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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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Dynamic changes in the intranuclear distribution of Hrb57A during recovery from heat shock. Stereo images of amnioserosa nuclei reconstructed from five optical sections separated by 2 μm. Intranuclear accumulations of Hrb57A are numbered (see text). (A) 6  min after reducing the temperature to 25°C. (B) 10 min into recovery. Bar, 5 μm.
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Figure 8: Dynamic changes in the intranuclear distribution of Hrb57A during recovery from heat shock. Stereo images of amnioserosa nuclei reconstructed from five optical sections separated by 2 μm. Intranuclear accumulations of Hrb57A are numbered (see text). (A) 6 min after reducing the temperature to 25°C. (B) 10 min into recovery. Bar, 5 μm.

Mentions: We were able to observe another type of dynamic behavior of Hrb57A in vivo after the transition from the high (37°C) to normal temperature (25°C), i.e., during recovery of the embryos from heat shock. Onset of recovery begins immediately, as reflected by the change in the disposition of Hrb57A at the 93D locus. The earliest recordings were made at 1 min after the temperature drop and subsequent images at 2 or 4 min intervals. The intensity of the Q18 staining of the 93D locus begins to decrease in almost all loci immediately. Many new weaker loci appear throughout the nuclear volume. There is large variability in the rate of appearance and number of resolvable new loci in individual nuclei. The rate of recovery also is dependent upon the extent and duration of the prior heat shock treatment. An example of recovery behavior is demonstrated in Fig. 8. Two stereo image pairs of the amnioserosa of an embryo are reconstructed from image stacks separated by 4 min in time after return to normal temperature. Local accumulations of mAb Q18 in the two fully visible nuclei in this field have been marked by numbers. The nucleus on the left shows small reorganizations of the Hrb57A loci at 6 and 10 min into heat shock recovery, and the composite loci are labeled 1 and 2 for simplicity. The nucleus on the right displayed dramatic changes in the Hrb57A pattern at these time points. One of the spots disappeared completely (3), another one became smaller (5), and two new accumulations appeared (6 and 7). Computer animations of the complete time series of this recovery show increasingly complex reorganization of the Hrb57A protein. These dynamic changes cannot be attributed to a rotation of the nucleus as a whole or to a simple translational mobility of most of the loci as is documented in Fig. 7, B–D. We have observed a large number of similar and more complicated dynamic redistributions of the Hrb57A protein during heat shock recovery in many embryos. We conclude that the redistribution of Hrb57A is causally related to the reactivation of gene expression that occurs in the cell nucleus during recovery from heat shock.


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)

Dynamic changes in the intranuclear distribution of Hrb57A during recovery from heat shock. Stereo images of amnioserosa nuclei reconstructed from five optical sections separated by 2 μm. Intranuclear accumulations of Hrb57A are numbered (see text). (A) 6  min after reducing the temperature to 25°C. (B) 10 min into recovery. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 8: Dynamic changes in the intranuclear distribution of Hrb57A during recovery from heat shock. Stereo images of amnioserosa nuclei reconstructed from five optical sections separated by 2 μm. Intranuclear accumulations of Hrb57A are numbered (see text). (A) 6 min after reducing the temperature to 25°C. (B) 10 min into recovery. Bar, 5 μm.
Mentions: We were able to observe another type of dynamic behavior of Hrb57A in vivo after the transition from the high (37°C) to normal temperature (25°C), i.e., during recovery of the embryos from heat shock. Onset of recovery begins immediately, as reflected by the change in the disposition of Hrb57A at the 93D locus. The earliest recordings were made at 1 min after the temperature drop and subsequent images at 2 or 4 min intervals. The intensity of the Q18 staining of the 93D locus begins to decrease in almost all loci immediately. Many new weaker loci appear throughout the nuclear volume. There is large variability in the rate of appearance and number of resolvable new loci in individual nuclei. The rate of recovery also is dependent upon the extent and duration of the prior heat shock treatment. An example of recovery behavior is demonstrated in Fig. 8. Two stereo image pairs of the amnioserosa of an embryo are reconstructed from image stacks separated by 4 min in time after return to normal temperature. Local accumulations of mAb Q18 in the two fully visible nuclei in this field have been marked by numbers. The nucleus on the left shows small reorganizations of the Hrb57A loci at 6 and 10 min into heat shock recovery, and the composite loci are labeled 1 and 2 for simplicity. The nucleus on the right displayed dramatic changes in the Hrb57A pattern at these time points. One of the spots disappeared completely (3), another one became smaller (5), and two new accumulations appeared (6 and 7). Computer animations of the complete time series of this recovery show increasingly complex reorganization of the Hrb57A protein. These dynamic changes cannot be attributed to a rotation of the nucleus as a whole or to a simple translational mobility of most of the loci as is documented in Fig. 7, B–D. We have observed a large number of similar and more complicated dynamic redistributions of the Hrb57A protein during heat shock recovery in many embryos. We conclude that the redistribution of Hrb57A is causally related to the reactivation of gene expression that occurs in the cell nucleus during recovery from heat shock.

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