Limits...
Mutant Rab24 GTPase is targeted to nuclear inclusions.

Maltese WA, Soule G, Gunning W, Calomeni E, Alexander B - BMC Cell Biol. (2002)

Bottom Line: Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form.If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope.However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43614, USA. wmaltese@mco.edu

ABSTRACT

Background: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity.

Results: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24.

Conclusion: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

Show MeSH

Related in: MedlinePlus

Disruption of nuclear architecture by Rab24(D123I) inclusions. NIH 3T3 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. (A) The inclusions detected by staining with the anti-myc monoclonal antibody appeared to occupy areas of the nucleus devoid of DAPI staining (arrows). In some cells (upper panels) the nucleus was well defined, and it was possible to distinguish between inclusions localized inside and outside the nucleus. In other cells (lower panels), the inclusions were more diffuse, the nucleus was distorted, and it was difficult to discern the boundaries between the nuclear and cytoplasmic compartments. (B) mycRab24(D123I) inclusions were localized with rabbit anti-myc polyclonal antibody followed by FITC-conjugated GAR IgG. The nuclear envelope was highlighted by co-staining with a monoclonal antibody against lamins A and B, followed by rhodamine-conjugated GAM IgG. In some cells with immunoreactive inclusions (upper panels) a well defined nuclear envelope was visible, whereas in others (bottom panels) the nuclear envelope appeared to be completely disrupted. Where both cytoplasmic and nuclear inclusions were present (top and middle panels), it appeared that the myc-reactive aggregates inside the nucleus contained lamins (arrows), while those outside the nucleus did not (arrowheads). For comparison, panel C shows the nuclear lamin staining typically observed in non-transfected cells. The scale bar represents 10 microns.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC130051&req=5

Figure 4: Disruption of nuclear architecture by Rab24(D123I) inclusions. NIH 3T3 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. (A) The inclusions detected by staining with the anti-myc monoclonal antibody appeared to occupy areas of the nucleus devoid of DAPI staining (arrows). In some cells (upper panels) the nucleus was well defined, and it was possible to distinguish between inclusions localized inside and outside the nucleus. In other cells (lower panels), the inclusions were more diffuse, the nucleus was distorted, and it was difficult to discern the boundaries between the nuclear and cytoplasmic compartments. (B) mycRab24(D123I) inclusions were localized with rabbit anti-myc polyclonal antibody followed by FITC-conjugated GAR IgG. The nuclear envelope was highlighted by co-staining with a monoclonal antibody against lamins A and B, followed by rhodamine-conjugated GAM IgG. In some cells with immunoreactive inclusions (upper panels) a well defined nuclear envelope was visible, whereas in others (bottom panels) the nuclear envelope appeared to be completely disrupted. Where both cytoplasmic and nuclear inclusions were present (top and middle panels), it appeared that the myc-reactive aggregates inside the nucleus contained lamins (arrows), while those outside the nucleus did not (arrowheads). For comparison, panel C shows the nuclear lamin staining typically observed in non-transfected cells. The scale bar represents 10 microns.

Mentions: The accumulation of nuclear inclusions in cells expressing Rab24(D123I) led to progressive degeneration of nuclear structure. Figure 4 shows a series of representative photographs of transfected 3T3 cells that were co-stained with antibody against the Rab24 myc epitope, combined with DAPI to visualize the chromatin (Fig. 4A), or an antibody against lamins A and B to visualize the nuclear envelope (Fig. 4B). At 24 h after transfection, we observed many cells with mycRab24-positive inclusions that showed fairly uniform staining of the nuclear envelope with no loss of chromatin or deformation of nuclear shape. However, in some cells with larger and more numerous inclusions we observed discontinuities in the anti-lamin staining of the nuclear envelope and some co-localization of lamin with mycRab24(D123I) in the intranuclear aggregates. The gaps in the nuclear membrane were clearly visible in electron micrographs of cells with large numbers of intranuclear aggregates (Fig. 3C). In the most extreme cases, the nuclear envelope was significantly distorted and fragmented and there was leakage of chromatin into the cytoplasm (Fig. 4A &4B, lower panels). The number of cells with obvious nuclear disruption increased with time, so that by 48-h this was seen in the majority of the transfected cells (not shown).


Mutant Rab24 GTPase is targeted to nuclear inclusions.

Maltese WA, Soule G, Gunning W, Calomeni E, Alexander B - BMC Cell Biol. (2002)

Disruption of nuclear architecture by Rab24(D123I) inclusions. NIH 3T3 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. (A) The inclusions detected by staining with the anti-myc monoclonal antibody appeared to occupy areas of the nucleus devoid of DAPI staining (arrows). In some cells (upper panels) the nucleus was well defined, and it was possible to distinguish between inclusions localized inside and outside the nucleus. In other cells (lower panels), the inclusions were more diffuse, the nucleus was distorted, and it was difficult to discern the boundaries between the nuclear and cytoplasmic compartments. (B) mycRab24(D123I) inclusions were localized with rabbit anti-myc polyclonal antibody followed by FITC-conjugated GAR IgG. The nuclear envelope was highlighted by co-staining with a monoclonal antibody against lamins A and B, followed by rhodamine-conjugated GAM IgG. In some cells with immunoreactive inclusions (upper panels) a well defined nuclear envelope was visible, whereas in others (bottom panels) the nuclear envelope appeared to be completely disrupted. Where both cytoplasmic and nuclear inclusions were present (top and middle panels), it appeared that the myc-reactive aggregates inside the nucleus contained lamins (arrows), while those outside the nucleus did not (arrowheads). For comparison, panel C shows the nuclear lamin staining typically observed in non-transfected cells. The scale bar represents 10 microns.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC130051&req=5

Figure 4: Disruption of nuclear architecture by Rab24(D123I) inclusions. NIH 3T3 cells expressing mycRab24(D123I) were processed for immunofluorescence microscopy 24 h after transfection. (A) The inclusions detected by staining with the anti-myc monoclonal antibody appeared to occupy areas of the nucleus devoid of DAPI staining (arrows). In some cells (upper panels) the nucleus was well defined, and it was possible to distinguish between inclusions localized inside and outside the nucleus. In other cells (lower panels), the inclusions were more diffuse, the nucleus was distorted, and it was difficult to discern the boundaries between the nuclear and cytoplasmic compartments. (B) mycRab24(D123I) inclusions were localized with rabbit anti-myc polyclonal antibody followed by FITC-conjugated GAR IgG. The nuclear envelope was highlighted by co-staining with a monoclonal antibody against lamins A and B, followed by rhodamine-conjugated GAM IgG. In some cells with immunoreactive inclusions (upper panels) a well defined nuclear envelope was visible, whereas in others (bottom panels) the nuclear envelope appeared to be completely disrupted. Where both cytoplasmic and nuclear inclusions were present (top and middle panels), it appeared that the myc-reactive aggregates inside the nucleus contained lamins (arrows), while those outside the nucleus did not (arrowheads). For comparison, panel C shows the nuclear lamin staining typically observed in non-transfected cells. The scale bar represents 10 microns.
Mentions: The accumulation of nuclear inclusions in cells expressing Rab24(D123I) led to progressive degeneration of nuclear structure. Figure 4 shows a series of representative photographs of transfected 3T3 cells that were co-stained with antibody against the Rab24 myc epitope, combined with DAPI to visualize the chromatin (Fig. 4A), or an antibody against lamins A and B to visualize the nuclear envelope (Fig. 4B). At 24 h after transfection, we observed many cells with mycRab24-positive inclusions that showed fairly uniform staining of the nuclear envelope with no loss of chromatin or deformation of nuclear shape. However, in some cells with larger and more numerous inclusions we observed discontinuities in the anti-lamin staining of the nuclear envelope and some co-localization of lamin with mycRab24(D123I) in the intranuclear aggregates. The gaps in the nuclear membrane were clearly visible in electron micrographs of cells with large numbers of intranuclear aggregates (Fig. 3C). In the most extreme cases, the nuclear envelope was significantly distorted and fragmented and there was leakage of chromatin into the cytoplasm (Fig. 4A &4B, lower panels). The number of cells with obvious nuclear disruption increased with time, so that by 48-h this was seen in the majority of the transfected cells (not shown).

Bottom Line: Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form.If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope.However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, OH 43614, USA. wmaltese@mco.edu

ABSTRACT

Background: Members of the Rab GTPase family regulate intracellular protein trafficking, but the specific function of Rab24 remains unknown. Several attributes distinguish this protein from other members of the Rab family, including a low intrinsic GTPase activity.

Results: The functions of other Rab proteins have been defined through the use of dominant-negative mutants with amino acid substitutions in the conserved N(T)KxD nucleotide binding motif. Surprisingly, when such Rab24 constructs were expressed in cultured cells, they accumulated in nuclear inclusions which disrupted the integrity of the nuclear envelope. The inclusions reacted positively with antibodies against ubiquitin and Hsp70, similar to protein aggregates observed in polyglutamine disorders. They also appeared to sequester importin-beta and GFP-coupled glucocorticoid receptor. Other Rab GTPases with similar mutations in the N(T)KxD motif were never found in inclusions, suggesting that the unusual localization of Rab24 is not related solely to misfolding of its nucleotide-free form. Studies with Rab24/Rab1B chimeras indicated that targeting of the mutant protein to inclusions requires the unique C-terminal domain of Rab24.

Conclusion: These studies demonstrate that mutations in Rab24 can trigger a cytopathic cellular response involving accumulation of nuclear inclusions. If the N(T)KxD mutants of Rab24 function as dominant suppressors, these studies may point to a unique role for Rab24 in degradation of misfolded cellular proteins or trafficking of proteins to the nuclear envelope. However, we cannot yet eliminate the possibility that these phenomena are related to unusual non-physiological protein interactions with the mutant form of Rab24.

Show MeSH
Related in: MedlinePlus