Volume 21, Issue 23, Pages 1951-1959 (December 2011) The P4-ATPase TAT-5 Inhibits the Budding of Extracellular Vesicles in C. elegans Embryos  Ann M. Wehman, Corey Poggioli, Peter Schweinsberg, Barth D. Grant, Jeremy Nance  Current Biology  Volume 21, Issue 23, Pages 1951-1959 (December 2011) DOI: 10.1016/j.cub.2011.10.040 Copyright © 2011 Elsevier Ltd Terms and Conditions

Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 TAT-5 Is Required for Cell Shape Changes and Gastrulation (A and B) Stills of 28-cell embryos from differential interference contrast (DIC) movies. E indicates endodermal precursors ingressing in control but not tat-5 embryos. (C and D) Endodermal precursors express end-1::GFP::CAAX during gastrulation in 26-cell embryos. P granules (red) localize to the germ cell precursor. (E and F) Stills of late 4-cell embryos from DIC movies. Arrowheads point to the anterior edge of EMS, which extends under ABa in control but not tat-5 embryos. (G and H) Stills of devitellinized late 4-cell embryos from DIC movies. Cells appear more rounded in tat-5 embryos as denoted by contour lines. All images lateral views with anterior to the left and dorsal up. Scale bar represents 15 μm. See also Figure S1, Movie S1, and Movie S2. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Extracellular Vesicles Are Present on Cell Surfaces and between Cell-Cell Contacts in tat-5 Embryos (A and B) PHPLC1∂1::mCherry in 4-cell embryos. Arrowheads point to thick patches of membrane in tat-5 embryos. Scale bar represents 15 μm. (C and D) Transmission electron micrograph (TEM) of the cell contact from 2-cell embryos. Black and white arrowheads point to the plasma membrane of different cells. (E and F) TEM of the surface of 1-cell embryos. Arrows point to extracellular vesicles or tubules. Scale bars in (C)–(F) represent 200 nm. See Figure S2 for additional TEMs. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Extracellular Vesicles Are Generated by Budding from the Plasma Membrane (A) Model based on a TEM tomogram depicting the range in vesicle (arrowhead) and tubulovesicle (arrow) shapes found in a 2-cell tat-5 embryo. Each color represents a distinct structure and only membranes are traced. (A′) Box from (A) depicting a vesicle (shaded) connected to the plasma membrane. See also Movie S3 for vesicle shapes. (B) Histogram of vesicle diameters from tat-5 intralumenal multivesicular body (MVB) vesicles (light gray) averaging 65 ± 15 nm, n = 110 from three embryos, and extracellular vesicles (dark gray) averaging 149 ± 35 nm, n = 89 extracellular vesicles (ECVs) from three embryos. (C and D) GFP::ZF1::SYX-4 (green) in 26-cell embryos. Dotted lines trace cells where ZF1 degradation has occurred. In insets, SAX-7 (red) labels the plasma membrane. Nuclei are blue. (E and F) Surface view of 26-cell embryos expressing med-1::GFP::CAAX. Oval outlines embryos. Arrowheads point to membrane thickening. See Figure S3 for exosome marker data. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 TAT-5 Localizes to the Plasma Membrane and Maintains Phosphatidylethanolamine Asymmetry (A and B) GFP::TAT-5 (A) and GFP::TAT-5(E246Q) (B) expression in 2-cell embryos. (C and D) GFP::TAT-5 (C) rescues (n = 164 embryos) and GFP::TAT-5(E246Q) (D) fails to rescue (n = 28 embryos) membrane thickenings (arrowhead) in 15-cell embryos from tat-5(tm1741) mothers. (E–G) Annexin V staining in 8- to 16-cell devitellinized embryos. (H) Graph of normalized Annexin V membrane labeling intensity (control: n = 20 embryos, tat-1: n = 14, tat-5: n = 14). (I–K) Duramycin staining in 44- to 46-cell devitellinized embryos. Arrowhead marks a cell contact. (L) Graph of normalized duramycin membrane labeling intensity (control: n = 15 embryos, tat-1: n = 12, tat-5: n = 7). Data are represented as mean ± standard deviation (SD). Asterisks mark a significant difference compared to control: ∗p < 0.05, ∗∗p < 0.01. See Figure S4 for GFP::TAT-5(D439E) localization and overall phospholipid content. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 5 Clathrin, RAB-11, and the ESCRT Complex Are Recruited to the Plasma Membrane in tat-5 Embryos (A and B) GFP::CHC-1 (clathrin) in 4-cell embryos. Arrowhead indicates cell contact. (C and D) GFP::RAB-11 in 4-cell embryos. Bright spot is midbody. (E and F) GFP::MVB-12 (ESCRT-I) in 4-cell embryos from vps-4 feeding RNA interference (fRNAi) and tat-5 + vps-4 double-stranded RNA (dsRNA) injections. (G and H) Projections of 2-cell embryos stained for ESCRT-III protein VPS-32 (green) from vps-4 fRNAi or tat-5 + vps-4 dsRNA injections (vps-4: 0.27 ± 0.11 puncta/μm, n = 7, 136 μm examined; tat-5 + vps-4: 0.62 ± 0.16 puncta/μm, n = 4, 97 μm examined; p < 0.01). PAR-3 (red) marks cell cortex. See Figure S5 for more endosomal markers. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 6 RAB-11 and the ESCRT Complex Mediate Extracellular Vesicle Production (A and B) Graphs displaying percentage of embryos ± SD with wild-type membranes, or mild or severe thickenings. Thickenings were judged mild if they only affected a subset of 3-cell junctions or severe if they affected 2- and 3-cell junctions. Asterisks mark a significant difference compared to tat-5: ∗p < 0.05, ∗∗p < 0.01. (C–H) Embryos expressing PHPLC1∂1::GFP from adults injected with the indicated dsRNAs. See Figure S6 for controls and Table S1 for n values. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 7 Two Models for the Role of TAT-5 and PE Externalization in Ectosome Production (A and B) TAT-5 normally internalizes phosphatidylethanolamine (PE). PE externalization by an unknown scramblase could lead to the recruitment or stabilization of the ESCRT complex to the plasma membrane, where the ESCRT complex buds out ectosomes. RAB-11 could deliver proteins needed for budding to the plasma membrane or could have a more direct role in the budding process. (A) Externalized PE results in microdomains of increased negative charge at the cytoplasmic face (from relatively increased phosphatidylserine and phosphatidylinositols density). (B) Externalized PE induces domains of negative membrane curvature that could act as hinge regions. These models are not exclusive. Current Biology 2011 21, 1951-1959DOI: (10.1016/j.cub.2011.10.040) Copyright © 2011 Elsevier Ltd Terms and Conditions