Limits...
Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.

Suhr ST, Senut MC, Whitelegge JP, Faull KF, Cuizon DB, Gage FH - J. Cell Biol. (2001)

Bottom Line: One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs).Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex.These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Show MeSH

Related in: MedlinePlus

Morphological changes and cell death in induced polyQ-expressing cells. (a–d) 5-d induced 96QN cells matched fluorescence (a and c) and phase–contrast images (b and d), revealing multinucleated syncitia-like cell structures. In a and c, nuclei are red, and polyQ–GFP IAs are green. In b and d, red arrowheads indicate the syncitia boundaries. (e) Quantification of the number of syncitia-like structures/103 cells in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. (f–i) TUNEL positivity in induced 96QN cells. (f) Fluorescent view; (g) bright-field view reveling DAB-stained TUNEL-positive profiles; (h) overlay of both views; and (i) corresponding phase–contrast view. Arrows in panel g indicate TUNEL profiles that were scored as positive for quantification. Overlay and phase–contrast views reveal that the TUNEL positive cells are often rounded up and resting just above the monolayer of surviving cells below. (j) Quantification of the number of TUNEL-positive profiles in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. The slight increase in apoptotic cells at 3 and 5 d for 13QN and 96Q cells is probably the consequence of high cell density near the end of the culture period. Bar: (a–d) 50 μm; (f–i) 250 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169460&req=5

Figure 2: Morphological changes and cell death in induced polyQ-expressing cells. (a–d) 5-d induced 96QN cells matched fluorescence (a and c) and phase–contrast images (b and d), revealing multinucleated syncitia-like cell structures. In a and c, nuclei are red, and polyQ–GFP IAs are green. In b and d, red arrowheads indicate the syncitia boundaries. (e) Quantification of the number of syncitia-like structures/103 cells in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. (f–i) TUNEL positivity in induced 96QN cells. (f) Fluorescent view; (g) bright-field view reveling DAB-stained TUNEL-positive profiles; (h) overlay of both views; and (i) corresponding phase–contrast view. Arrows in panel g indicate TUNEL profiles that were scored as positive for quantification. Overlay and phase–contrast views reveal that the TUNEL positive cells are often rounded up and resting just above the monolayer of surviving cells below. (j) Quantification of the number of TUNEL-positive profiles in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. The slight increase in apoptotic cells at 3 and 5 d for 13QN and 96Q cells is probably the consequence of high cell density near the end of the culture period. Bar: (a–d) 50 μm; (f–i) 250 μm.

Mentions: Study of induced cells revealed no obvious time-dependent morphological changes in any of the induced lines with the exception of 96QN, which at time points >4 d exhibited irregularities in nuclear morphology, including nuclear hypertrophy, the formation of small satellite structures of nuclear material, and an increase in the formation of multinucleated syncitia. Fig. 2, a–d, shows examples of the occasional multinucleated syncytia that form at 5 d after induction. One hallmark of these syncytia is the frequent presence of large cytoplasmic IAs at the center of a ring of nuclei (Fig. 2, a and c). The number of syncytia within representative cell populations at different times after induction is shown in Fig. 2 e. At the 5-d time point, 96QN cells develop over fivefold more multinucleated syncytia than 5-d 13QN cells, 96Q cells, or 96QN cells at the earlier time points.


Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.

Suhr ST, Senut MC, Whitelegge JP, Faull KF, Cuizon DB, Gage FH - J. Cell Biol. (2001)

Morphological changes and cell death in induced polyQ-expressing cells. (a–d) 5-d induced 96QN cells matched fluorescence (a and c) and phase–contrast images (b and d), revealing multinucleated syncitia-like cell structures. In a and c, nuclei are red, and polyQ–GFP IAs are green. In b and d, red arrowheads indicate the syncitia boundaries. (e) Quantification of the number of syncitia-like structures/103 cells in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. (f–i) TUNEL positivity in induced 96QN cells. (f) Fluorescent view; (g) bright-field view reveling DAB-stained TUNEL-positive profiles; (h) overlay of both views; and (i) corresponding phase–contrast view. Arrows in panel g indicate TUNEL profiles that were scored as positive for quantification. Overlay and phase–contrast views reveal that the TUNEL positive cells are often rounded up and resting just above the monolayer of surviving cells below. (j) Quantification of the number of TUNEL-positive profiles in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. The slight increase in apoptotic cells at 3 and 5 d for 13QN and 96Q cells is probably the consequence of high cell density near the end of the culture period. Bar: (a–d) 50 μm; (f–i) 250 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169460&req=5

Figure 2: Morphological changes and cell death in induced polyQ-expressing cells. (a–d) 5-d induced 96QN cells matched fluorescence (a and c) and phase–contrast images (b and d), revealing multinucleated syncitia-like cell structures. In a and c, nuclei are red, and polyQ–GFP IAs are green. In b and d, red arrowheads indicate the syncitia boundaries. (e) Quantification of the number of syncitia-like structures/103 cells in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. (f–i) TUNEL positivity in induced 96QN cells. (f) Fluorescent view; (g) bright-field view reveling DAB-stained TUNEL-positive profiles; (h) overlay of both views; and (i) corresponding phase–contrast view. Arrows in panel g indicate TUNEL profiles that were scored as positive for quantification. Overlay and phase–contrast views reveal that the TUNEL positive cells are often rounded up and resting just above the monolayer of surviving cells below. (j) Quantification of the number of TUNEL-positive profiles in a typical experiment in each induced cell population at 0, 3, and 5 d. Numbers indicate fold change relative to day 0. The slight increase in apoptotic cells at 3 and 5 d for 13QN and 96Q cells is probably the consequence of high cell density near the end of the culture period. Bar: (a–d) 50 μm; (f–i) 250 μm.
Mentions: Study of induced cells revealed no obvious time-dependent morphological changes in any of the induced lines with the exception of 96QN, which at time points >4 d exhibited irregularities in nuclear morphology, including nuclear hypertrophy, the formation of small satellite structures of nuclear material, and an increase in the formation of multinucleated syncitia. Fig. 2, a–d, shows examples of the occasional multinucleated syncytia that form at 5 d after induction. One hallmark of these syncytia is the frequent presence of large cytoplasmic IAs at the center of a ring of nuclei (Fig. 2, a and c). The number of syncytia within representative cell populations at different times after induction is shown in Fig. 2 e. At the 5-d time point, 96QN cells develop over fivefold more multinucleated syncytia than 5-d 13QN cells, 96Q cells, or 96QN cells at the earlier time points.

Bottom Line: One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs).Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex.These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.

ABSTRACT
Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycle-regulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.

Show MeSH
Related in: MedlinePlus