Limits...
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.

Show MeSH

Related in: MedlinePlus

Detection and mobility of the 93D locus in  vivo. Living embryos were  injected with rhodaminecoupled mAb Q18. After development at room temperature, embryos were heat  shocked at 37°C directly on  the stage of the microscope  to mark the 93D locus. (A) A  field of epidermal interphase  nuclei from an embryo 75  min after the beginning of  heat shock. (B) Time series  of 2-D projections of a single  amnioserosa nucleus from a  heat-shocked embryo during germ band elongation.  The original data set consisted of seven 1-μm Z-axis  sections per time point. The  schematic drawing in C  shows the location of the nucleolus (N) and the 93D loci  (a–d) within the nucleus in B.  (D) A plot of the relative  minimal distances between  the loci of the nucleus in B  against time. The distances  are given relative to the first  image of the series. (E) 10  two-body distance plots  against time demonstrating  the time-dependent changes  in the distances between 93D  loci during heat shock. The  distances were measured  from 2-D projections of image stacks from seven different interphase nuclei in several embryos.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139770&req=5

Figure 7: Detection and mobility of the 93D locus in vivo. Living embryos were injected with rhodaminecoupled mAb Q18. After development at room temperature, embryos were heat shocked at 37°C directly on the stage of the microscope to mark the 93D locus. (A) A field of epidermal interphase nuclei from an embryo 75 min after the beginning of heat shock. (B) Time series of 2-D projections of a single amnioserosa nucleus from a heat-shocked embryo during germ band elongation. The original data set consisted of seven 1-μm Z-axis sections per time point. The schematic drawing in C shows the location of the nucleolus (N) and the 93D loci (a–d) within the nucleus in B. (D) A plot of the relative minimal distances between the loci of the nucleus in B against time. The distances are given relative to the first image of the series. (E) 10 two-body distance plots against time demonstrating the time-dependent changes in the distances between 93D loci during heat shock. The distances were measured from 2-D projections of image stacks from seven different interphase nuclei in several embryos.

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)

Detection and mobility of the 93D locus in  vivo. Living embryos were  injected with rhodaminecoupled mAb Q18. After development at room temperature, embryos were heat  shocked at 37°C directly on  the stage of the microscope  to mark the 93D locus. (A) A  field of epidermal interphase  nuclei from an embryo 75  min after the beginning of  heat shock. (B) Time series  of 2-D projections of a single  amnioserosa nucleus from a  heat-shocked embryo during germ band elongation.  The original data set consisted of seven 1-μm Z-axis  sections per time point. The  schematic drawing in C  shows the location of the nucleolus (N) and the 93D loci  (a–d) within the nucleus in B.  (D) A plot of the relative  minimal distances between  the loci of the nucleus in B  against time. The distances  are given relative to the first  image of the series. (E) 10  two-body distance plots  against time demonstrating  the time-dependent changes  in the distances between 93D  loci during heat shock. The  distances were measured  from 2-D projections of image stacks from seven different interphase nuclei in several embryos.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Detection and mobility of the 93D locus in vivo. Living embryos were injected with rhodaminecoupled mAb Q18. After development at room temperature, embryos were heat shocked at 37°C directly on the stage of the microscope to mark the 93D locus. (A) A field of epidermal interphase nuclei from an embryo 75 min after the beginning of heat shock. (B) Time series of 2-D projections of a single amnioserosa nucleus from a heat-shocked embryo during germ band elongation. The original data set consisted of seven 1-μm Z-axis sections per time point. The schematic drawing in C shows the location of the nucleolus (N) and the 93D loci (a–d) within the nucleus in B. (D) A plot of the relative minimal distances between the loci of the nucleus in B against time. The distances are given relative to the first image of the series. (E) 10 two-body distance plots against time demonstrating the time-dependent changes in the distances between 93D loci during heat shock. The distances were measured from 2-D projections of image stacks from seven different interphase nuclei in several embryos.
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