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

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

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

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

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

The item banking service as a part of a national system of evaluation

<div><p>CAV1 (caveolin 1, caveolae protein, 22kDa) is well known as a principal scaffolding protein of caveolae, a specialized plasma membrane structure. Relatively, the caveolae-independent function of CAV1 is less studied. Autophagy is a process known to involve various membrane structures, including autophagosomes, lysosomes, and autolysosomes for degradation of intracellular proteins and organelles. Currently, the function of CAV1 in autophagy remains largely elusive. In this study, we demonstrate for the first time that CAV1 deficiency promotes both basal and inducible autophagy. Interestingly, the promoting effect was found mainly in the late stage of autophagy via enhancing lysosomal function and autophagosome-lysosome fusion. Notably, the regulatory function of CAV1 in lysosome and autophagy was found to be caveolae-independent, and acts through lipid rafts. Furthermore, the elevated autophagy level induced by CAV1 deficiency serves as a cell survival mechanism under starvation. Importantly, downregulation of CAV1 and enhanced autophagy level were observed in human breast cancer cells and tissues. Taken together, our data reveal a novel function of CAV1 and lipid rafts in breast cancer development via modulation of lysosomal function and autophagy.</p></div