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

In Drosophila, the NF-κB/REL family transcription factor, Dorsal, redistributes from the cytoplasm to nuclei, forming a concentration gradient across the dorsoventral axis of the embryo. Using live imaging techniques in conjunction with embryos expressing a chimeric Dorsal-GFP, we demonstrate that the redistribution of Dorsal from cytoplasm to nucleus is an extremely dynamic process. Nuclear Dorsal concentration changes continuously over time in all nuclei during interphase. While Dorsal appears to be nuclearly localized primarily in ventral nuclei, it is actively shuttling into and out of all nuclei, including nuclei on the dorsal side. Nuclear export is blocked by leptomycin B, a potent inhibitor of Exportin 1 (CRM1)-mediated nuclear export. We have developed a novel in vivo assay revealing the presence of a functional leucine-rich nuclear export signal within the carboxyterminal 44 amino acids of Dorsal. We also find that diffusion of Dorsal is partially constrained to cytoplasmic islands surrounding individual syncitial nuclei. A model is proposed in which the generation and maintenance of the Dorsal gradient is a consequence of an active process involving both restricted long-range diffusion and the balancing of nuclear import with nuclear export.

[1]  G. Rubin,et al.  Genetic transformation of Drosophila with transposable element vectors. , 1982, Science.

[2]  B. Alberts,et al.  Studies of nuclear and cytoplasmic behaviour during the five mitotic cycles that precede gastrulation in Drosophila embryogenesis. , 1983, Journal of cell science.

[3]  J. Sedat,et al.  Nuclear structure: determination of the fate of the nuclear envelope in Drosophila during mitosis using monoclonal antibodies. , 1983, Journal of cell science.

[4]  D. Baltimore,et al.  Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-κB transcription factor , 1988, Cell.

[5]  R. Steward,et al.  The dorsal protein is distributed in a gradient in early drosophila embryos , 1988, Cell.

[6]  D. Baltimore,et al.  Oligonucleotide that binds nuclear factor NF-kappa B acts as a lymphoid-specific and inducible enhancer element. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Levine,et al.  The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila , 1989, Cell.

[8]  R. Steward.  Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function , 1989, Cell.

[9]  D. Baltimore,et al.  A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. , 1989, Genes & development.

[10]  C. Nüsslein-Volhard,et al.  A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo , 1989, Cell.

[11]  D. Baltimore,et al.  Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. , 1990, Nature.

[12]  M. Leptin.  twist and snail as positive and negative regulators during Drosophila mesoderm development. , 1991, Genes & development.

[13]  K. Anderson,et al.  Plasma membrane localization of the Toll protein in the syncytial Drosophila embryo: importance of transmembrane signaling for dorsal-ventral pattern formation. , 1991, Development.

[14]  J. Boulay,et al.  The snail gene required for mesoderm formation in Drosophila is expressed dynamically in derivatives of all three germ layers. , 1991, Development.

[15]  C. Nüsslein-Volhard,et al.  cactus, a maternal gene required for proper formation of the dorsoventral morphogen gradient in Drosophila embryos. , 1991, Development.

[16]  Dorsal, a Drosophila Rel-like protein, is phosphorylated upon activation of the transmembrane protein Toll. , 1994, Molecular and cellular biology.

[17]  R. Delotto,et al.  Ventralizing signal determined by protease activation in Drosophila embryogenesis , 1994, Nature.

[18]  M. Levine,et al.  Regulation of the dorsal morphogen by the Toll and torso signaling pathways: a receptor tyrosine kinase selectively masks transcriptional repression. , 1994, Genes & development.

[19]  K. Anderson,et al.  Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo. , 1995, Annual review of genetics.

[20]  S A Wasserman,et al.  A gradient of cactus protein degradation establishes dorsoventral polarity in the Drosophila embryo. , 1996, Developmental biology.

[21]  Robert Geisler,et al.  A gradient of cytoplasmic Cactus degradation establishes the nuclear localization gradient of the dorsal morphogen in Drosophila , 1996, Mechanisms of Development.

[22]  Minoru Yoshida,et al.  CRM1 is responsible for intracellular transport mediated by the nuclear export signal , 1997, Nature.

[23]  R. Steward,et al.  A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[24]  P. Rørth.  Gal4 in the Drosophila female germline , 1998, Mechanisms of Development.

[25]  S. Wasserman,et al.  Recruitment of Tube and Pelle to signaling sites at the surface of the Drosophila embryo. , 1998, Development.

[26]  David M. Rothwarf,et al.  The NF-κB Activation Pathway: A Paradigm in Information Transfer from Membrane to Nucleus , 1999, Science's STKE.

[27]  R. Steward,et al.  Nuclear import of the Drosophila Rel protein Dorsal is regulated by phosphorylation. , 1999, Genes & development.

[28]  R. Steward,et al.  Cactus-independent regulation of Dorsal nuclear import by the ventral signal , 2000, Current Biology.

[29]  G. Odell,et al.  Microtubules and mitotic cycle phase modulate spatiotemporal distributions of F-actin and myosin II in Drosophila syncytial blastoderm embryos. , 2000, Development.

[30]  Eva E. Qwarnstrom,et al.  Dynamic Shuttling of Nuclear Factor κB between the Nucleus and Cytoplasm as a Consequence of Inhibitor Dissociation* , 2000, The Journal of Biological Chemistry.

[31]  Robert DeLotto.  Gastrulation defective, a complement factor C2/B‐like protease, interprets a ventral prepattern in Drosophila , 2001, EMBO reports.

[32]  Tom Misteli,et al.  Kinetic modelling approaches to in vivo imaging , 2001, Nature Reviews Molecular Cell Biology.

[33]  E. Kiseleva,et al.  Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos. , 2001, Journal of cell science.

[34]  M. Karin,et al.  Missing Pieces in the NF-κB Puzzle , 2002, Cell.

[35]  G. Stark,et al.  NFkappaB-dependent signaling pathways. , 2002, Experimental hematology.

[36]  Michael Levine,et al.  Dorsal gradient networks in the Drosophila embryo. , 2002, Developmental biology.

[37]  G. Stark,et al.  NFκB-dependent signaling pathways , 2002 .

[38]  B. Binder,et al.  Signaling molecules of the NF-kappa B pathway shuttle constitutively between cytoplasm and nucleus. , 2002, The Journal of biological chemistry.

[39]  Bernd R. Binder,et al.  Signaling Molecules of the NF-κB Pathway Shuttle Constitutively between Cytoplasm and Nucleus* , 2002, The Journal of Biological Chemistry.

[40]  R. Steward,et al.  Tamo selectively modulates nuclear import in Drosophila , 2003, Genes to cells : devoted to molecular & cellular mechanisms.

[41]  C. Murphy,et al.  Steady-state dynamics of Cajal body components in the Xenopus germinal vesicle , 2003, The Journal of cell biology.

[42]  Bernhard Schmierer,et al.  Analysis of Smad nucleocytoplasmic shuttling in living cells , 2004, Journal of Cell Science.

[43]  Søren Brunak,et al.  Analysis and prediction of leucine-rich nuclear export signals. , 2004, Protein engineering, design & selection : PEDS.

[44]  S. Ghosh,et al.  Cytosolic, nuclear and nucleolar localization signals determine subcellular distribution and activity of the NF-κB inducing kinase NIK , 2004, Journal of Cell Science.

[45]  S. Roth,et al.  Dorsoventral Axis Formation in the Drosophila Embryo—Shaping and Transducing a Morphogen Gradient , 2005, Current Biology.

[46]  J. Lippincott-Schwartz,et al.  The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei , 2006, The Journal of cell biology.