Immunolocalisation of Endogenous and Overexpressed Rabankyrin-5 in NIH3T3 Cells

<div><p>Untransfected NIH3T3 cells or cells transfected with Rabankyrin-5 were labelled with antibodies to Rabankyrin-5 followed by 10 nm protein A gold.</p> <p>(A) Transfected cell showing labelling of a group of vesicular structures underlying the plasma membrane (pm).</p> <p>(B and C) In control (untransfected) cells, low but specific labelling for Rabankyrin-5 (arrowheads) is associated with compartments close to the pm. Scale bars represent 200 nm.</p></div

Inhibition of Rab5 Activity Decreases Fluid-Phase Uptake

<div><p>NIH3T3 cells transiently transfected for either (A) GFP-Rab5wt or (B) RN-tre were subjected to a 30-min uptake of rhodamine-conjugated transferrin (1 μg/ml) or dextran (MW, 70.000; 3 mg/ml) at 37 °C and further processed for confocal imaging with indicated antibodies.</p> <p>(A) Rab5wt transfected cells show colocalisation of Rabankyrin-5–labelled macropinocytic structures, indicated by the lack of transferrin accumulation, with Rab5.</p> <p>(B) Cells transiently transfected for RN-tre (asterisk) show a significant reduction of fluid-phase uptake compared to nontransfected cells.</p> <p>(C) Fluid-phase dextran quantification of single cells transfected for RN-tre by measuring internalised fluorescence intensity (<i>p</i> > 0,001). Values shown are means ± standard deviation of at least 15 cells. Scale bars represent 10 μm.</p></div

Rabankyrin-5 Localises to Macropinosomes in NIH3T3 Cells

<div><p>(A) NIH3T3 cells directly fixed and immunostained for endogenous Rabankyrin-5 and EEA1 reveal segregation of Rabankyrin-5 from EEA1-containing structures (arrows) in the cell periphery, while there is colocalisation in the cell centre (arrowheads).</p> <p>(B) Overexpression of Rabankyrin-5 increases the number of peripheral enlarged structures devoid of EEA1. NIH3T3 cells were infected with recombinant adenovirus for Rabankyrin-5 for 18 h. Dextran (2,5 mg/ml) uptake was performed for 3 min at 37 °C, fixed, and immunostained for the indicated antigens.</p> <p>(C and D) Formation of enlarged Rabankyrin-5 structures requires PI3-K activity. NIH3T3 cells either (C) DMSO treated or (D) pretreated for 20 min with wortmannin (WM; 100 nM) were incubated for 8 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 10.000), fixed, processed for immunofluorescence, and analysed by confocal scanning microscopy.</p> <p>(E) NIH3T3 cells transiently transfected for YFP-Rabankyrin-5 and CFP-actin were imaged using time-lapse video microscopy to visualise the formation of macropinosomes by actin-driven membrane ruffles. Images were taken for the indicated time points. The arrowhead points towards Rabankyrin-5 association to an enlarged vesicle driven by actin dynamics over time. Scale bars represent 10 μm.</p></div

Rabankyrin-5 Overexpression Increases, whereas Knock-Down Decreases Fluid-Phase Uptake, Specifically

<div><p>(A–C) NIH3T3 cells were either mock-infected or infected with recombinant adenovirus encoding Rabankyrin-5. Simultaneous uptake of HRP (5 mg/ml) and biotinylated transferrin (2 μg/ml) was performed at 37 °C for the indicated time points.</p> <p>(C) NIH3T3 cells were pulsed with HRP (10 mg/ml) for 10 min. Recycled HRP into the medium was determined after the indicated time points.</p> <p>(D–F) A431 cells were treated with esiRNA against Rabankyrin-5 or control treated for 4 d.</p> <p>(D) Western blot analysis revealed a 50% reduction of Rabankyrin-5 in whole-cell lysate.</p> <p>(E and F) Serum-starved cells (1 h) were stimulated with 50 ng/ml EGF in complete medium for the indicated time points in the presence of 5 mg/ml HRP and 2 μg/ml biotinylated transferrin. Values shown are means ± standard deviation and were performed in duplicates. The results are representatives of at least two independent experiments.</p></div

A Protein of 130 kDa Is a New Rab5 Effector

<div><p>(A) GST-Rab5-GDP and GST-Rab-GTPγS were loaded on beads and incubated with bovine brain cytosol. Bound proteins were eluted and analysed by SDS-PAGE followed by Coommasie Blue staining. The positions of the already known Rab5 effectors (EEA1, Rabaptin-5, hVps34, p110β, and Rabenosyn-5) and of the new Rab5 effector are indicated.</p> <p>(B) Schematic representation of the domain organisation in Rabankyrin-5. ANK, ankyrin repeats.</p> <p>(C) Bovine brain cytosol or HeLa cell cytosol was incubated with GST-Rab5-GDP– or GST-Rab5-GTPγS–loaded beads. Subsequently the beads were washed, and bound proteins were eluted and analysed by Western blotting using anti–Rabankyrin-5 antibodies.</p> <p>(D) GST-Rab4, -5, -7, and -11 fusion proteins were preloaded with GDP or GTPγS and incubated with in vitro-translated ³⁵S-methionine–labelled Rabankyrin-5 full-length protein. As a control, bound and unbound material was analysed by SDS-PAGE followed by phosphoimager analysis.</p> <p>(E) Rabankyrin-5 binds most strongly to PI(3)P. Recombinant full-length Rabankyrin-5 was incubated with liposomes containing 2% of the indicated phosphoinositide. Bound Rabankyrin-5 was detected by Western blotting.</p></div

Rabankyrin-5 Associates with Two Types of Rab5-Containing Vesicles in A431 Cells, Early Endosomes, and Macropinosomes

<div><p>(A) A431 cells stably transfected for GFP-Rab5wt were immunostained for endogenous Rabankyrin-5 and EEA1. While there is perinuclear overlap between Rab5, Rabankyrin-5, and EEA1 in nontransfected cells (arrowheads), some smaller peripheral structures are devoid of EEA1 (arrows).</p> <p>(B) Overexpression of Rabankyrin-5 in A431 by using a recombinant adenovirus construct of Rabankyrin-5 causes an accumulation of peripheral, enlarged Rab5-positive structures, costained mainly by Rabankyrin-5 (arrows) but not detectable for EEA1.</p> <p>(C) Rabankyrin-5 localises on EGF-induced and -enriched macropinosomes. Serum-starved A431 cells (16 h) were incubated for 7 min with 100 ng/ml rhodamine-conjugated EGF to induce macropinocytosis and 1 μg/ml Cy5-labelled transferrin. Endogenous Rabankyrin-5 localises to enlarged EGF-containing macropinosomes, indicated by the lack of transferrin labelling (arrows), but also to EGF- and transferrin-containing endosomes (arrowheads).</p> <p>(D) Rabankyrin-5 structures contain tyrosine-phosphorylated proteins. A431 cells, stably transfected for GFP-Rab5, were stimulated with 50 ng/ml EGF for 7 min and immediately processed for immunofluorescence. Costaining of Rabankyrin-5 and tyrosine-phosphorylated proteins (α-4G10) reveal the localisation of Rabankyrin-5 to plasma membrane ruffles. Scale bars represent 10 μm.</p></div