QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 November 2004
doi: 10.1242/jcs.01560

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Simpson, J. C. Articles by Jones, A. T. Search for Related Content PubMed PubMed Citation Articles by Simpson, J. C. Articles by Jones, A. T.

A role for the small GTPase Rab21 in the early endocytic pathway

Jeremy C. Simpson¹, Gareth Griffiths¹, Marianne Wessling-Resnick², Jack A. M. Fransen³, Holly Bennett^* and Arwyn T. Jones^4,

¹ Cell Biology and Biophysics Programme, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany
² Department of Nutrition, Harvard School of Public Health, Boston, MA 02115, USA
³ Department of Cell Biology, NCMLS, UMC St. Radboud, University of Nijmegen, 6500 HB, Nijmegen, The Netherlands
⁴ Welsh School of Pharmacy, Cardiff University, Redwood Building, Cardiff, CF10 3XF, Wales, UK

View larger version (76K): [in a new window]   Fig. 1. Characterisation of GFP-Rab21 variants. (A-C) Lysates from HeLa cells expressing GFP-Rab21 wt, GFP-Rab21 T33N, GFP-Rab21 Q78L or GFP alone were immunoprecipitated using anti-GFP antibodies and subjected to SDS-PAGE and western analysis before detection of GFP by ECL (A) or GTP binding using 32P-GTP (B). Graph in C shows quantification of radioactivity from GFP-Rab21 bands in B and shows maximum GTP binding by GFP-Rab21 Q78L and baseline GTP binding by GFP-Rab21 T33N. (D-F) HeLa cells transfected using calcium phosphate with GFP-Rab21 wt (D), GFP-Rab21 T33N (E) and GFP-Rab21 Q78L (F) plasmids were fixed and processed for fluorescence microscopy. GFP-Rab21 wt and Rab21 Q78L predominantly label the perinuclear region, in addition to peripheral vesicular structures. Rab21 Q78L is also localised to a reticular network emanating from the perinuclear area to the cell periphery. Rab21 T33N labels the perinuclear region in addition to small cytoplasmic vesicles (E). Bars, 10 µm. Video microscopy of HeLa cells expressing GFP-Rab21 variants can be seen as QuickTime Movies 1-3 in supplementary material.

View larger version (85K): [in a new window]   Fig. 2. Subcellular localisation of GFP-Rab21. HeLa cells, transfected using calcium phosphate with GFP-Rab21 wt (A-C,G-O) or GFP-Rab21 T33N (D-F) plasmids were either directly fixed and labelled with anti-p230 (A-F), anti-Lamp-1 (G-I) and anti-transferrin receptor (J-L) antibodies, followed by rhodamine-conjugated secondary antibodies (red in merge), or were first incubated for 6 minutes at 37°C with 75 ng/ml Rh-EGF (M-O) before fixation. Confocal fluorescence microscopy shows some colocalisation between Rab21 wt and p230 in the more intensely labelled perinuclear area but GFP-Rab21 peripheral structures are free of this marker (A-C, arrowheads). Rab21 T33N overlaps extensively with p230 (D-F). There is very little overlap between Rab21 and Lamp-1 (G-I) but some overlap in peripheral structures with the transferrin receptor (J-L) and internalised Rh-EGF (M-O). Arrows indicate colocalising structures, and arrowheads indicate structures containing only Rab21. Bars, 10 µm.

View larger version (84K): [in a new window]   Fig. 3. Colocalisation of YFP-Rab21 with other CFP-Rab proteins. (A-G) HeLa cells double transfected with YFP-Rab21 wt and other CFP-tagged Rab proteins were fixed and processed for fluorescence microscopy and colocalisation analysis. Examples of Rab21 wt with Rab5a (A-C) and Rab21 wt with Rab11a (D-F) are shown. Arrows indicate colocalising structures, and arrowheads indicate structures containing only Rab21. Bar, 10 µm. Graph in G shows quantitative analysis of colocalisation of discrete Rab21 structures with other Rabs. Error bars represent mean and standard deviation.

View larger version (119K): [in a new window]   Fig. 4. Expression of GFP-Rab21 wt causes enlargement of EEA1-positive endosomes. HeLa cells transfected with GFP-Rab21 wt (A-C), GFP-Rab21 Q78L (D-F) or GFP-Rab21 T33N (G-I) plasmids using calcium phosphate were fixed and labelled with anti-EEA1 antibodies and Texas Red-conjugated secondary antibodies (red in merge) before analysis by confocal fluorescence microscopy. GFP-Rab21 wt expression results in the formation of swollen endosomes that label intensely with anti-EEA1 antibodies (A-C). GFP-Rab21 Q78L expression also causes increased prominence of EEA1-positive endosomes (D-F) but these are not as distinct as those in cells expressing GFP-Rab21 wt. EEA1 labels smaller vesicles in nontransfected cells and those expressing Rab21 T33N (G-I). Arrows indicate colocalisation of GFP-Rab21 and EEA1, and asterisks represent transfected cells. Bars, 10 µm.

View larger version (92K): [in a new window]   Fig. 5. Endogenous Rab21 and GFP-Rab21 distribution in Hep3B cells. Hep3B cells were either directly fixed and stained with anti-Rab21 antibodies (A-C), or first transfected with GFP-Rab21 wt (D-F), GFP-Rab21 Q78L (G-I) or GFP-Rab21 T33N (J-L) plasmids using calcium phosphate before fixing and labelling with anti-EEA1 antibodies and Texas Red-conjugated secondary antibodies (red in merge) before analysis by confocal fluorescence microscopy. Endogenous Rab21 was seen to localise to punctate structures, many of which also contained EEA1 (A-C, arrows). In addition, many EEA1-positive, but Rab21-negative structures were observed (arrowheads). GFP-Rab21 wt expression results in the formation of swollen endosomes that label intensely with anti-EEA1 antibodies (D-F). GFP-Rab21 Q78L expression also causes slight enlargement of EEA1-positive endosomes (G-I). EEA1 labels smaller vesicles in nontransfected cells and those expressing Rab21 T33N (J-L). Arrows indicate colocalisation of GFP-Rab21 and EEA1, and asterisks represent transfected cells. Bars, 10 µm.

View larger version (132K): [in a new window]   Fig. 6. Localisation of GFP-Rab21 on Golgi and endosomal membranes by immunogold labelling and electron microscopy. HeLa cells transfected with GFP-Rab21 plasmids using calcium phosphate were fixed and prepared for cryosectioning and immunolabelling with anti-GFP, and anti-EEA1, TfR or Hrs antibodies. (A) Single labelling of GFP-Rab21 in a section across an elongated cell process showing the presence of GFP-Rab21 on the plasma membrane and the periphery of internal vesicles. (B) Peripheral membranes of a swollen endosome labelled with GFP-Rab21. (C,D) Colocalisation of GFP-Rab21 and EEA1 on endosomal membranes. Cryosections were double labelled with anti-GFP (arrowheads) and anti-EEA1 (arrows) antibodies. The images show examples of vesicular and tubular structures labelled with both antibodies. The localisation of Rab21 and EEA1 is most apparent on peripheral membranes of early endosomes (marked EE) close to the plasma membrane (marked PM). In D, both Rab21 and EEA1 are in close proximity to a caveolae invagination on the plasma membrane (marked Cv). (E-I) Examples of the extent of colocalisation of GFP-Rab21 with the two endosomal markers TfR and Hrs. Note in E, that Golgi (marked Go) labelling is unique to Rab21; however, endosomes (marked E) and tubular structures are labelled with both antibodies. (H,I) GFP-Rab21 and Hrs are often colocalised on endosomal structures close to the plasma membrane (marked PM). Note, however, the Hrs-positive, Rab21-negative early endosome (marked EE) in I. Some labelling of the plasma membrane (PM) was also observed with both antibodies.

View larger version (84K): [in a new window]   Fig. 7. Endocytosis of TxR-Tf is inhibited in cells expressing GFP-Rab21 T33N. Hep3B cells transfected with GFP-Rab21 plasmids were incubated for 15 minutes at 37°C with 50 nM TxR-Tf, washed on ice, fixed and analysed by fluorescence microscopy. There was no visible difference in the levels or distribution pattern of internalised TxR-Tf (red in merge) in nontransfected cells and those expressing GFP-Rab21 wt (A-C) or GFP-Rab21 Q78L (D-F). Levels of internalised Tf were significantly lower in cells expressing GFP-Rab21 T33N and the label was confined to relatively small vesicles (G-I). Arrows in A-C point to Rab21 wt colocalisation with internalised TxR-Tf. (J-M) HeLa cells transfected with GFP-Rab21 wt plasmid were incubated for 4 minutes at 37°C with 50 nM TxR-Tf, washed on ice, fixed and labelled with anti-EEA1-and Alexa647-conjugated secondary antibodies, followed by confocal fluorescence microscopy. Arrows point to Rab21 wt, EEA1 and TxR-Tf colocalising structures. Asterisks represent transfected cells. Bars, 10 µm. (N) HeLa cells transfected with GFP-Rab21 wt plasmids were incubated with 50 nM TxR-Tf, for either 4 minutes at 37°C, 1 hour at 16°C or for 30 minutes at 37°C followed by washing and chasing for a further 30 minutes at 37°C in the absence of TxR-Tf, before fixing and analysis by fluorescence microscopy. Quantification of the number of TxR-Tf structures colocalising with Rab21 was determined for the different internalisation conditions. Error bars show mean and standard deviation between individual cells. Asterisk indicates statistical significance of P<0.001 compared with 4 minutes internalisation at 37°C.

View larger version (86K): [in a new window]   Fig. 8. Endocytosis of Rh-EGF is inhibited in cells expressing GFP-Rab21 T33N. HeLa cells transfected with GFP-Rab21 plasmids using calcium phosphate were incubated for 30 minutes at 37°C with 150 ng/ml Rh-EGF, washed on ice, fixed and analysed by confocal fluorescence microscopy. There was no visible difference in the levels or distribution pattern of internalised Rh-EGF (red in merge) in nontransfected cells and those expressing GFP-Rab21 wt (A-C) or GFP-Rab21 Q78L (D-F). Levels of internalised EGF were significantly lower in cells expressing GFP-Rab21 T33N and the label was confined to relatively small vesicles (G-I). Arrows in A-F point to Rab21 wt and Rab21Q78L colocalisation with internalised Rh-EGF. Asterisks represent transfected cells. Bars, 10 µm. Video microscopy of HeLa cells expressing GFP-Rab21 wt, GFP-Rab21 Q78L and GFP-Rab21 T33N following 15 minutes internalisation of Rh-EGF can be seen as QuickTime Movies 4-6 in supplementary material.

View larger version (54K): [in a new window]   Fig. 9. GFP-Rab21 T33N expression inhibits lysosomal delivery of Rh-EGF. HeLa cells transfected with GFP-Rab21 plasmids were incubated for 60 minutes with 150 ng/ml Rh-EGF before washing on ice and incubating (chase) for a further 120 minutes in Rh-EGF-free medium. The cells were fixed and analysed by fluorescence microscopy. Shown is the almost complete lack of Rh-EGF fluorescence in cells expressing moderate levels of GFP-Rab21 wt (A,B) and Rab21 Q78L (E,F). By contrast, undegraded EGF was clearly observed in distinct perinuclear structures close to the nucleus in cells expressing very high levels of GFP-Rab21 wt (C,D). Extensive Rh-EGF fluorescence in small diffuse punctate structures in GFP-Rab21 T33N expressing cells is also visible (G,H). Arrows point to colocalising GFP-Rab21 wt and Rh-EGF. HeLa cells expressing GFP-Rab21 T33N were also fixed and stained for Lamp-1 after this chase period (I-L). None of the undegraded Rh-EGF colocalises with Lamp-1 (arrowheads). Bars, 10 µm.