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Subcellular localization of SUN2 is regulated by lamin A and Rab5.

Liang Y, Chiu PH, Yip KY, Chan SY - PLoS ONE (2011)

Bottom Line: We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2.Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process.These findings support a role of SUN2 in endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong SAR, China.

ABSTRACT
SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis.

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Endogenous SUN2 and Rab5 colocalize.(A) Western blot analysis of HeLa cell fractions separated by sucrose density gradient centrifugation with the antibodies indicated on the left. Twelve 1 ml fractions were collected from the top (fraction 1) to the bottom of the gradient. SUN2 and Rab5 coexisted in fractions 2 and 3. (B) HeLa cells were serum starved for 3 hours and then serum was added to induce endocytosis. Confocal images of double staining for SUN2 and Rab5 were taken before and 10 minutes after serum addition. Colocalization is indicated by arrows. Scale bar, 10 µm.
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pone-0020507-g007: Endogenous SUN2 and Rab5 colocalize.(A) Western blot analysis of HeLa cell fractions separated by sucrose density gradient centrifugation with the antibodies indicated on the left. Twelve 1 ml fractions were collected from the top (fraction 1) to the bottom of the gradient. SUN2 and Rab5 coexisted in fractions 2 and 3. (B) HeLa cells were serum starved for 3 hours and then serum was added to induce endocytosis. Confocal images of double staining for SUN2 and Rab5 were taken before and 10 minutes after serum addition. Colocalization is indicated by arrows. Scale bar, 10 µm.

Mentions: We moved on to investigate whether endogenous SUN2 and Rab5 colocalize. In subcellular fractionation experiment using sucrose float-up gradients, the early endosome marker EEA1 is concentrated in the dense fractions 10–12 whereas the top fractions 1–3 are highly enriched in Golgi apparatus, endosomes and associated vesicles. Localization of SUN2 and Rab5 overlapped in fractions 2 and 3 of nontransfected HeLa cells, indicating that partial colocalization of the two proteins in vesicles is possible (Fig. 7A). To initiate endocytosis, we serum starved the cells for 3 hours and then added 10% serum. Interestingly, the rim-like NE SUN2 staining decreased and prominent vesicle SUN2 staining appeared when cells were serum starved (Fig. 7B). Shortly after feeding with 10% serum, colocalization between endogenous SUN2 and Rab5 could be found in some vesicles (Fig. 7B, arrows).


Subcellular localization of SUN2 is regulated by lamin A and Rab5.

Liang Y, Chiu PH, Yip KY, Chan SY - PLoS ONE (2011)

Endogenous SUN2 and Rab5 colocalize.(A) Western blot analysis of HeLa cell fractions separated by sucrose density gradient centrifugation with the antibodies indicated on the left. Twelve 1 ml fractions were collected from the top (fraction 1) to the bottom of the gradient. SUN2 and Rab5 coexisted in fractions 2 and 3. (B) HeLa cells were serum starved for 3 hours and then serum was added to induce endocytosis. Confocal images of double staining for SUN2 and Rab5 were taken before and 10 minutes after serum addition. Colocalization is indicated by arrows. Scale bar, 10 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105078&req=5

pone-0020507-g007: Endogenous SUN2 and Rab5 colocalize.(A) Western blot analysis of HeLa cell fractions separated by sucrose density gradient centrifugation with the antibodies indicated on the left. Twelve 1 ml fractions were collected from the top (fraction 1) to the bottom of the gradient. SUN2 and Rab5 coexisted in fractions 2 and 3. (B) HeLa cells were serum starved for 3 hours and then serum was added to induce endocytosis. Confocal images of double staining for SUN2 and Rab5 were taken before and 10 minutes after serum addition. Colocalization is indicated by arrows. Scale bar, 10 µm.
Mentions: We moved on to investigate whether endogenous SUN2 and Rab5 colocalize. In subcellular fractionation experiment using sucrose float-up gradients, the early endosome marker EEA1 is concentrated in the dense fractions 10–12 whereas the top fractions 1–3 are highly enriched in Golgi apparatus, endosomes and associated vesicles. Localization of SUN2 and Rab5 overlapped in fractions 2 and 3 of nontransfected HeLa cells, indicating that partial colocalization of the two proteins in vesicles is possible (Fig. 7A). To initiate endocytosis, we serum starved the cells for 3 hours and then added 10% serum. Interestingly, the rim-like NE SUN2 staining decreased and prominent vesicle SUN2 staining appeared when cells were serum starved (Fig. 7B). Shortly after feeding with 10% serum, colocalization between endogenous SUN2 and Rab5 could be found in some vesicles (Fig. 7B, arrows).

Bottom Line: We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2.Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process.These findings support a role of SUN2 in endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong SAR, China.

ABSTRACT
SUN2 is an inner nuclear membrane protein with a conserved Sad1/UNC-84 homology SUN-domain at the C-terminus. Intriguingly, SUN2 has also been reported to interact with Rab5, which localizes in early endosomes. To clarify the dual subcellular localization of SUN2, we investigated its localization in lamin A/C deficient cells rescued with lamin A or lamin C isoform, and in HeLa cells transfected with Rab5 or its mutants. We found that expression of lamin A but not lamin C partly restored the nuclear envelope localization of SUN2. SUN2 was redistributed to endosomes upon overexpression of Rab5, but remained on the nuclear envelope when the SUN domain was deleted. To explore the physiological function of SUN2 in vesicle trafficking and endocytosis, we demonstrated the colocalization of endogenous SUN2 and Rab5. Moreover, overexpression of SUN2 stimulated the uptake of transferrin while suppression of SUN2 expression attenuated the process. These findings support a role of SUN2 in endocytosis.

Show MeSH
Related in: MedlinePlus