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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 3-D intranuclear distribution of Hrb57A  in situ and in vivo. (A) Stereo  image showing the distribution in discrete loci of the  Hrb57A protein in a fixed interphase nucleus of a wholemount embryo reconstructed  from 25 optical sections with  a Z axis distance of 0.25 μm.  (B) Stereo image of a part of  a similar nucleus in a living  embryo, reconstructed from  6 optical sections separated  by 0.4 μm. Both image stacks  have been recorded with a  Plan-neofluar oil immersion  objective 63×, NA 1.4, with a  0.14-mm coverslip. Bars, 2 μm.
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Figure 3: The 3-D intranuclear distribution of Hrb57A in situ and in vivo. (A) Stereo image showing the distribution in discrete loci of the Hrb57A protein in a fixed interphase nucleus of a wholemount embryo reconstructed from 25 optical sections with a Z axis distance of 0.25 μm. (B) Stereo image of a part of a similar nucleus in a living embryo, reconstructed from 6 optical sections separated by 0.4 μm. Both image stacks have been recorded with a Plan-neofluar oil immersion objective 63×, NA 1.4, with a 0.14-mm coverslip. Bars, 2 μm.

Mentions: Using the high spatial resolution of the fluorescence CLSM we have analyzed the 3-D distribution of Hrb57A in the nuclei of numerous fixed and living embryos. Fig. 3 A shows a stereo image of the protein distribution in a nucleus from the amnioserosa of a fixed embryo. Hrb57A was localized in a number of optically resolvable spots throughout the nuclear volume excluding the nucleoli, which can be identified as unstained domains. The number of discrete protein loci in such interphase nuclei was determined by intensity segmentation of the 3-D data sets and generation of labeled binary objects. The representative nucleus of Fig. 3 A contains about 200 Hrb57A spots. This number is close to the number of Hrb57A-bound transcriptional loci on polytene chromosomes and confirms our supposition that the protein denotes gene loci sensitive to activation and binding of the hnRNP K in the embryonic nuclei.


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 3-D intranuclear distribution of Hrb57A  in situ and in vivo. (A) Stereo  image showing the distribution in discrete loci of the  Hrb57A protein in a fixed interphase nucleus of a wholemount embryo reconstructed  from 25 optical sections with  a Z axis distance of 0.25 μm.  (B) Stereo image of a part of  a similar nucleus in a living  embryo, reconstructed from  6 optical sections separated  by 0.4 μm. Both image stacks  have been recorded with a  Plan-neofluar oil immersion  objective 63×, NA 1.4, with a  0.14-mm coverslip. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139770&req=5

Figure 3: The 3-D intranuclear distribution of Hrb57A in situ and in vivo. (A) Stereo image showing the distribution in discrete loci of the Hrb57A protein in a fixed interphase nucleus of a wholemount embryo reconstructed from 25 optical sections with a Z axis distance of 0.25 μm. (B) Stereo image of a part of a similar nucleus in a living embryo, reconstructed from 6 optical sections separated by 0.4 μm. Both image stacks have been recorded with a Plan-neofluar oil immersion objective 63×, NA 1.4, with a 0.14-mm coverslip. Bars, 2 μm.
Mentions: Using the high spatial resolution of the fluorescence CLSM we have analyzed the 3-D distribution of Hrb57A in the nuclei of numerous fixed and living embryos. Fig. 3 A shows a stereo image of the protein distribution in a nucleus from the amnioserosa of a fixed embryo. Hrb57A was localized in a number of optically resolvable spots throughout the nuclear volume excluding the nucleoli, which can be identified as unstained domains. The number of discrete protein loci in such interphase nuclei was determined by intensity segmentation of the 3-D data sets and generation of labeled binary objects. The representative nucleus of Fig. 3 A contains about 200 Hrb57A spots. This number is close to the number of Hrb57A-bound transcriptional loci on polytene chromosomes and confirms our supposition that the protein denotes gene loci sensitive to activation and binding of the hnRNP K in the embryonic nuclei.

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