Data supplements
Index of Online Supplemental Material for
J. Cell Biol. 10.1083/jcb.200601156
Frescas et al.•Video 13D rendering of ER membranes in a Lys-GFP-KDEL-expressing embryo.
• Video 2Nuclear migration during nuclear cycle 10 interphase in a Lys-GFP-KDEL-expressing embryo as viewed from the embryo surface.
• Video 3Divisions of ER membranes during nuclear cycles 10-13 in a Lys-GFP-KDEL-expressing embryo as viewed from the embryo surface.
• Video 4Dynamics of Golgi puncta during nuclear cycles 11-13 at a cross section of a GalT-GFP-expressing embryo.
• Video 5Movement of Golgi structures around individual syncytial interphase nuclei in a GalT-GFP-expressing embryo as viewed from the embryo surface.
• Video 6Movement of Golgi structures during nuclear cycles 12-13 in a GalT-GFP-expressing embryo as viewed at a cross section of the embryo.
Article Figures & Data
Figures
- Figure 1.
ER organization before nuclear migration. (A) The distribution of ER in a Lys-GFP-KDEL–expressing preblastoderm embryo. The ER was restricted to the periphery of the embryo during all nuclear divisions before cellularization (see Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1). (B) Confocal sections of a Lys-GFP-KDEL–expressing preblastoderm embryo at z = 0, 2, and 6 μm beneath the plasma membrane. ER (green) forms an interconnected network of membrane sheets and accumulates in clusters directly beneath the plasma membrane (z = 0 and 2 μm) and forms a loose network of membrane tubules deeper into the periplasm (z = 6 μm), with yolk granules (red) filling the space between the tubules. (C) Confocal section of a GFP-tubulin–expressing embryo showing that tubulin is concentrated within 20 μm of the plasma membrane. (D) Confocal section of a GFP-tubulin–expressing embryo 6 μm beneath the plasma membrane. Filamentous, tubulin-rich structures surrounding spherical particles that excluded fluorescence can be seen (arrows). (E) ER membranes beneath the plasma membrane organize in tight clusters that are interconnected with membrane tubules (left). After nocodazole microinjection, ER clusters disappear and are replaced by long strands of ER (right).
- Figure 2.
ER membranes form an interconnected membrane network before nuclear migration. (A) Lys-GFP-KDEL molecules freely diffuse within the ER lumen of the preblastoderm as determined by FRAP. A small ROI (outlined circle) was photobleached, and fluorescence recovery was monitored. Lys-GFP-KDEL fluorescence significantly recovered within 38 s of photobleaching. (B and C) FLIP of Lys-GFP-KDEL in the preblastoderm reveals the continuity of ER membranes. Repetitive photobleaching of a small ROI (red box) was performed. Fluorescence was depleted exponentially in distant regions (blue and green boxes) surrounding the bleached ROI, indicating that the ER membranes were interconnected.
- Figure 3.
Reorganization of ER membranes after nuclear migration is mediated by centrosomally derived microtubules. (A) Time-lapse of nuclear migration in Lys-GFP-KDEL embryos. Once nuclei (N) reach the periphery, individual nuclei reorganize the maternal ER around themselves. The tight ER clusters (arrows) are sequestered to form individual ER units surrounding a given nuclei, and, by the end of migration, they have completely unwound (see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1). (B) Time-lapse images of nuclear migration in Lys-GFP-KDEL embryos injected with rhodamine-tubulin. Note that the concentration of ER (green) around nuclei correlates with the distribution of astral microtubules (red). (C and D) Confocal sections of a Lys-GFP-KDEL–expressing syncytial blastoderm embryo showing the tight association of ER with individual nuclei when viewed from the embryo surface (C, top) or at a cross section (D, top). This tight association is lost upon nocodazole microinjection (C and D, bottom). (E) A schematic diagram showing the movement of a nucleus to the surface of the embryo. Microtubules (red) help direct nuclei from the embryo interior toward the periphery during the interphase of cycle 10, at the same time displacing yolk granules (gray) into the interior. Interactions between centrosome-nucleated microtubules and the ER (blue) then lead to the recruitment of ER membranes around individual nuclei. Preexisting tight ER clusters unwind as their membranes are organized around each nucleus.
- Figure 4.
Upon nuclear migration, the ER becomes compartmentalized around individual nuclei by a process that is dependent on astral microtubules. (A) Top view of the ER during nuclear division 11. ER membranes surrounding individual nuclei (N) appeared as distinct units at the periphery during each interphase and through each nuclear division (see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1). (B and C) Lys-GFP-KDEL molecules freely diffuse within the ER lumen of the syncytial blastoderm as determined by FRAP. A small ROI (red circle) was photobleached, and fluorescence recovery was monitored. Lys-GFP-KDEL fluorescence significantly recovered within 20 s of photobleaching. Images shown in B were inverted. (D and E) Top view FLIP of Lys-GFP-KDEL in the syncytial blastoderm shows compartmentalization of ER membranes. Repetitive photobleaching of a small ROI (red box) adjacent to one nucleus was performed. Fluorescence was depleted exponentially in the region directly surrounding the bleached ROI (blue box), whereas the fluorescence of neighboring ER–nuclei systems (orange, magenta, blue, green, and yellow outlines) was minimally affected, indicating a loss in ER membrane continuity. (F and G) Top view FLIP of Lys-GFP-KDEL in the syncytial blastoderm after nocodazole microinjection results in the loss of ER compartmentalization. Embryos were injected with nocodazole as described in Materials and methods. Repetitive photobleaching of a small ROI (red box) was performed. Fluorescence was depleted exponentially in distant regions (blue and green boxes) surrounding the bleached ROI, indicating long-range diffusion of the Lys-GFP-KDEL molecules.
- Figure 5.
Compartmentalization of ER around nuclei is not dependent on plasma membrane invaginations. (A) Plasma membrane (PM) invaginations in the syncytial blastoderm during interphase were visualized with Spider-GFP (left; see Materials and methods). ER membranes visualized during the same nuclear cycle with Lys-GFP-KDEL extend much deeper than the plasma membrane (right). (B and C) Side view FLIP of Lys-GFP-KDEL over a row of nuclei (N). Repetitive photobleaching of a small ROI (red box) was performed. Fluorescence was depleted exponentially in the region directly below the bleached ROI (blue box), whereas neighboring regions (green and black boxes) were minimally affected. (D and E) Side view FLIP of Lys-GFP-KDEL below a row of nuclei. Repetitive photobleaching of a small ROI (red box) was performed. Fluorescence was depleted exponentially in the region directly above the bleached ROI (blue box), whereas neighboring regions (green and black boxes) were minimally affected. (F and G) Side view FLIP of Lys-GFP-KDEL in a preblastoderm embryo. Repetitive photobleaching of a ROI (red box) was performed. Fluorescence was depleted exponentially in the region directly adjacent to the bleached ROI (blue box), indicating long-range diffusion across the embryo periphery. Images shown in B, D, and F were inverted.
- Figure 6.
Golgi distribution and dynamics. (A) Golgi membranes labeled with GalT-GFP appeared as several thousand structures located at the periphery of the embryo (see Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200601156/DC1). The images shown were inverted. (B and C) The dynamics of Golgi puncta were observed for 20 s during a syncytial interphase. Golgi puncta merged (B, follow arrow in sequence) and separated (C, follow arrow in sequence) in the immediate area around each nucleus (N; see Videos 5 and 6). The images shown were inverted.
- Figure 7.
Golgi enzymes recycle through an interconnected ER system before nuclear migration. Recycling is restricted to individual ER–nuclei units after nuclear migration. (A and B) Top view FLIP of GalT-GFP in the preblastoderm shows recycling of Golgi enzymes through an interconnected ER membrane network. Repetitive photobleaching of a small ROI (red box) was performed. Fluorescence was depleted exponentially in distant regions (blue box) surrounding the bleached ROI, indicating long-range recycling of the enzymes through ER membranes. (C and D) Top view FLIP of GalT-GFP in the syncytial blastoderm shows compartmentalization of Golgi units. Repetitive photobleaching of a small ROI (red box) adjacent to one nucleus was performed. Fluorescence was depleted exponentially in the region directly surrounding the bleached ROI (green box), whereas the fluorescence of neighboring Golgi–nuclei systems (blue box) was minimally affected, indicating that the recycling of Golgi enzymes is now restricted to individual ER–nuclei units. (E and F) Side view FLIP of GalT-GFP below a row of nuclei (N). Repetitive photobleaching in a small ROI (red box) was performed. Fluorescence was depleted exponentially in the region directly above the bleached ROI (green box), whereas neighboring regions (blue box) were minimally affected. The images shown in E were inverted.
- Figure 8.
Evidence that plasma membrane–bound material originates from localized secretory units around individual nuclei. (A) Injection of BFA (arrowhead) in embryos expressing a plasma membrane marker (Spider-GFP; see Materials and methods) resulted in a dramatic slowdown of membrane invagination at the injection site, suggesting that BFA-induced impairment of secretory units at the injection site is not compensated for by adjacent secretory units. (B) Side view FRAP of a Spider-GFP–expressing embryo during early cellularization. ROIs encompassing plasma membrane–bound Spider-GFP (blue box) or both plasma membrane–bound and intracellular Spider-GFP pools (red box) were simultaneously photobleached, and fluorescence recovery was monitored. Spider-GFP fluorescence on the plasma membrane recovered very little in the case where the intracellular Spider-GFP pool was depleted (red box), indicating that secretion is taking place in a localized manner and not randomly through an extensive secretory system. The images shown were inverted.
- Figure 9.
Schematic model for restricted ER/Golgi secretory units in the Drosophila syncytial blastoderm. ER and Golgi associate with individual nuclei, and this association is dependent on an intact microtubule network emanating from the centrosomes of each nucleus. Despite the absence of plasma membrane boundaries between nuclei, proteins within the ER lumen and Golgi membranes do not significantly exchange with molecules localized in the ER lumen and Golgi membranes surrounding adjacent nuclei. Instead, their movement is restricted to individual ER/Golgi units associated with individual nuclei. Secretory carriers moving from these ER/Golgi units to the plasma membrane likewise undergo restricted movement and insert into plasma membrane areas that are directly adjacent to the ER/Golgi system from which they were generated.
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