Limits...
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.

Show MeSH

Related in: MedlinePlus

Knockdown of SUN2 expression attenuates transferrin uptake.(A) HeLa cells were transiently transfected with either pEGFP or pSUPER-EGFP-SUN2. SUN2 expression was significantly downregulated in cells transfected with pSUPER-EGFP-SUN2 as shown by Western blotting. (B) Texas Red labeled-Tf accumulation in HeLa cells transfected with the indicated plasmids. Fluorescent Tf in pEGFP transfected HeLa cells (arrows, top panel) was at a similar intensity as in nontransfected cells. Tf accumulation increased remarkably in cells transfected with pEGFP-SUN2 (open arrows, middle panel) and decreased in some but not all cells transfected with pSUPER-EGFP-SUN2 (arrowheads, bottom panel). Scale bar, 20 µm. (C) Statistical analysis was used to compare the effect of SUN2 on Tf uptake. Each group represents the mean ± S.E.M. of three independent experiments. *P<0.05, ***P<0.001, one way ANOVA with post Tukey test.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3105078&req=5

pone-0020507-g008: Knockdown of SUN2 expression attenuates transferrin uptake.(A) HeLa cells were transiently transfected with either pEGFP or pSUPER-EGFP-SUN2. SUN2 expression was significantly downregulated in cells transfected with pSUPER-EGFP-SUN2 as shown by Western blotting. (B) Texas Red labeled-Tf accumulation in HeLa cells transfected with the indicated plasmids. Fluorescent Tf in pEGFP transfected HeLa cells (arrows, top panel) was at a similar intensity as in nontransfected cells. Tf accumulation increased remarkably in cells transfected with pEGFP-SUN2 (open arrows, middle panel) and decreased in some but not all cells transfected with pSUPER-EGFP-SUN2 (arrowheads, bottom panel). Scale bar, 20 µm. (C) Statistical analysis was used to compare the effect of SUN2 on Tf uptake. Each group represents the mean ± S.E.M. of three independent experiments. *P<0.05, ***P<0.001, one way ANOVA with post Tukey test.

Mentions: As Rab5 is a key molecule in endocytosis, we determined what role SUN2 plays in endocytosis. In a previous report, SUN2 has been shown to stimulate horseradish peroxidase accumulation when overexpressed. Conversely, addition of antiserum against SUN2 inhibited in vitro endosome fusion [16]. Here we transfected pSUPER-EGFP-SUN2 for expressing shRNA to knockdown SUN2 expression. Western blot analysis revealed that SUN2 expression was inhibited to a significant extent in transiently transfected HeLa cells (Fig. 8A). Next, transferrin uptake assay was employed. As shown in Fig. 8B, HeLa cells transfected with pEGFP-SUN2 showed a marked increase in Tf uptake, while most cells transfected with pSUPER-EGFP-SUN2 did not show an obvious decrease in the uptake of Tf compared to nontransfected cells. The immunofluorescence intensity of Tf inside each cell was further calculated by the confocal microscope software. Statistical analysis showed that overexpression of SUN2 significantly stimulated Tf internalization (P<0.001), while suppression of SUN2 expression attenuated the process (P<0.05) (Fig. 8C). Ideally, the experiment should be repeated in SUN2 knockout cells to see if a more significant effect than knockdown is revealed. The effects of forced and suppressed SUN2 expression on Tf uptake strongly suggest that SUN2 enhances receptor mediated endocytosis.


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

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

Knockdown of SUN2 expression attenuates transferrin uptake.(A) HeLa cells were transiently transfected with either pEGFP or pSUPER-EGFP-SUN2. SUN2 expression was significantly downregulated in cells transfected with pSUPER-EGFP-SUN2 as shown by Western blotting. (B) Texas Red labeled-Tf accumulation in HeLa cells transfected with the indicated plasmids. Fluorescent Tf in pEGFP transfected HeLa cells (arrows, top panel) was at a similar intensity as in nontransfected cells. Tf accumulation increased remarkably in cells transfected with pEGFP-SUN2 (open arrows, middle panel) and decreased in some but not all cells transfected with pSUPER-EGFP-SUN2 (arrowheads, bottom panel). Scale bar, 20 µm. (C) Statistical analysis was used to compare the effect of SUN2 on Tf uptake. Each group represents the mean ± S.E.M. of three independent experiments. *P<0.05, ***P<0.001, one way ANOVA with post Tukey test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020507-g008: Knockdown of SUN2 expression attenuates transferrin uptake.(A) HeLa cells were transiently transfected with either pEGFP or pSUPER-EGFP-SUN2. SUN2 expression was significantly downregulated in cells transfected with pSUPER-EGFP-SUN2 as shown by Western blotting. (B) Texas Red labeled-Tf accumulation in HeLa cells transfected with the indicated plasmids. Fluorescent Tf in pEGFP transfected HeLa cells (arrows, top panel) was at a similar intensity as in nontransfected cells. Tf accumulation increased remarkably in cells transfected with pEGFP-SUN2 (open arrows, middle panel) and decreased in some but not all cells transfected with pSUPER-EGFP-SUN2 (arrowheads, bottom panel). Scale bar, 20 µm. (C) Statistical analysis was used to compare the effect of SUN2 on Tf uptake. Each group represents the mean ± S.E.M. of three independent experiments. *P<0.05, ***P<0.001, one way ANOVA with post Tukey test.
Mentions: As Rab5 is a key molecule in endocytosis, we determined what role SUN2 plays in endocytosis. In a previous report, SUN2 has been shown to stimulate horseradish peroxidase accumulation when overexpressed. Conversely, addition of antiserum against SUN2 inhibited in vitro endosome fusion [16]. Here we transfected pSUPER-EGFP-SUN2 for expressing shRNA to knockdown SUN2 expression. Western blot analysis revealed that SUN2 expression was inhibited to a significant extent in transiently transfected HeLa cells (Fig. 8A). Next, transferrin uptake assay was employed. As shown in Fig. 8B, HeLa cells transfected with pEGFP-SUN2 showed a marked increase in Tf uptake, while most cells transfected with pSUPER-EGFP-SUN2 did not show an obvious decrease in the uptake of Tf compared to nontransfected cells. The immunofluorescence intensity of Tf inside each cell was further calculated by the confocal microscope software. Statistical analysis showed that overexpression of SUN2 significantly stimulated Tf internalization (P<0.001), while suppression of SUN2 expression attenuated the process (P<0.05) (Fig. 8C). Ideally, the experiment should be repeated in SUN2 knockout cells to see if a more significant effect than knockdown is revealed. The effects of forced and suppressed SUN2 expression on Tf uptake strongly suggest that SUN2 enhances receptor mediated endocytosis.

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