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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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Nuclear import and  mitotic distribution of  Hrb57A in living embryos.  A shows selected frames  from a time series of confocal  images from a single blastoderm embryo during the nuclear cycles 11–14. The cycle  number is given at the bottom of each panel. M denotes  mitosis. Arrows indicate the  nonfluorescent mitotic chromatin (first panel) and a  weak accumulation of  Hrb57A in cycle 11 interphase nuclei (second panel).  (B) Plot of the mean nuclear  fluorescence intensity measured in a single focal plane  for a series of images measured at 1 min intervals during cycle 13. An import of  Hrb57A is evident for the 10  min during interphase while  the sharp decline of fluorescence falls together with nuclear division. C and D are  time series of confocal images showing nuclear division in blastoderm (C) and  after gastrulation (D). I, interphase; M, metaphase; A,  anaphase. Arrows denote the  location of the dividing chromatin which contains a detectable amount of Hrb57A  after (D) but not before (C)  gastrulation. Bar, 10 μm.
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Figure 2: Nuclear import and mitotic distribution of Hrb57A in living embryos. A shows selected frames from a time series of confocal images from a single blastoderm embryo during the nuclear cycles 11–14. The cycle number is given at the bottom of each panel. M denotes mitosis. Arrows indicate the nonfluorescent mitotic chromatin (first panel) and a weak accumulation of Hrb57A in cycle 11 interphase nuclei (second panel). (B) Plot of the mean nuclear fluorescence intensity measured in a single focal plane for a series of images measured at 1 min intervals during cycle 13. An import of Hrb57A is evident for the 10 min during interphase while the sharp decline of fluorescence falls together with nuclear division. C and D are time series of confocal images showing nuclear division in blastoderm (C) and after gastrulation (D). I, interphase; M, metaphase; A, anaphase. Arrows denote the location of the dividing chromatin which contains a detectable amount of Hrb57A after (D) but not before (C) gastrulation. Bar, 10 μm.

Mentions: Images (8 bit) were acquired with an appropriate scanning time and frame averaging. For double staining, the images of the two fluorophore distributions were recorded separately and saved to separate channels of an RGB image. Reconstructions of stereo images were performed using the projection functions of the LSM310 software or NIH-Image (National Institutes of Health, Bethesda, Maryland). Additional image processing was performed on some images which included contrast stretching, uniform filtering, bit plane masking, and intensity quantitation using Scil Image (Technical University, Delft, The Netherlands), NIH-Image (National Institutes of Health), Photoshop 3.0 (Adobe Systems, Mountain View, CA), and Imaris 2.2.6 (Bitplane AG, Zürich, Switzerland). Quantitative image processing was performed on confocal laser scanning microscopy (CLSM) data on a Silicon Graphics (Mountain View, CA) workstation using Scil Image or the depth analyzer module of Imaris 2.2.6. The latter program permits the interactive definition of polygons in three dimensional (3-D) stacks of sequential sections in up to three different fluorescence channels and calculates volume, mean, and integrated greyvalues for each of the channels in three dimensions. Nuclear/chromosomal and cellular volumes were outlined by such polygons for the determination of the mean pixel intensities as calculated in Fig. 2 B. Masks for the 93D subnuclear region were generated from the Fl-P2 oligonucleotide fluorescence image stacks by intensity thresholds and used to calculate the distribution of the Hrb57A protein coincident signal in comparison to the protein signal in the total nucleus. Minimal translational distance measurements such as those shown in Fig. 7, D and E were calculated from 2-D maximal intensity 3-D projection images using NIH-Image on a Macintosh Power PC.


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)

Nuclear import and  mitotic distribution of  Hrb57A in living embryos.  A shows selected frames  from a time series of confocal  images from a single blastoderm embryo during the nuclear cycles 11–14. The cycle  number is given at the bottom of each panel. M denotes  mitosis. Arrows indicate the  nonfluorescent mitotic chromatin (first panel) and a  weak accumulation of  Hrb57A in cycle 11 interphase nuclei (second panel).  (B) Plot of the mean nuclear  fluorescence intensity measured in a single focal plane  for a series of images measured at 1 min intervals during cycle 13. An import of  Hrb57A is evident for the 10  min during interphase while  the sharp decline of fluorescence falls together with nuclear division. C and D are  time series of confocal images showing nuclear division in blastoderm (C) and  after gastrulation (D). I, interphase; M, metaphase; A,  anaphase. Arrows denote the  location of the dividing chromatin which contains a detectable amount of Hrb57A  after (D) but not before (C)  gastrulation. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 2: Nuclear import and mitotic distribution of Hrb57A in living embryos. A shows selected frames from a time series of confocal images from a single blastoderm embryo during the nuclear cycles 11–14. The cycle number is given at the bottom of each panel. M denotes mitosis. Arrows indicate the nonfluorescent mitotic chromatin (first panel) and a weak accumulation of Hrb57A in cycle 11 interphase nuclei (second panel). (B) Plot of the mean nuclear fluorescence intensity measured in a single focal plane for a series of images measured at 1 min intervals during cycle 13. An import of Hrb57A is evident for the 10 min during interphase while the sharp decline of fluorescence falls together with nuclear division. C and D are time series of confocal images showing nuclear division in blastoderm (C) and after gastrulation (D). I, interphase; M, metaphase; A, anaphase. Arrows denote the location of the dividing chromatin which contains a detectable amount of Hrb57A after (D) but not before (C) gastrulation. Bar, 10 μm.
Mentions: Images (8 bit) were acquired with an appropriate scanning time and frame averaging. For double staining, the images of the two fluorophore distributions were recorded separately and saved to separate channels of an RGB image. Reconstructions of stereo images were performed using the projection functions of the LSM310 software or NIH-Image (National Institutes of Health, Bethesda, Maryland). Additional image processing was performed on some images which included contrast stretching, uniform filtering, bit plane masking, and intensity quantitation using Scil Image (Technical University, Delft, The Netherlands), NIH-Image (National Institutes of Health), Photoshop 3.0 (Adobe Systems, Mountain View, CA), and Imaris 2.2.6 (Bitplane AG, Zürich, Switzerland). Quantitative image processing was performed on confocal laser scanning microscopy (CLSM) data on a Silicon Graphics (Mountain View, CA) workstation using Scil Image or the depth analyzer module of Imaris 2.2.6. The latter program permits the interactive definition of polygons in three dimensional (3-D) stacks of sequential sections in up to three different fluorescence channels and calculates volume, mean, and integrated greyvalues for each of the channels in three dimensions. Nuclear/chromosomal and cellular volumes were outlined by such polygons for the determination of the mean pixel intensities as calculated in Fig. 2 B. Masks for the 93D subnuclear region were generated from the Fl-P2 oligonucleotide fluorescence image stacks by intensity thresholds and used to calculate the distribution of the Hrb57A protein coincident signal in comparison to the protein signal in the total nucleus. Minimal translational distance measurements such as those shown in Fig. 7, D and E were calculated from 2-D maximal intensity 3-D projection images using NIH-Image on a Macintosh Power PC.

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