Taunton J , Rowning BA , Coughlin ML , Wu M , Moon RT , Mitchison TJ , Larabell CA .

GM35126 NIGMS NIH HHS , R01 GM035126 NIGMS NIH HHS

	Figure 1. Actin comet tail formation in an activated Xenopus egg. (A) Vegetal pole of an egg, 2 min after activating the animal hemisphere by pricking with a glass micropipet. This region of the egg, viewed with a scanning laser confocal microscope, has not yet undergone activation-induced modifications. A diffuse distribution of rhodamine-actin and XPKCα-GFP is seen amidst the secretory cortical granules (1–3-μm-diam circular shadows) just beneath the plasma membrane. (B) Same region of the egg, 4 min after activation. The cortical secretory granules have undergone exocytosis and larger vesicles enriched in XPKCα-GFP (3–5-μm-diam) are now visible. As the egg reaches maximal contraction, large spherical accumulations of rhodamine-actin form amidst the field of vesicles. (C) A similar region of the egg after contraction (8 min after activation) showing numerous rhodamine-actin comet tails in a field of XPKCα-GFP vesicles of varying sizes. This is a tangential optical section, with the far right edge of the image showing microvillar tips on the egg surface and deeper regions of the egg cortex revealed on the left. (D) Time-lapse sequence (3.5-s intervals) showing the birth of rhodamine-actin comet tails. Actin assembly appears to be initiated by a PKC-enriched vesicle and ultimately effects a 10-μm displacement of that vesicle. See Online Supplemental Material, Video 1 (http://www.jcb. org/cgi/content/full/148/3/519/ DC1). Bars: (A–C) 10 μm; (D) 5 μm.
	Figure 2. Comet tail formation in vivo induced by fertilization and PMA treatment. (A) Gallery of confocal images showing actin comet tails in eggs fixed 8 min after fertilization. F-actin was visualized by fluorescein-phalloidin staining. (B) In vivo time-lapse sequence (16-s intervals) showing rhodamine-actin comet tails (indicated by arrowheads) 20 min after treatment with 2 μM PMA. See Online Supplemental Material, Video 2 (http://www.jcb.org/cgi/content/full/148/3/519/DC1). Bars, 10 μm.
	Figure 3. Reconstitution of PMA-stimulated actin assembly in vitro. (A) Time-lapse sequences showing actin comet tails in Xenopus egg extracts. Cytosol was supplemented with a crude membrane fraction, rhodamine-actin, and 1 μM PMA. After incubating at room temperature for 20 min, samples were viewed by fluorescence (left) or phase contrast (right; note higher magnification) microscopy. Arrowheads (left) indicate a moving comet tail. Arrow (bottom right) indicates a phase dense particle at the tip of the comet tail. (B) In vitro actin assembly reactions as in (A) performed in the presence or absence of the PKC inhibitor, BIM-I (40 μM). Fluorescence images from a control reaction without PMA are also presented. Low magnification (20× objective) images were acquired from ten random fields, two of which are depicted for each condition. (C) Quantitation of data presented in B. The total number of pixels with fluorescence intensities above the background were summed over 10 random fields. Data (mean ± SD) from three experiments are presented. Bars: (A) 10 μm; (B) 20 μm.
	Figure 4. PMA stimulates the recruitment of N-WASP to vesicles associated with actin comet tails. Cell-free vesicle motility assays containing Xenopus cytosol, membranes, and 1 μM PMA were fixed in perfusion chambers and immunolabeled with affinity-purified anti–N-WASP antibodies followed by Texas red secondary antibodies and FITC-phalloidin. N-WASP and F-actin recruitment occurred in the presence of 75 μM latrunculin A, but comet tails were not observed. ToxB treatment prevented both N-WASP and F-actin recruitment, although nonspecific labeling of fixed membranes could be observed with long exposure times. Bar, 5 μm.
	Figure 6. Thin-section electron microscopy analysis of vesicles associated with comet tails. Cell-free reactions were fixed in perfusion chambers and processed for electron microscopy (see Materials and Methods). A gallery of representative images was assembled to highlight the odd shapes, tubular processes, and multivesicular lumens of vesicles associated with tails. Note the clear circular profiles and mitochondria, none of which are associated with comet tails. Bars, 500 nm.
	Figure 5. Accumulation of acridine orange, a marker for endosomes and lysosomes, in motile vesicles. Cell-free reactions (without rhodamine-actin) were monitored by phase contrast and fluorescence microscopy in the presence of 20 μg/ml acridine orange. A gallery of merged images (phase contrast and Texas red filter set) from three experiments is presented. 80% of the vesicles associated with comet tails strongly accumulated acridine orange (n = 417 comet tails). Bar, 10 μm.
	Figure 7. Actin-dependent propulsion of exogenous endosomes in Xenopus egg cytosol. HeLa cells were treated with Texas red transferrin (40 μg/ml) for 20 min at 37°C, and a postnuclear supernatant (PNS) was prepared. The PNS was diluted 60-fold with Xenopus egg cytosol containing 0.5 μM Alexa(488)-actin and 1 μM PMA. Alexa(488) and Texas red epifluorescence images were acquired every 10 s. Approximately half of the vesicles associated with actin comet tails contained internalized Texas red transferrin. Bar, 5 μm.
	Figure 8. Endosomes and lysosomes preferentially nucleate actin assembly and move in vitro. HeLa postnuclear supernatants were prepared identically from unlabeled cells (A and D) and cells that had been treated with Texas red transferrin at 37°C (B) or 4°C (C). Cell-free motility reactions were fixed and processed for epifluorescence detection of organelle markers (red). F-actin (green, A–D) was detected with FITC-phalloidin. (A) ER membranes were immunodetected with affinity-purified anti-Sec61β antibodies followed by Texas red secondary antibodies. (B) Early and recycling endosomes were detected by labeling cells with Texas red transferrin for 20 min at 37°C. (C) Plasma membranes were detected by labeling cells with Texas red transferrin for 20 min at 4°C. (D) Lysosomes were immunodetected with anti–Lamp-1 H4A3 mAb followed by Texas red secondary antibodies. Bar, 5 μm. (E) Quantitation of comet tails associated with a labeled vesicle in the experiments described in A–D. The n value refers to the total number of actin comet tails counted under each condition from 8–10 random fields.
	Figure 9. Gallery of electron micrographs depicting N-WASP immunogold-labeled HeLa vesicles associated with actin comet tails. Cell-free motility reactions containing unlabeled HeLa membranes were fixed in perfusion chambers, immunolabeled with affinity-purified N-WASP antibodies followed by 15 nm protein A–gold, and processed for electron microscopy. Gold particles are occasionally visible in the comet tails, but the vast majority are associated with the membrane. Note the similarity in vesicle morphology to the Xenopus egg vesicles shown in Fig. 6. Bar, 200 nm.

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