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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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SUN2 retention on the NE is dependent on lamin A.(A) Immunofluorescence analysis of SUN2 in embryonic fibroblasts derived from wild-type (+/+) and Lmna knockout (−/−) mice. SUN2 appeared as a rim-like shape around the nucleus in wild-type cells (left). While in Lmna−/− cells, aberrant distribution of SUN2 was detected in the cytoplasm (middle, arrow) and the irregularly shaped nucleus (right, arrowhead). (B) In the EGFP-Lamin A positive Lmna−/− embryonic fibroblast, SUN2 (red) restored its rim-like shape around the nucleus (arrow), whereas SUN2 localized predominantly in the cytoplasm of the lamin A negative cell (arrowhead). (C) SUN2 expression (green) remained aberrant in the Lmna−/− cell transfected with pDsRed-laminC (arrow). Nuclei were stained with DAPI. Scale bars, 20 µm.
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pone-0020507-g002: SUN2 retention on the NE is dependent on lamin A.(A) Immunofluorescence analysis of SUN2 in embryonic fibroblasts derived from wild-type (+/+) and Lmna knockout (−/−) mice. SUN2 appeared as a rim-like shape around the nucleus in wild-type cells (left). While in Lmna−/− cells, aberrant distribution of SUN2 was detected in the cytoplasm (middle, arrow) and the irregularly shaped nucleus (right, arrowhead). (B) In the EGFP-Lamin A positive Lmna−/− embryonic fibroblast, SUN2 (red) restored its rim-like shape around the nucleus (arrow), whereas SUN2 localized predominantly in the cytoplasm of the lamin A negative cell (arrowhead). (C) SUN2 expression (green) remained aberrant in the Lmna−/− cell transfected with pDsRed-laminC (arrow). Nuclei were stained with DAPI. Scale bars, 20 µm.

Mentions: Lamins are composed of A and B types [22]. A-type lamins, including lamin A and C, are alternative splicing products of Lmna at its 3′end [23]. Lamin A/C is required for the NE localization of SUN2 and we substantiate this by analyzing the distribution patterns of SUN2 in primary embryonic fibroblasts derived from wild-type and homozygous Lmna−/− mice [24]. In wild-type cells, SUN2 was localized mainly on the NE (Fig. 2A, left). In Lmna−/− cells, we observed three aberrant distribution patterns of SUN2, (1) streaks of SUN2 in the cytoplasm (Fig. 2A, arrow); (2) nuclear SUN2 was expressed but lost from one pole of the irregularly shaped nucleus (Fig. 2A, arrowhead); (3) same intensity of SUN2 staining in the nucleus and cytoplasm (not shown). The loss of expression from one pole of the nucleus in Lmna−/− cells has also been reported for lamin-associated protein 2, lamin B and Nup153 [24]. From three independent experiments, the percentage distribution of these three patterns was 56±7.6 %, 17±2.8 % and 14±3.1 % (100 cells were scored each time). In the remaining 13±2.6 % of the cells, the typical NE localization of SUN2 was observed, but the expression level was generally weaker than that observed in the wild-type. We further determined whether lamin A/C could reverse the aberrant distribution of SUN2 in Lmna−/− cells. In around 50 % of lamin A positive cells after transfection, SUN2 displayed the typical NE localization pattern (Fig. 2B, arrow), while the displacement of SUN2 was not reverted in the lamin A negative cell (Fig. 2B, arrowhead). In contrast, exogenous lamin C failed to restore the NE localization of SUN2 or itself in the lamin A negative cell (Fig. 2C, arrow).


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

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

SUN2 retention on the NE is dependent on lamin A.(A) Immunofluorescence analysis of SUN2 in embryonic fibroblasts derived from wild-type (+/+) and Lmna knockout (−/−) mice. SUN2 appeared as a rim-like shape around the nucleus in wild-type cells (left). While in Lmna−/− cells, aberrant distribution of SUN2 was detected in the cytoplasm (middle, arrow) and the irregularly shaped nucleus (right, arrowhead). (B) In the EGFP-Lamin A positive Lmna−/− embryonic fibroblast, SUN2 (red) restored its rim-like shape around the nucleus (arrow), whereas SUN2 localized predominantly in the cytoplasm of the lamin A negative cell (arrowhead). (C) SUN2 expression (green) remained aberrant in the Lmna−/− cell transfected with pDsRed-laminC (arrow). Nuclei were stained with DAPI. Scale bars, 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105078&req=5

pone-0020507-g002: SUN2 retention on the NE is dependent on lamin A.(A) Immunofluorescence analysis of SUN2 in embryonic fibroblasts derived from wild-type (+/+) and Lmna knockout (−/−) mice. SUN2 appeared as a rim-like shape around the nucleus in wild-type cells (left). While in Lmna−/− cells, aberrant distribution of SUN2 was detected in the cytoplasm (middle, arrow) and the irregularly shaped nucleus (right, arrowhead). (B) In the EGFP-Lamin A positive Lmna−/− embryonic fibroblast, SUN2 (red) restored its rim-like shape around the nucleus (arrow), whereas SUN2 localized predominantly in the cytoplasm of the lamin A negative cell (arrowhead). (C) SUN2 expression (green) remained aberrant in the Lmna−/− cell transfected with pDsRed-laminC (arrow). Nuclei were stained with DAPI. Scale bars, 20 µm.
Mentions: Lamins are composed of A and B types [22]. A-type lamins, including lamin A and C, are alternative splicing products of Lmna at its 3′end [23]. Lamin A/C is required for the NE localization of SUN2 and we substantiate this by analyzing the distribution patterns of SUN2 in primary embryonic fibroblasts derived from wild-type and homozygous Lmna−/− mice [24]. In wild-type cells, SUN2 was localized mainly on the NE (Fig. 2A, left). In Lmna−/− cells, we observed three aberrant distribution patterns of SUN2, (1) streaks of SUN2 in the cytoplasm (Fig. 2A, arrow); (2) nuclear SUN2 was expressed but lost from one pole of the irregularly shaped nucleus (Fig. 2A, arrowhead); (3) same intensity of SUN2 staining in the nucleus and cytoplasm (not shown). The loss of expression from one pole of the nucleus in Lmna−/− cells has also been reported for lamin-associated protein 2, lamin B and Nup153 [24]. From three independent experiments, the percentage distribution of these three patterns was 56±7.6 %, 17±2.8 % and 14±3.1 % (100 cells were scored each time). In the remaining 13±2.6 % of the cells, the typical NE localization of SUN2 was observed, but the expression level was generally weaker than that observed in the wild-type. We further determined whether lamin A/C could reverse the aberrant distribution of SUN2 in Lmna−/− cells. In around 50 % of lamin A positive cells after transfection, SUN2 displayed the typical NE localization pattern (Fig. 2B, arrow), while the displacement of SUN2 was not reverted in the lamin A negative cell (Fig. 2B, arrowhead). In contrast, exogenous lamin C failed to restore the NE localization of SUN2 or itself in the lamin A negative cell (Fig. 2C, arrow).

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