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Figure 1. ER network formation in crude interphase X. laevis egg extracts. Demembranated sperm was added to a crude interphase X. laevis egg extract containing Alexa fluor 488âlabeled tubulin and the hydrophobic dye DiIC18. The formation of MT asters (A) and ER network (B) was followed over time by confocal fluorescence microscopy (for full time course, see Videos 1 and 2). Sperm was added at time zero. Bars, 30 µm. (C) As in A and B, but after 60 min, a time point at which the NE has formed. Bars, 20 µm.
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Figure 2. Dynein is required for movement of ER membranes toward the minus end of MTs. (A) Demembranated sperm was added after 10 min to a crude interphase X. laevis egg extract containing Alexa fluor 488âlabeled tubulin and the hydrophobic dye DiIC18. The extension of membrane tubules was followed over time. Arrowheads indicate the tip of extending ER membrane tubule. The kymograph shows the movement of the same tubule. Bars, 2 µm. (B) As in A, but the extract was incubated for 30 min with and without 6 µM of the CC1 of p150 glued to inhibit dynein function. Network formation was visualized by confocal fluorescence microscopy. Bars, 10 µm.
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Figure 3. The leading ends of ER tubules track with the plus ends of MTs. (A) Demembranated sperm was added for 30 min to a crude interphase X. laevis egg extract containing the hydrophobic dye DiIC18 and a GFP fusion of the plus end tracking protein EB-1. The movements of ER tubules and of the plus ends of MTs were followed over time by confocal fluorescence microscopy (for full time course, see Video 4). Yellow arrows indicate the leading ends of ER tubules (red) that track with the plus ends of growing microtubules (green). The blue arrow indicates the site of attachment between one of the extending ER tubules and another tubule to form a three-way junction. Bars, 2 µm. (B) Quantification of experiments such as shown in A. The percentage of outward moving ER tubules that tracked with EB-1 is shown. Error bars indicate the range within three independent experiments, with 50 tubule-extension events analyzed in each experiment.
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Figure 4. The ER network undergoes tubule-to-sheet conversion during the transition from interphase to meiosis and mitosis. (A) A crude CSF extract was driven into interphase by addition of Ca2+ ions. The ER network was stained with the hydrophobic dye DiIC18 and visualized by confocal fluorescence microscopy. Bar, 10 µm. (B) As in A, but the meiotic (CSF) state of the extract was maintained by omitting Ca2+. Bar, 20 µm. (C and D) As in B, but with addition of demembranated sperm. C shows the MTs stained with Alexa fluor 488âlabeled tubulin, and D shows the membranes stained with DiIC18. Bars, 10 µm. (E) Interphase cytosol, light membranes, and an energy regenerating system were mixed with cyclin BÎ90 to generate a mitotic state. A control was performed with buffer. The membranes were stained with octadecyl rhodamine and visualized by confocal microscopy. The bottom panels show magnified views of the three-way junctions highlighted in the boxed area. Bars: (top) 20 µm; (bottom) 10 µm. (F) The number of three-way junctions in E was counted and expressed as a percentage of the control. The data plotted are the mean ± SD of three independent experiments. ***, P < 0.001, Studentâs t test.
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Figure 5. ATL is required for ER network formation in crude interphase and mitotic extracts. (A and B) Demembranated sperm was added to a crude interphase X. laevis egg extract containing Alexa fluor 488âlabeled tubulin and the hydrophobic dye DiIC18. The extract also contained 2 µM of the cytoplasmic fragment of X. laevis ATL2 (cytATL) to inhibit ATL-mediated membrane fusion. Labeled MT asters (A) and ER membranes (B) were visualized by confocal fluorescence microscopy. Bars, 20 µm. (C and D) As in A and B, but with 2 µM of a point mutant of cytATL (cytATL(R232Q)) that does not inhibit fusion. Bars, 20 µm. (E and F) Inhibition of ATL-mediated fusion in mitotic extracts. 2 µM cytATL(R232Q) (E) or cytATL (F) were added to CSF extracts and the membranes were stained with DiIC18. Bars, 10 µm.
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Figure 6. ATL is required for ER network formation in a fractionated system. (A) Interphase cytosol, light membranes, and an energy regenerating system were mixed and incubated for 30 min with buffer, 2 µM of the cytoplasmic fragment of X. laevis ATL2 (cytATL), or 2 µM of mutant fragment (cytATL(R232Q)). The membranes were stained with octadecyl rhodamine and visualized by confocal microscopy. (B) As in A, but the mitotic state was generated by addition of cyclin BÎ90. (C) Interphase ER network formation with DiOC18 prelabeled light membranes was performed at different GTP concentrations for 15 min at room temperature. Bars, 20 µm.
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Figure 7. ATL mediates the fusion of membranes into an ER network. (A) A crude interphase extract was preincubated for 5 min with 2 µM of an inactive mutant of the cytosolic fragment of ATL (ATL(R232Q)). Then, membranes were added that were separately prestained with DiIC18 (red) and DiOC18 (green). The sample was imaged after 30-min incubation by confocal microscopy with a short exposure time (40 ms). (B) As in A, but with wild-type cytosolic fragment of ATL (cytATL). Insets in A and B show a magnified view of the boxed areas. (C) As in A and B, but with membrane-depleted interphase cytosol and a mixture of light membranes that were prestained with either DiIC18 or DiOC18. Insets show a magnified view of the boxed areas. Bars: (A and B) 20 µm; (A and B, insets) 10 µm; (C) 10 µm; (C, insets) 5 µm.
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Figure 8. ATL is required for NE fusion. (A) Interphase cytosol and light membranes were mixed with buffer, 2 µM of the cytoplasmic fragment of X. laevis ATL2 (cytATL), or 2 µM of mutant fragment (cytATL(R232Q)). Demembranated sperm and an energy regenerating system were added. The samples were incubated at room temperature for 1.5 h and mixed with fluorescently labeled 70-kD dextran for 5 min on ice. After fixation with a solution containing Hoechst to stain chromatin and DHCC to stain membranes, the samples were analyzed by confocal microscopy. Bar, 20 µm. The number of closed nuclei (CNE) was counted based on membrane continuity and exclusion of dextran. Fusion is expressed as the percentage of the control. At least 100 nuclei were counted for each sample. The data plotted are the mean ± SD of three independent experiments. ***, P < 0.001; NS (not significant), P > 0.05; Studentâs t test. (B) Interphase cytosol, light membranes, sperm, and an energy regenerating system were mixed. After 10, 30, or 50 min of incubation at room temperature, buffer or 2 µM cytATL was added. Samples were fixed after 1.5 h and analyzed by confocal microscopy. Bar, 20 µm. NS, not significant (P > 0.05, Studentâs t test).
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Figure 9. ER SNARE function is required for NE fusion. NE assembly was examined by mixing interphase cytosol, light membranes, sperm, and an energy regenerating system with buffer, 50 µM dominant-negative αSNAP mutant (αSNAP(L294A)), 20 µM of the cytoplasmic fragment of Bnip1 (cytBnip1), 30 µM of the cytoplasmic fragment of syntaxin 18 (cytStx18), or 70 µM of the cytoplasmic fragment of Use1 (cytUse1). Fluorescently labeled dextran was added to detect nuclei with closed NE. The samples were also stained for membranes and DNA. Bar, 20 µm. The percentage of NE fusion was determined as described in Fig. 8 A and the data plotted are the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.005; and ***, P < 0.001; Studentâs t test.
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