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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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Fields of interphase nuclei in the amnioserosa of a fixed  (A) and a living (B) embryo during stage 11 (elongated germ  band) stained for Hrb57A. Both images are single confocal sections from stacks recorded as in Fig. 3. Width of the total field is  40 μm.
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Figure 4: Fields of interphase nuclei in the amnioserosa of a fixed (A) and a living (B) embryo during stage 11 (elongated germ band) stained for Hrb57A. Both images are single confocal sections from stacks recorded as in Fig. 3. Width of the total field is 40 μm.

Mentions: A nucleus from the same tissue of a living embryo 5 h after the injection of rhodamine-labeled mAb Q18 is shown in Fig. 3 B. In this case the unstained nucleoli were surrounded by a positively fluorescent nuclear volume punctated by discrete loci with higher concentrations of Hrb57A. These loci are somewhat larger and fewer in number than those resolved in the fixed embryos. Both stereo images in Fig. 3 have been reconstructed from confocal sections acquired under optical conditions as nearly identical as possible. Fig. 4 shows overviews of a field of amnioserosa cells from another fixed (A) and another living (B) embryo which demonstrate that these differences in the distribution of Hrb57A between fixed and living embryos are highly reproducible and visible already in single confocal sections. We attribute the differences to a differential extraction of the protein as well as to changes in the chromatin structure introduced by the fixation procedure and/or to the dynamic movement of transcription sites in living embryos (see Discussion).


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)

Fields of interphase nuclei in the amnioserosa of a fixed  (A) and a living (B) embryo during stage 11 (elongated germ  band) stained for Hrb57A. Both images are single confocal sections from stacks recorded as in Fig. 3. Width of the total field is  40 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Fields of interphase nuclei in the amnioserosa of a fixed (A) and a living (B) embryo during stage 11 (elongated germ band) stained for Hrb57A. Both images are single confocal sections from stacks recorded as in Fig. 3. Width of the total field is 40 μm.
Mentions: A nucleus from the same tissue of a living embryo 5 h after the injection of rhodamine-labeled mAb Q18 is shown in Fig. 3 B. In this case the unstained nucleoli were surrounded by a positively fluorescent nuclear volume punctated by discrete loci with higher concentrations of Hrb57A. These loci are somewhat larger and fewer in number than those resolved in the fixed embryos. Both stereo images in Fig. 3 have been reconstructed from confocal sections acquired under optical conditions as nearly identical as possible. Fig. 4 shows overviews of a field of amnioserosa cells from another fixed (A) and another living (B) embryo which demonstrate that these differences in the distribution of Hrb57A between fixed and living embryos are highly reproducible and visible already in single confocal sections. We attribute the differences to a differential extraction of the protein as well as to changes in the chromatin structure introduced by the fixation procedure and/or to the dynamic movement of transcription sites in living embryos (see Discussion).

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