spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02607

Journal of Cell Science 118, 4823-4832 (2005)
Published by The Company of Biologists 2005
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salas-Cortes, L.
Right arrow Articles by Coudrier, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salas-Cortes, L.
Right arrow Articles by Coudrier, E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes

Laura Salas-Cortes1,*, Fei Ye2,*, Danièle Tenza1, Claire Wilhelm3, Alexander Theos4, Daniel Louvard1, Graça Raposo1 and Evelyne Coudrier1,{ddagger}

1 Institut Curie, CNRS UMR144, 26 rue d'Ulm, 75248, Paris, Cedex 05, France
2 E363 INSERM, Faculté de Médecine, Necker, 156 Rue de Vaugirard, 75015, Paris, France
3 Laboratoire des milieux désordonnés et hétérogènes, Universite Pierre et Marie Curie, Boite 86, 4 Place Jussieu, 75252 Paris Cedex 05, France
4 Department of Pathology and Laboratory Medicine, University of Pennsylvania, 3451 Walnut Street, Philadelphia, PA 19104, USA



View larger version (131K):

[in a new window]
  		Fig. 1. MYO1B co-distributes with actin filaments at the cell periphery and partially with endosomes in MNT1 cells. (A-C) MNT-1 cells were co-labelled with affinity-purified polyclonal anti-Myo1b antibodies (SE5237) and phalloidin and analysed using confocal microscopy. Same optical section throughout the focal plane of the nucleus of one MNT-1 cell is shown for (A) Myo1b and (B) actin. Overlay of A and B is shown in C. Arrows show membrane areas enriched in actin filaments. Bar, 2.2 µm. (D-F) MNT-1 cells were co-labelled with affinity-purified polyclonal anti-Myo1b antibody (SE5237) and anti-EEA1 antibody. Same optical section throughout the focal plane of the nucleus of one MNT-1 cell for (D) Myo1b and (E) EEA1. An overlay of D and E and is shown in F. Arrows imdicate the partial co-distribution between endosomes and Myo1b. Bar, 1.9 µm.

 


View larger version (103K):

[in a new window]
  		Fig. 2. Expression of EGFP-Myo1b induces the re-distribution of endosomes in the perinuclear region of MNT-1 cells. (A) 20 µg of the membrane-enriched fraction P2, as well as pellets and supernatants that had been collected after high-speed centrifugation of 20 µg P2 treated with 1%Triton X-100 or 1%Triton X-100 with 5 mM ATP for 30 minutes at 4°C, were separated by 7% SDS-PAGE and transferred on nitrocellulose membranes. The membrane was probed with anti-Myo1b antibody (SE7995). Endogenous MYO1B and EGFP-Myo1b are both released after treatment with Triton X-100 supplemented with ATP. (B-J) EGFP-Myo1b-expressing and EGFP-expressing cells (B-G and H-J, respectively) were labelled with (B-D) phalloidin, (E-J) anti-EEA1 antibody and analysed by confocal microscopy. Same confocal section throughout the focal plane of the nucleus for the expression of (B) EGFP-Myo1b and (C) actin, (E) EGFP-Myo1b and (F) EEA1, (H) EGFP and (I) EEA1. (D) Overlay of B and C. (G) Overlay of E and F. (J) Overlay of H and I. The arrows show a membrane area enriched in actin filaments. Notice that overexpression of EGFP-Myo1b affects the cellular distribution of the endocytic compartments. Bars, 2.2 µm (B,E) and 1.9 µm (H).

 


View larger version (152K):

[in a new window]
  		Fig. 3. Expression of EGFP-Myo1b and cytochalasin D affect the distribution and morphology of endosomes. (A) Ultra-thin section of Epon-embedded EGFP-Myo1b cells. Notice the clustered vacuolar endosomes with an electron-dense coat (stars). Ultra-thin cryosections of EGFP-Myo1b cells singly immunogold-labelled for EEA1 (B) or doubly immunogold-labelled for EEA1 and EGFP (C). Notice the numerous vesicular and tubular extensions at the opposite site of the coats in B and C (large arrows). (D) EGFP-Myo1b was visualized on whole-mounted cells. An endosome filled with HRP and immunogold-labelled for GFP is shown. Labelling is observed on the vacuolar domain and mostly concentrated in an extending tubule (arrows). (E) Ultra-thin cryosections of nontransfected cells immunogold-labelled for actin. Actin (long arrows) is detected on small tubulovesicular elements close to the endosomal vacuoles (stars) at the opposite site of the coated areas (short arrows). (F) Ultra-thin cryosection of MNT-1 cells immunogold-labelled for actin and treated with 0.4 µM cytochalasin D for 90 minutes. Actin is detected at the cytosolic side of tubular structures (arrows). Bars, 200 nm.

 


View larger version (138K):

[in a new window]
  		Fig. 4. Cytochalasin D induces the re-distribution of endosomes in the perinuclear region of MNT-1 cells. (A-F) MNT-1 cells that had been treated (D,E,F) or not (A,B,C) with cytochalasin D were co-labelled with anti-EEA1 antibody (A,D), and phalloidin (B,E), and then analysed using confocal microscopy. (A,B) and (D,E) show the same confocal sections throughout the nucleus. (C,F) Overlay of (C) A,B and (F) D,E. Arrows show co-distribution of EEA1-labelled compartments with actin filaments. Bar, 2.8 µm.

 


View larger version (140K):

[in a new window]
  		Fig. 5. Expression of EGFP-Myo1b perturbs the cellular distribution of Pmel17. (A,B) EGFP-Myo1b-expressing (A) or EGFP-expressing (B) MNT-1 cells labelled with anti-Pmel17 antibody (HMB50) and analysed by confocal microscopy. Overlay of the optical section through the nucleus shows the distribution of Pmel17 and EGFP-Myo1b (A). Overlay of the optical sections throughout the nucleus showing the distribution of Pmel17 and EGFP (B). (C,D) Pmel17-expressing HeLa cells that had been transiently transfected with cDNA encoding (C) EGFP-Myo1b and (D) EGFP, were labelled with anti-Pmel17 antibody (HMB50) and analysed by confocal microscopy. Overlay of the optical sections through the nucleus show the distribution of (C) Pmel17 and EGFP-Myo1b, and (D) Pmel17 and EGFP. Notice the re-distribution of Pmel17 (arrows) towards the nucleus in EGFP-Myo1b-expressing cells. Bars, 1.9 µm.

 


View larger version (56K):

[in a new window]
  		Fig. 6. Expression of EGFP-Myo1b delays the processing of Pme17. (A) Schematic representation of Pmel17 and the processed forms proposed by Berson et al. (Berson et al., 2001Go). Pmel17 is synthesized as type-I integral membrane protein precursor P1 in the endoplasmic reticulum, glycosylated to precursor P2 in the Golgi, and cleaved into two disulfide-linked subunits: a large lumenal subunit M{alpha} and an integral membrane subunit Mß. Antibodies HMB45 and HMB50 recognize the N-terminus of P1, P2 and M{alpha}. HMB50 immunoprecipitates both M{alpha} and Mß, because of their disulfide-link. {alpha}PEP13 antibody recognizes the C-terminus of P1, P2 and Mß. (B) Lysates from five million EGFP-expressing or EGFP-Myo1b-expressing cells were immunoprecipitated with HMB50. Precipitates were then analysed by western blotting with HMB45 to detect M{alpha}, and {alpha}PEP13 to detect P1, P2 and Mß. The same membrane was used for the detection of the different polypeptides. One of four representative experiments is shown. (C) EGFP-expressing cells and EGFP-Myo1b cells were metabolically pulse-labelled with [35S]methionine/cysteine for 10 minutes and then chased for 0 minutes, 30 minutes, 1 hour and 2 hours. 25 µCi of cell lysate were then immunoprecipitated with HMB50. Precipitated proteins were analysed by SDS-PAGE and detected by autoradiography. (D) The relative intensities of Mß in EGFP and EGFP-Myo1b cells were quantified and expressed in arbitrary units. (C,D) One of three representative experiments is shown.

 


View larger version (183K):

[in a new window]
  		Fig. 7. Morphology of pre-melanosomes is altered upon expression of EGFP-Myo1b. (A) Ultra-thin section of Epon-embedded nontransfected MNT1 cells. Several melanosomal stages (II, III and IV) as well as coated sorting MVEs (CE) are shown. (B) Ultra-thin section of Epon-embedded EGFP-Myo1b cells. Stage-II and stage-III melanosomes show disrupted intraluminal fibrils and abnormal deposits of melanin (stars). Bars, 200 nm.

 


View larger version (32K):

[in a new window]
  		Fig. 8. EGFP-Myo1b and MYO1B co-immunoprecipitate with the precursor P2 of Pmel17. (A) Schematic representation of recombinant EGFP-Myo1b and Myo1b-truncated domains. Myo1b has a motor domain with ATP-and actin-binding sites at the N-terminus and a tail with IQ motifs and a cluster of basic amino acids at the C-terminus. The motor domain was deleted in EGFP-Myo1b-Tail. (B) EGFP or EGFP-Myo1b cell lysates were immunoprecipitated with anti-GFP antibodies and analysed by SDS-PAGE, and western blotting with anti-GFP antibody and the anti-Pmel17 antibody {alpha}Pep13. (C) EGFP, EGFP-Myo1b and EGFP-Myo1b-Tail cell lysates were immunoprecipitated with the anti-Pmel17 antibody (HMB50) and analysed by SDS-PAGE, and western blotting with anti-GFP antibody. EGFP and EGFP-Myo1b cell lysates immunoprecipitated with HMB50 antibody were also analysed by SDS-PAGE, and western blotting with anti-Myo1b antibody (SE7995). These experiments were repeated three and four times for B and C, respectively.

 


View larger version (43K):

[in a new window]
  		Fig. 9. EGFP-Myo1b and Pmel17 associated to magnetic-endosome co-immunoprecipitate. (A) Post-nuclear supernatant (PNS), non-magnetic (NMF) and magnetic fractions (MF) isolated from wild-type or EGFP-Myo1b cells as described in Materials and Methods, were analysed by SDS-PAGE, and western blotting with anti-Rab5 and anti-Rab7antibodies in the case of wild-type cells, anti-Myo1b (SE7995) and anti-Pmel17 (Pep13h) antibodies for the magnetic fraction of both cell types, and anti-GFP antibodies for the magnetic fraction isolated from EGFP-Myo1b cells. Note that MYO1B as well as P1, P2 and Mß polypeptides of Pmel17 are present in the magnetic fraction enriched for Rab5, and that EGFP-Myo1b is detected on the magnetic fraction isolated from EGFP-Myo1b cells. (B) Lysate of the magnetic fraction isolated from EGFP-Myo1b cells was immunoprecipitated with anti-GFP antibody and analysed by SDS-PAGE, and western blotting with anti-Pmel17 (Pep13h) antibody. The molecular mass of the immunoprecipitated proteins was compared to the molecular mass of the Pmel17 precursor forms P1 and P2, which had been detected with the same antibody in the post nuclear supernatant (PNS). A polypeptide of the size comparable with P2 co-immunoprecipitates with EGFP-Myo1b.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2005