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Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle.

Liang Y, Häring M, Roughley PJ, Margolis RK, Margolis RU - J. Cell Biol. (1997)

Bottom Line: In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons.Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle.Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, New York University Medical Center, New York 10016, USA.

ABSTRACT
We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

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Transport of HSV-tagged glypican to the nuclei of  transfected COS-1 cells. Cells were transfected with full-length  glypican cDNA containing an HSV epitope tag and stained with  propidium iodide (A and C) or an mAb to the HSV tag (B and  D). Staining is seen in the nuclei and in the surrounding ER and  Golgi membranes. Bars, 50 μm.
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Figure 13: Transport of HSV-tagged glypican to the nuclei of transfected COS-1 cells. Cells were transfected with full-length glypican cDNA containing an HSV epitope tag and stained with propidium iodide (A and C) or an mAb to the HSV tag (B and D). Staining is seen in the nuclei and in the surrounding ER and Golgi membranes. Bars, 50 μm.

Mentions: When the basic KRRRAK sequence of the glypican– β-galactosidase fusion protein was mutated by PCR to non-basic amino acids and the cDNA transfected into 293 cells, the nuclear localization of the resulting fusion protein was greatly decreased although not entirely eliminated (Fig. 12 H). The presence of some residual nuclear immunoreactivity probably does not result from effects of the remainder of the glypican sequence but rather to the nature of the β-galactosidase fusion protein itself, insofar as a second mutant in which most of the remaining glypican sequence was deleted from the nonbasic mutant fusion protein (Fig. 11 B) also showed the same small degree of nuclear immunoreactivity (Fig. 12 J). Although full-length glypican was not transported to the nucleus of 293 cells (data not shown), we did find nuclear expression of glypican in COS-1 cells transfected with an HSV epitope-tagged glypican cDNA (Fig. 13).


Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle.

Liang Y, Häring M, Roughley PJ, Margolis RK, Margolis RU - J. Cell Biol. (1997)

Transport of HSV-tagged glypican to the nuclei of  transfected COS-1 cells. Cells were transfected with full-length  glypican cDNA containing an HSV epitope tag and stained with  propidium iodide (A and C) or an mAb to the HSV tag (B and  D). Staining is seen in the nuclei and in the surrounding ER and  Golgi membranes. Bars, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139971&req=5

Figure 13: Transport of HSV-tagged glypican to the nuclei of transfected COS-1 cells. Cells were transfected with full-length glypican cDNA containing an HSV epitope tag and stained with propidium iodide (A and C) or an mAb to the HSV tag (B and D). Staining is seen in the nuclei and in the surrounding ER and Golgi membranes. Bars, 50 μm.
Mentions: When the basic KRRRAK sequence of the glypican– β-galactosidase fusion protein was mutated by PCR to non-basic amino acids and the cDNA transfected into 293 cells, the nuclear localization of the resulting fusion protein was greatly decreased although not entirely eliminated (Fig. 12 H). The presence of some residual nuclear immunoreactivity probably does not result from effects of the remainder of the glypican sequence but rather to the nature of the β-galactosidase fusion protein itself, insofar as a second mutant in which most of the remaining glypican sequence was deleted from the nonbasic mutant fusion protein (Fig. 11 B) also showed the same small degree of nuclear immunoreactivity (Fig. 12 J). Although full-length glypican was not transported to the nucleus of 293 cells (data not shown), we did find nuclear expression of glypican in COS-1 cells transfected with an HSV epitope-tagged glypican cDNA (Fig. 13).

Bottom Line: In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons.Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle.Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, New York University Medical Center, New York 10016, USA.

ABSTRACT
We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

Show MeSH
Related in: MedlinePlus