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First published online 31 October 2007
doi: 10.1242/dev.010934

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Nucleocytoplasmic shuttling mediates the dynamic maintenance of nuclear Dorsal levels during Drosophila embryogenesis

¹ Department of Molecular Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
² Department of Molecular Biology and Biochemistry, Rutgers University, Waksman Institute, 190 Frelinghuysen Rd, Piscataway, NJ 08854-8020, USA.
³ Cell Biology and Metabolism Branch, NICHD, NIH, Bldg. 1, 8T, 18 Library Drive, Bethesda, MD 20892, USA.

View larger version (64K): [in this window] [in a new window]   Fig. 1. Breakdown during mitosis and reformation of the Dorsal gradient during interphase. (A) Cross-sectional views of a live Drosophila embryo, showing the distribution of Dorsal-GFP from nuclear cycles 11 to 14; orientation is ventral down, dorsal up. (B) Saggital views, showing interphase nuclear cycle 13, mitosis (prophase) and interphase nuclear cycle 14; the orientation in all panels is ventral down, dorsal up. (C) A quantification of the relative nuclear fluorescence of one nucleus at a fixed position as a function of time from nuclear cycles 12 to 14. Nuclear fluorescence intensity was calculated as spot intensity within nuclei minus the spot intensity of the adjacent cytoplasm plotted against time in seconds. Relative periods of interphase and mitosis (shown by hatched bars) were determined by visualizing mitotic spindles that are readily apparent on the dorsal side via their transient interaction with Dorsal-GFP.

View larger version (133K): [in this window] [in a new window]   Fig. 2. Local redistribution of Dorsal as nuclei proceed through mitosis in Drosophila. (A) A lateral view of the transition between relative nuclear inclusion and exclusion (for orientation, see arrow in fourth panel of Fig. 1A). On the dorsolateral side, Dorsal transiently enters nuclei that previously excluded it at the start of mitosis (early prophase) and shortly afterward redistributes in a diffuse relatively uniform pattern (telophase, bottom panel). (B) A side view of the ventral surface showing a transient particulate distribution at the plasma membrane surface at the end of mitosis (white arrow). (C) A surface view (image inverted), showing the distribution of Dorsal-GFP in the particulate compartment on the ventral side (VENTRAL). Dorsal-GFP is never observed in a particulate compartment on dorsal plasma membranes, where nuclei do not take up Dorsal (DORSAL). (D) Dorsal is present within late ventral nuclei diffusely in the nucleoplasm and enriched in the chromosomal compartment.

View larger version (44K): [in this window] [in a new window]   Fig. 3. In Drosophila Dorsal is highly mobile and shuttles from nucleus to cytoplasm in both ventral and dorsal nuclei. (A) A fluorescence recovery after photobleaching (FRAP) reveals that Dorsal-GFP is highly mobile within the nucleus. A bleached area (red circle) before and after a 3 second photobleach, showing extensive and uniform loss of nuclear fluorescence. This indicates high mobility of Dorsal-GFP within nuclei. However, the extensive fluorescence recovery by 154 seconds postbleach shows that nuclear Dorsal-GFP must exchange with the cytoplasmic pool and thus undergoes nucleocytoplasmic shuttling. (B) Dorsal is present in extreme dorsal nuclei and shuttles from nucleus to cytoplasm. An identical FRAP conducted on a nucleus located on the extreme dorsal side, showing both initial loss and subsequent recovery of nuclear fluorescence over time. (C) Quantification of the data from the ventral nuclear FRAP. To compesate for any change in nuclear levels over time, internal nuclear fluorescence intensity was normalized to that of a ventral nucleus at the same relative DV position. (D) Quantification of the data from the dorsal nuclear FRAP. As in C, internal nuclear fluorescence intensity normalized to a nearby (dorsal) nucleus at the same relative DV position. Bleach boxes in both experiments are shown in red.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Dorsal is exported from nuclei by a CRM1-mediated process and has a carboxyterminal LRNES. (A) A saggital confocal section of a wild-type Drosophila embryo, showing the normal polarized nuclear Dorsal distribution. (B) A saggital confocal section of an embryo treated with LMB during nuclear cycle 14, showing Dorsal protein within dorsal nuclei, which normally do not accumulate Dorsal. (C) A dorsal view of embryo at the beginning of gastrulation. (D) A dorsal view of an LMB-treated embryo, showing perdurance of high levels of nuclear Dorsal during gastrulation. (E) Anti-Twist staining of an LMB-treated embryo, showing expansion of mesodermal cell fates. (F) Putative LRNESs within the carboxyterminus of Dorsal. (G) Schematic of tandem GFP constructs used in an in vivo assay for nuclear export. (H) Localization of Tandem GFP driven by nanos-GAL4. (I) Localization of tandem GFP with Dorsal's carboxyterminal 44 amino acids driven by nanos-GAL4. (J) Alignment of Putative LRNES sequences at the carboxyterminus with other Drosophila NF-B proteins and several mammalian LRNESs.

View larger version (84K): [in this window] [in a new window]   Fig. 5. Dorsal partitions into nucleus-associated cytoplasmic domains in Drosophila. (A) Photobleaching of the cytoplasm preferentially reduces nuclear Dorsal levels in one nearby nucleus. FLIP of the common cytoplasm results in the preferential reduction of nuclear fluorescence in one associated nucleus, suggesting compartmentalization of the syncitial cytoplasm. Bleach box is shown in red. (B) A dorsal surface view, showing high cytoplasmic levels of Dorsal adhering to nuclear-associated domains. (C) A side confocal view, showing dorsal nuclear domains. (D) A drawing of the domains. (E) A FRAP of both nucleus and cytoplasm on the ventral side at the beginning of mitosis that preferentially bleaches one cytoplasmic domain. Rapid recovery in the 25 and 37.5 second panels shows that Dorsal within the domain can exchange with a mobile pool from elsewhere within the embryo. (F) A false color image of an FLIP of peripheral and deep cytoplasm on the dorsal side (bleach box in white), indicating that Dorsal is partially constrained in its mobility near the plasma membrane surface but is more freely diffusing in the lower, deep cytoplasm. Relative linear intensities are indicated by the color bar at the bottom left. (G) Dorsal is partially constrained in its diffusion to the part of the cytoplasm between the nucleus and the plasma membrane. A 5 second FRAP of nucleus (bleach box in red) transiently reduces fluorescence preferentially in the blue zone. In subsequent images (data not shown), cytoplasmic fluorescence is recovered with delayed kinetics by flow from the deep cytoplasm.

View larger version (19K): [in this window] [in a new window]   Fig. 6. A model for redistribution of Dorsal between cytoplasm and nucleus as a dynamic equilibrium between the activated Toll receptors and the nucleus in Drosophila. Dorsal and Cactus are recruited and phosphorylated by active Toll membrane receptor complexes on the ventral plasma membrane. Phosphorylated Cactus is degraded and phosphorylated Dorsal translocates to the nucleus. Within the nucleus it becomes dephosphorylated and is exported, re-entering the cytoplasmic pool binding de novo synthesized Cactus. Dorsal is again recruited to active receptor complexes and the cycle repeats itself. Partial compartmentalization of the cytoplasm assures integration of the signal from only local active signaling complexes and buffering from signaling complexes associated with the plasma membrane in close proximity to adjacent nuclei.