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The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

Buchenau P, Saumweber H, Arndt-Jovin DJ - J. Cell Biol. (1997)

Bottom Line: Injection of antibody into living embryos had no apparent deleterious effects on further development.The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos.These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

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The distribution of  Hrb57A in fixed and living  embryos. The overall distribution of Hrb57A during  embryogenesis was observed  in fixed embryos (A–C) using  indirect antibody staining or  followed directly in vivo by  microinjection of rhodaminecoupled mAb Q18 into living  embryos (D). (A) Preferential cytoplasmic localization of  Hrb57A, syncytial blastoderm,  nuclear cycle 13. (B) Nuclear  localization, cellular blastoderm, cycle 14. (C) Hrb57A  is nuclear in all tissues during  later embryogenesis, as shown  here for an embryo of stage  10, at the time of full germ  band elongation. (D) The similar complete localization of  Hrb57A in late embryogenesis occurs in the microinjected embryos. Two optical  sections through a living embryo of stage 12 during germ  band retraction, showing the  in vivo labeling pattern of  Hrb57A. All images are single confocal sections.
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Figure 1: The distribution of Hrb57A in fixed and living embryos. The overall distribution of Hrb57A during embryogenesis was observed in fixed embryos (A–C) using indirect antibody staining or followed directly in vivo by microinjection of rhodaminecoupled mAb Q18 into living embryos (D). (A) Preferential cytoplasmic localization of Hrb57A, syncytial blastoderm, nuclear cycle 13. (B) Nuclear localization, cellular blastoderm, cycle 14. (C) Hrb57A is nuclear in all tissues during later embryogenesis, as shown here for an embryo of stage 10, at the time of full germ band elongation. (D) The similar complete localization of Hrb57A in late embryogenesis occurs in the microinjected embryos. Two optical sections through a living embryo of stage 12 during germ band retraction, showing the in vivo labeling pattern of Hrb57A. All images are single confocal sections.

Mentions: Hrb57A showed a dynamic redistribution from the cytoplasm to the nuclei at the time of the initiation of massive zygotic transcription in early embryos. Fixed embryos of the nuclear cycles 10–12 were characterized by strong cytoplasmic staining with very few accumulations of Hrb57A within the interphase nuclei (Fig. 1 A). In contrast, embryos of the cycle 14 showed an exclusively nuclear Hrb57A signal (Fig. 1 B) whereas cycle 13 embryos were clearly in transition, demonstrating examples of both patterns (data not shown). From cycle 14 throughout the rest of embryogenesis, the protein was found in all interphase nuclei of fixed embryos without any preferential tissue distribution (Fig. 1 C). In living embryos we observed similar cytoplasmic antibody fluorescence in the early nuclear cycles and an almost exclusively nuclear staining at later stages. Fig. 1 D shows an example of two optical sections through a living embryo during germ band retraction. By this time of development, about 12 h after microinjection, most of the nuclei had divided two to three times after cellularization and have gone through six to eight cell cycles since microinjection.


The dynamic nuclear redistribution of an hnRNP K-homologous protein during Drosophila embryo development and heat shock. Flexibility of transcription sites in vivo.

Buchenau P, Saumweber H, Arndt-Jovin DJ - J. Cell Biol. (1997)

The distribution of  Hrb57A in fixed and living  embryos. The overall distribution of Hrb57A during  embryogenesis was observed  in fixed embryos (A–C) using  indirect antibody staining or  followed directly in vivo by  microinjection of rhodaminecoupled mAb Q18 into living  embryos (D). (A) Preferential cytoplasmic localization of  Hrb57A, syncytial blastoderm,  nuclear cycle 13. (B) Nuclear  localization, cellular blastoderm, cycle 14. (C) Hrb57A  is nuclear in all tissues during  later embryogenesis, as shown  here for an embryo of stage  10, at the time of full germ  band elongation. (D) The similar complete localization of  Hrb57A in late embryogenesis occurs in the microinjected embryos. Two optical  sections through a living embryo of stage 12 during germ  band retraction, showing the  in vivo labeling pattern of  Hrb57A. All images are single confocal sections.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139770&req=5

Figure 1: The distribution of Hrb57A in fixed and living embryos. The overall distribution of Hrb57A during embryogenesis was observed in fixed embryos (A–C) using indirect antibody staining or followed directly in vivo by microinjection of rhodaminecoupled mAb Q18 into living embryos (D). (A) Preferential cytoplasmic localization of Hrb57A, syncytial blastoderm, nuclear cycle 13. (B) Nuclear localization, cellular blastoderm, cycle 14. (C) Hrb57A is nuclear in all tissues during later embryogenesis, as shown here for an embryo of stage 10, at the time of full germ band elongation. (D) The similar complete localization of Hrb57A in late embryogenesis occurs in the microinjected embryos. Two optical sections through a living embryo of stage 12 during germ band retraction, showing the in vivo labeling pattern of Hrb57A. All images are single confocal sections.
Mentions: Hrb57A showed a dynamic redistribution from the cytoplasm to the nuclei at the time of the initiation of massive zygotic transcription in early embryos. Fixed embryos of the nuclear cycles 10–12 were characterized by strong cytoplasmic staining with very few accumulations of Hrb57A within the interphase nuclei (Fig. 1 A). In contrast, embryos of the cycle 14 showed an exclusively nuclear Hrb57A signal (Fig. 1 B) whereas cycle 13 embryos were clearly in transition, demonstrating examples of both patterns (data not shown). From cycle 14 throughout the rest of embryogenesis, the protein was found in all interphase nuclei of fixed embryos without any preferential tissue distribution (Fig. 1 C). In living embryos we observed similar cytoplasmic antibody fluorescence in the early nuclear cycles and an almost exclusively nuclear staining at later stages. Fig. 1 D shows an example of two optical sections through a living embryo during germ band retraction. By this time of development, about 12 h after microinjection, most of the nuclei had divided two to three times after cellularization and have gone through six to eight cell cycles since microinjection.

Bottom Line: Injection of antibody into living embryos had no apparent deleterious effects on further development.The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos.These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
The Drosophila protein Hrb57A has sequence homology to mammalian heterogenous nuclear ribonucleoprotein (hnRNP) K proteins. Its in vivo distribution has been studied at high resolution by confocal laser scanning microscopy (CLSM) in embryos injected with fluorescently labeled monoclonal antibody. Injection of antibody into living embryos had no apparent deleterious effects on further development. Furthermore, the antibody-protein complex could be observed for more than 7 cell cycles in vivo, revealing a dynamic redistribution from the nucleus to cytoplasm at each mitosis from blastoderm until hatching. The evaluation of two- and three-dimensional CLSM data sets demonstrated important differences in the localization of the protein in the nuclei of living compared to fixed embryos. The Hrb57A protein was recruited to the 93D locus upon heat shock and thus serves as an in vivo probe for the activity of the gene in diploid cells of the embryo. Observations during heat shock revealed considerable mobility within interphase nuclei of this transcription site. Furthermore, the reinitiation as well as the down regulation of transcriptional loci in vivo during the recovery from heat shock could be followed by the rapid redistribution of the hnRNP K during stress recovery. These data are incompatible with a model of the interphase nucleus in which transcription complexes are associated with a rigid nuclear matrix.

Show MeSH
Related in: MedlinePlus