative to an axis running from the cell body along the extension, and various parameters of the movement are shown. In each case a mean value is given, along with the standard error of mean. A `displacement' is defined as the movement between successive frames. A `transport event' is defined as a displacement of 0.15 µm or more per second, because this is typically the minimum speed of microtubule-dependent transport in vivo (Gross et al., 2000Go). A `static episode' is a period where the displacement between each successive frame does not exceed 0.15 µm/second. A `long episode of continuous fast motion' is when the net direct displacement over eight successive frames exceeds 1 µm. The length of an `episode of continuous motion' is defined for objects that have made a transport event, and is the net direct displacement before the object reverses direction relative to the axis, or does not make a transport event (i.e. moves less than 0.15 µm/second between frames). Perinuclear clusters of lysosomes were not included in the analysis as the software could not recognise every lysosome within these clusters.

 

Figure 7
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  		Fig. 7. Localisation of Arl8b in mitotic cells. Projection images of a stack of confocal slices from NRK cells transfected with a plasmid expressing Arl8b-GFP. After fixation and permeabilisation the cells were stained with antibodies to lysosomes and tubulin. Mitotic cells were identified in the total population by tubulin staining. Arl8b-GFP is localised to lysosomes, and is not detectable on the spindle mid-zone (A) or the midbody (B). Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Endogenous Arl8 is present on lysosomes in Drosophila cells. (A) Protein blot of total cellular proteins from D.Mel-2 cells probed with a rabbit antiserum raised against Drosophila Arl8 (Dm Arl8, CG7891). The cells were treated with dsRNA from GFP (con) or Drosophila Arl8. (B) Confocal micrographs of D.Mel-2 cells treated as in (A) with dsRNA from GFP (control) or Drosophila Arl8. After fixation and permeabilisation, cells were labelled with anti-Drosophila Arl8, and imaged with identical settings. (C) Confocal micrographs of S2 cells expressing a GFP fusion to the C-terminus of Drosophila LAMP1 (CG3305) and labelled with anti-Drosophila Arl8. (D-E) Confocal projection stacks of mitotic D.Mel-2 cells labelled with antibodies against Drosophila Arl8 and tubulin. Arl8 does not detectably concentrate in the spindle mid-zone in late anaphase (D), or in the midbodies (E) during late telophase. (F) Confocal micrographs of COS cells transfected with a plasmid expressing a native (i.e. un-tagged) form of Drosophila Arl8, and labelled with antibodies to Drosophila Arl8 and CD63. The Drosophila protein is localised to lysosomes, and alters the perinuclear distribution of lysosomes seen in the adjacent untransfected cell. Bars, 5 µm.

 

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