Whalen K et al. (2012), Actin polymerization controls the activation of...

ECB-ART-42520

Mol Biol Cell 2012 Sep 01;2318:3663-72. doi: 10.1091/mbc.E12-06-0438.

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Actin polymerization controls the activation of multidrug efflux at fertilization by translocation and fine-scale positioning of ABCB1 on microvilli.

Whalen K , Reitzel AM , Hamdoun A .

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Fertilization changes the structure and function of the cell surface. In sea urchins, these changes include polymerization of cortical actin and a coincident, switch-like increase in the activity of the multidrug efflux transporter ABCB1a. However, it is not clear how cortical reorganization leads to changes in membrane transport physiology. In this study, we used three-dimensional superresolution fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing actin filaments, and we show that efflux activity is established after ABCB1a translocates to the tips of the microvilli. Inhibition of actin polymerization or bundle formation prevents tip localization, resulting in the patching of ABCB1a at the cell surface and decreased efflux activity. In contrast, enhanced actin polymerization promotes tip localization. Finally, interference with Rab11, a regulator of apical recycling, inhibits activation of efflux activity in embryos. Together our results show that actin-mediated, short-range traffic and positioning of transporters at the cell surface regulates multidrug efflux activity and highlight the multifaceted roles of microvilli in the spatial distribution of membrane proteins.

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Species referenced: Echinodermata
Genes referenced: abcb1 LOC100887844 LOC115919910 LOC577676 LOC586591 LOC590297 LOC590502 rab11
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	FIGURE 1:. S. purpuratus (Sp) ABCB1a is a homologue of human P-gp and is present in its mature form in the unfertilized egg prior to activation at fertilization. (A) Phylogenetic relationships of Sp ABCB family transporters with human (Hs), Ciona intestinalis (Ci), Strongylocentrotus franciscanus (Sf), Allocentrotus fragilis (Af), Patiria miniata (Pm), Drosophila melanogaster (Dm), Nematostella vectensis (Nv), Daphnia pulex (Dp), Amphimedon queenslandica (Aq), and Saccharomyces cerevisiae (Sc). (B) Immunoblot of ABCB1a in sea urchin egg and embryo cell lysates probed with Ab1-C1 shows that ABCB1a does not increase in abundance or change in glycoform profile at fertilization.
	FIGURE 2:. Characterization of actin filament elongation and ABCB1a translocation after fertilization, using confocal microscopy. (A) F-actin (green) and ABCB1a (red) in eggs and embryos. Left, MIP; right, cross-sections. Scale bars: 5 and 3 μm, respectively. (B) Bars represent mean length (± SD) of F-actin filaments (n = 3 eggs or embryos × 10 filaments per time) extending into the cortex and of microvillar projections. Zero (y-axis) denotes the base of the microvilli.
	FIGURE 3:. ABCB1a translocates to the microvillar tips and has a bimodal distribution at 60 min PF, as visualized by 3D-SIM. Cross-sections of an egg (A) and embryos (B–D) show the localization of ABCB1a in relation to actin filaments. Scale bar: 3 μm. ABCB1a distribution shown as mean percent of total (± SE; black line) and cumulative percent of total (± SE; red line) along an actin filament, as represented by an intensity profile in the egg (E) and embryos (F–H). (I) A summary of the change in ABCB1a distribution over time shown as mean percent of total for egg at 15, 45, and 60 min PF. Zero (y-axis) indicates the base of the microvilli.
	FIGURE 4:. Three-dimensional analysis of ABCB1a dispersal relative to F-actin filaments indicates efflux activation is mediated by short-range, actin-dependent translocation. F-actin–associated ABCB1a advances tip-ward after fertilization (A–D). Bars indicate percent of ABCB1a spots within 200 nm of actin surface, and zero (y-axis) indicates the tip of the microvillus (n = 6 eggs or embryos × 10 filaments per time). Red line indicates the average microvillar length at each time. Insets, representative actin filament isosurfaces and associated ABCB1a spots (red). Scale bar: 1 μm.
	FIGURE 5:. Perturbation of actin polymerization and F-actin bundle formation disrupts ABCB1a translocation and causes partial loss of transporter-mediated calcein efflux. Embryos treated with 2 μM jasplakinolide, cytochalasin D, latrunculin A, and DMSO (control) at 10 min PF and imaged at 60 min PF. (A) Cytochalasin D and latrunculin A eliminate ABCB1a accumulation in the cortex, while jasplakinolide increases accumulation of ABCB1a on microvilli. Scale bar: 2 μm. (B) Embryos (MIPs and cross-sections) activated in A23187 and grown in NaFFSW until 60 min PF were stained for actin and ABCB1a. Scale bar: 8 μm. (C) Average (± SE) intracellular calcein fluorescence (120 and 180 min PF) activated with A23187 and cultured in either NaFFSW or NaFFSW+NaCl (n = 10 embryos × 10 batches). A significant increase in calcein accumulation was observed in A23187-activated embryos cultured in NaFFSW (1.8-fold) at both 120 and 180 min PF. Letters denote significance (analysis of variance [ANOVA], p < 0.001).
	FIGURE 6:. Rab11 is required for activation of MDR activity. (A) Fold change in calcein fluorescence in Rab effector peptide–injected embryos relative to a two-cell control embryo. The average calcein fluorescence of 20 embryos (± SEM) is shown. Asterisks indicate significant differences from a two-cell control embryo (ANOVA, , p = 0.0016, *p < 0.0001). (B) Representative egg and two-cell embryos (150 min PF) showing differences in calcein accumulation after injection with 200 μM Rab11 effector peptide.
	FIGURE 7:. Rab11 colocalizes with ABCB1a in unfertilized eggs. MIP (four left panels) and cross-sections (right-most panels) of an egg and embryos depict the localization of Rab11 in relation to ABCB1a and actin. Scale bars: 5 and 2 μm, respectively.

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