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. 2011 Dec;12(12):1911-22.
doi: 10.1111/j.1600-0854.2011.01280.x. Epub 2011 Oct 9.

Coordination of the Rab5 cycle on macropinosomes

William D Feliciano  1 , Sei Yoshida, Samuel W Straight, Joel A Swanson
Affiliations

Coordination of the Rab5 cycle on macropinosomes

William D Feliciano et al. Traffic. 2011 Dec.

Abstract

The GTPase Rab5a regulates the homotypic and heterotypic fusion of membranous organelles during the early stages of endocytosis. Many of the molecules which regulate the Rab5a cycle of association with membranes, activation, deactivation and dissociation are known. However, the extent to which these molecular scale activities are coordinated on membranes to affect the behavior of individual organelles has not been determined. This study used novel Förster resonance energy transfer (FRET) microscopic methods to analyze the Rab5a cycle on macropinosomes, which are large endocytic vesicles that form in ruffled regions of cell membranes. In Cos-7 cells and mouse macrophages stimulated with growth factors, Rab5a activation followed immediately after its recruitment to newly formed macropinosomes. Rab5a activity increased continuously and uniformly over macropinosome membranes then decreased continuously, with Rab5a deactivation preceding dissociation by 1-12 min. Although the maximal levels of Rab5a activity were independent of organelle size, Rab5a cycles were longer on larger macropinosomes, consistent with an integrative activity governing Rab5a dynamics on individual organelles. The Rab5a cycle was destabilized by microtubule depolymerization and by bafilomycin A1. Overexpression of activating and inhibitory proteins indicated that active Rab5a stabilized macropinosomes. Thus, overall Rab5a activity on macropinosomes is coordinated by macropinosome structure and physiology.

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Figures

Figure 1
Figure 1. Visualization of the Rab5a activation cycle on macropinosomes
(A) A Cos-7 cell transfected with CFP-Rab5a, YFP-RBD and mCherry, stimulated with EGF and viewed by phase-contrast microscopy (PC). The enlarged region shows a macropinosome labeled with CFP-Rab5a (B) and YFP-RBD (C), and the distribution of cytoplasm indicated by mCherry (D). (E) A time series showing a region of the cell shown in panels A–D, processed for ratiometric imaging of CFP-Rab5a (D = ID + EDA*ξ) and for total fluorescence due to FRET between CFP-Rab5a and YFP-RBD (EDA = IF -α*IA -β*ID). In the bottom row, EAVG values are presented as pseudocolor overlaid on the phase-contrast images (EAVG = (EA + ED)/2). Color scale indicates EAVG values. EGF (100 ng/mL) was added after acquisition of the first frame. (F) BMM expressing CFP-Rab5a, YFP-RBD and mCherry, stimulated with M-CSF. Image processing for FRET stoichiometry obtained the CFP-Rab5a distribution (D), total FRET signal (EDA) and EAVG. For both cells, CFP-Rab5a recruitment to macropinosomes coincided with the increase in FRET, but FRET decreased before CFP-Rab5a dissociated from the macropinosome. Scale bars = 10 μm.
Figure 2
Figure 2. The Rab5a cycle is slower on larger macropinosomes
(A–B) Quantification of the CFP-Rab5a activation cycle on individual macropinosomes of Cos-7 (closed circles) and BMM (open circles). (A) CFP-Rab5a association and dissociation from macropinosomes. The larger macropinosome in the Cos-7 cell was labeled longer with CFP-Rab5a. (B) FRET from macropinosome-associated CFP-Rab5a and YFP-RBD (EDA). (C) Comparison of CFP-Rab5a association (closed symbols, from panel A) and activation (open symbols, from panel B) profiles on a single macropinosome from Cos-7 cells (circles) and BMM (triangles), normalized to the maximum and minimum values and plotted as Fraction of the Maximal Signal versus Time. Measured parameters were: a, for CFP-Rab5a association (D), the interval between the time the macropinosome reached half-maximal value ascending to the time it reached half-maximal value descending; b, for CFP-YFP FRET signals (EDA), the interval between the time the macropinosome reached half-maximal value ascending to the time it reached half-maximal value descending; c, for the ascending phase of D and EDA curves, the interval between the time of half-maximal D and half-maximal EDA; d, for the descending phases of the D and EDA curves, the interval between the time of half-maximal D and half-maximal EDA. (D) Maximal FRET signals (EAVGMax) on individual macropinosomes, plotted vs. macropinosome diameter. Although maximal FRET signals were lower in BMM (triangles) than in Cos-7 cells (circles) for macropinosomes of comparable sizes, maximal FRET signals did not correlate with macropinosome size. (E) Half-maximal widths for Rab5a cycles were measured in macropinosomes of Cos-7 cells (circles) and BMM (triangles). CFP-Rab5a FRET cycles (a, closed symbols) were shorter than CFP-Rab5a association cycles (b, open symbols). Cycles were generally longer on larger macropinosomes. (F) For both Cos-7 cells (circles) and BMM (triangles), CFP-Rab5a activation coincided with association for all sizes of macropinosomes (c, closed symbols), whereas deactivation preceded dissociation by up to 12 min, with larger macropinosomes exhibiting longer lag times (d, open symbols).
Figure 3
Figure 3. Rab5a GEFs increase formation of macropinosome-associated tubules
(A) Cos-7 cell expressing FRET probes and pIRES2-mCherry-Rabex-5. (B) Cos-7 cell expressing FRET probes and pIRES2-mCherry-Rin1. Top row, phase-contrast; middle row, D (CFP-Rab5a); bottom row, EO (EAVG Overlay). Overexpression of both GEFs increased CFP-Rab5a FRET on macropinosomes and remained higher than control. Macropinosomes extended tubules with high FRET signals. Color scale indicates EAVG FRET signals. Scale bar = 10 μm.
Figure 4
Figure 4. Bafilomycin A1 increases tubule formation and destabilizes the Rab5a cycle on macropinosomes
Macropinosomes in control cells (A) showed a single cycle of Rab5a activation and deactivation, whereas bafilomycin A1-treated cells (B) showed erratic patterns of CFP-Rab5a association, activation and deactivation and multiple tubular extensions with high Rab5a activity. Top row, phase-contrast; middle row, D (CFP-Rab5a); bottom row, EO [phase-contrast with overlay showing inactive CFP-Rab5a (red) and active CFP-Rab5a (green)], with the threshold for CFP-Rab5a activation set at EAVG = 8%. (C) Top row, phase-contrast; middle row, TO, phase-contrast with overlay showing active Rab5 on tubular extensions (green) of cell treated with bafilomycin A1; bottom row; EO, EAVG values presented as pseudocolor overlaid on phase-contrast. Scale bars = 10 μm. (D–I) Individual traces from macropinosomes in control (D–F) and bafilomycin A1-treated (G–I) Cos-7 cells showing CFP-Rab5 association (closed circles) and CFP-Rab5 FRET signals (open circles).
Figure 5
Figure 5. Nocodazole destabilizes the Rab5a cycle on macropinosomes
(A) Rab5a activation on a macropinosome of a cell in nocodazole; top, phase-contrast; bottom, EO, phase-contrast with overlay showing inactive CFP-Rab5a (red) and active CFP-Rab5a (green), with the threshold for CFP-Rab5a activation set at EAVG = 10%. Rab5a activity fluctuates on the macropinosome. Scale bar = 5 μm. (B–E) Individual traces from macropinosomes in nocodazole-treated cells.
Figure 6
Figure 6. RabGAP-5 and Rab5a(S34N) destabilize macropinosomes
(A) Cos-7 cells expressing YFP-RBD, CFP-Rab5a(S34N) and mCherry were stimulated by addition of EGF after the first frame was acquired. Small macropinosomes formed rarely. Most macropinosomes showed no association with CFP-Rab5a(S34N), no FRET and did not persist. (B) Cos-7 cells expressing YFP-RBD, CFP-Rab5a and pIRES2-mCherry-RabGAP-5 formed macropinosomes, which showed low CFP-Rab5 association and low FRET signals. The vast majority of the macropinosomes were unstable and fused back with the membrane. Some small macropinosomes that were able to form showed low FRET signals. (C) Macrophages expressing CFP-Rab5a(S34N) (black bars) formed smaller macropinosomes in response to M-CSF than macrophages expressing CFP-Rab5a (white bars). (D) Macrophages expressing CFP-Rab5a(S34N) formed unstable macropinosomes more often than did macrophages expressing CFP-Rab5a.
Figure 7
Figure 7. The Rab5a cycle is modulated by GEFs and GAPs
Cos-7 cells were transfected with YFP-RBD, CFP-Rab5a and pIRES2-mCherry co-expressing GEFs (Rin1 and Rabex-5), GAP (RabGAP-5) or nothing (control). Cells were stimulated and macropinosomes were imaged and analyzed as in Figures 1 and 2. For each condition, the maximum EAVG values were calculated for >9 macropinosomes and plotted as functions of macropinosome diameter. Relative to control cells expressing YFP-RBD, CFP-Rab5a and mCherry only, the GEFs, Rabex-5 and Rin1, increased EAVGMax, whereas the GAP, RabGAP-5, decreased EAVGMax. Treatment of control cells with bafilomycin A1 or nocodazole showed lower EAVGMax levels, but these were not significant.
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References

    1. Rink J, Ghigo E, Kalaidzidis Y, Zerial M. Rab conversion as a mechanism of progression from early to late endosomes. Cell. 2005;122:735–749. - PubMed
    1. Perskvist N, Roberg K, Kulyte A, Stendahl O. Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils. J Cell Sci. 2002;115:1321–1330. - PubMed
    1. Bucci C, Lutcke A, Steele-Mortimer O, Olkkonen VM, Dupree P, Chiariello M, Bruni CB, Simons K, Zerial M. Co-operative regulation of endocytosis by three Rab5 isoforms. FEBS Lett. 1995;366:65–71. - PubMed
    1. Stenmark H, Vitale G, Ullrich O, Zerial M. Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995;83:423–432. - PubMed
    1. Mu FT, Callaghan JM, Steele-Mortimer O, Stenmark H, Parton RG, Campbell PL, McCluskey J, Yeo JP, Tock EP, Toh BH. EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine “fingers” and contains a calmodulin-binding IQ motif. J Biol Chem. 1995;270:13503–13511. - PubMed

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