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First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02384

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RhoB regulates endosome transport by promoting actin assembly on endosomal membranes through Dia1

¹ The Netherlands Cancer Institute, Division of Tumour Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
² Sanquin Research at CLB, Department of Molecular Cell Biology, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands

View larger version (110K): [in a new window]   Fig. 1. Effect of activated RhoB on endosome distribution and the actin cytoskeleton. Cells were microinjected with myc-RhoBG14V (a-c), myc-RhoBwt (d-f) or myc-RhoAG14V (g-i). After 4 hours incubation, cells were fixed and stained for myc, the endosomal protein EEA1 and F-actin. RhoB proteins largely co-localised with EEA1 on endosomes (a,b and d,e, arrowheads) while RhoAG14V was cytosolic (g). EEA1-positive endosomes were located at the cell centre in control non-injected cells and in cells expressing RhoBwt (e) or RhoAG14V (h). In cells expressing RhoBG14V, EEA1-positive endosomes were peripheral and often appeared aligned on actin filaments (b,c, arrowheads). Bars: 10 µm.

View larger version (9K): [in a new window]   Fig. 2. The activated forms of RhoB and RhoA have opposite effects on cell morphology. The long and short perpendicular axes of control cells (n=30), RhoBG14V (n=30) and RhoAG14V (n=25) expressing cells were measured and the ratio between the two axes ±s.e.m. was calculated.

View larger version (89K): [in a new window]   Fig. 3. Activated RhoB induces the assembly of an F-actin coat around early endosomes, which appears associated to actin filaments. (A) High magnification microscopical analysis of endosomes of cells expressing myc-RhoBwt (a-c) or myc-RhoBG14V (d-f) and stained for Myc, EEA1 and F-actin. Both RhoBwt and RhoBG14V localised to EEA1-positive endosomes (a,b and d,e). RhoBwt-positive endosomes showed polymerized actin on a discrete site on their membranes (arrows in a-c and merge). RhoBG14V-positive endosomes were coated with F-actin and apparently associated with actin cables (arrows in d-f and merge). Bars: 2.5 µm. (B) Cells expressing GFP-actin were microinjected with myc-RhoBG14V plasmids and stained for Myc. High magnification images of the RhoBG14V-positive vesicles (indicated by arrowheads) show that these vesicles were coated with actin and apparently associated with actin fibres. Bar: 10 µm.

View larger version (62K): [in a new window]   Fig. 4. Activated RhoB inhibits transport of receptor-mediated and fluid-phase endocytosed cargo to the juxtanuclear area and impairs endosome motility. The effect of RhoBG14V on receptor-mediated endocytosis was studied by incubating cells with 50 µg ml–1 Texas Red-labelled human transferrin for 15 minutes. Myc-RhoBG14V was detected with 9E10 and FITC-labelled secondary antibodies (a). Fluid-phase endocytosis was analysed by 15 minutes incubation with the fluid-phase endocytic marker sulforhodamine 101 (SR101), followed by a 60 minutes chase (b). The effect of RhoBG14V on endosome motility was analysed on live cells transfected with pEGFP-FYVE (c). Myc-RhoBG14V cDNA was microinjected together with a 70 kDa Texas-Red-dextrane to mark the injected cells. Endosome dynamics was observed by time-lapse fluorescence confocal microscopy and analysed by projection of the time-lapse images (36 images with a time interval of 15 seconds) where vesicle movement is seen as green tracks. The projected image was then combined with a red-coloured image corresponding to t=0. In the RhoBG14V expressing cell (asterisk) vesicles form much shorter tracks (c, arrows) than in control non-injected cells (c, arrowheads). Nuclei of cells microinjected with myc-RhoBG14V are marked with an asterisk. Nuclei of control non-injected cells are marked with a circle. Bars: 10 µm.

View larger version (42K): [in a new window]   Fig. 5. Activated RhoB prevents Rab5-dependent endosome fusion. Cells were microinjected with the cDNA for the activated mutant of Rab5 (myc-Rab5Q79L) alone (a) or in combination with myc-RhoBG14V (b,b'). Rab5 was detected with the mouse 4F11 monoclonal antibody and RhoB with the rabbit sc-119 polyclonal antibody. Expression of myc-Rab5Q79L induced the formation of large endosomes that localised to the juxtanuclear area (a). Co-expressed myc-Rab5Q79L and myc-RhoBG14V co-localised in smaller endosomes scattered over the cell (b,b', arrows). Bars: 10 µm.

View larger version (88K): [in a new window]   Fig. 6. Involvement of Rho-effector proteins in the regulation of endosome transport downstream of RhoB. Cells expressing myc-RhoBG14V showed peripheral RhoBG14V-positive endosomes (a) and diffuse actin filaments (b) after 1h treatment with the ROCK inhibitor Y-27632. Co-expression of myc-RhoBG14V and a GFP-tagged dominant negative Dia1-N1 Dia1 (Dia1-N1) blocked the dispersion of RhoB-positive vesicles (c). Dia1-N1 partially co-localised with RhoB (merge of c and d). Bars: 10 µm.

View larger version (36K): [in a new window]   Fig. 7. Activated RhoB recruits mDia1 to endosomes. GFP-mDia1 (Dia1) localises to the cytoplasm of injected cells (a). Co-expression of myc-RhoBG14V and GFP-mDia1 results in the recruitment of Dia1 to peripheral RhoBG14V-positive vesicles (b,b'). The area within the square shows multiple vesicles positive for both Dia1 and RhoB. Bars 10 µm.

View larger version (162K): [in a new window]   Fig. 8. Activated RhoB effects on endosome distribution and actin coat assembly on endosomes are blocked by the dominant-negative Dia1-N1 mutant. GFP-mDia1-N1 (Dia1-N1) expression did not cause alteration of endosome distribution or the actin cytoskeleton (a-c). Dia1-N1 was recruited to endosomes by RhoBG14V (compare cell co-expressing both proteins with cell expressing only Dia1-N1 (d,e)). Co-expression of Dia1-N1 with myc-RhoBG14V blocked the effects of RhoBG14V on the distribution of EEA1-positive endosomes, which localised to the juxtanuclear area as in non-injected cells (d-f). Inset in 8f shows a magnification of the merged picture of the injected cell in 8d-f showing white-blue vesicles positive for Dia1-N1 (green), RhoB (red) and EEA1 (blue). Dia1-N1 also blocked the assembly of an actin coat around RhoBG14V-positive vesicles which now showed F-actin on a discrete site on their membranes (g-i). Inset in i shows vesicles labelled for RhoB (green) and F-actin (red). Bars: 10 µm.

View larger version (168K): [in a new window]   Fig. 9. An active mutant of Dia1 mimics RhoB effects. Expression of GFP-mDia1-N3 (Dia1-N3) induces the dispersion of EEA1-positive endosomes and the formation of thin parallel actin fibres (a,b, asterisk marks injected cell). Dia1-N3 is targeted to vesicles and induces the assembly of an actin coat around them (c,d and high magnification insets). Bars: 10 µm.

View larger version (33K): [in a new window]   Fig. 10. Model for the regulation of endosome transport by RhoB-Dia1. (a) RhoB-GDP is internalised from the plasma membrane and activated on endosomes by an exchange factor, for example Vav2 (Gampel and Mellor, 2002). (b) RhoB-GTP recruits Dia1 and PRK1 (Mellor et al., 1998) to endosomes. RhoB activates Dia1 by relieving the autoinhibitory intramolecular interaction between the DAD domain and the Rho-binding domain (RBD). (c) Activated Dia1 interacts with unknown factors on endosomes and promotes actin assembly/elongation on endosomes through FH2-dependent actin nucleation and/or FH1-dependent recruitment of profilin-ATP-actin. Endosomal actin associates with actin fibres that run underneath the plasma membrane, by an as yet unknown mechanism, preventing the transfer of endosomes to microtubules and inhibiting further transport. (d) Finally, inactivation of RhoB prevents further actin polymerisation, the actin coat depolymerises and endosomes can then bind to microtubules via the minus-end directed motor dynein for transport toward the microtubule minus-end.

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