Chapter 3 Sox2 interacts with Grouchos
3.2 Sox2 changes Groucho subcellular localization
Interaction between proteins can be tested by many methods including protein affinity chromatography, immunoprecipitation and yeast two hybrid assays. One rapid assay of protein interactions is to observe the subcellular localization change after co-expression in cells. Sox2 has three nuclear localization signals in its HMG DNA binding domain, therefore the majority of overexpressed Sox2 locate in the nucleus (Figure 3.2.1). The long forms of Groucho (Groucho 1-4) have casein kinase II/cdc2 phosphorylation sites and a nuclear localization sequence (CcN) which allows them also to locate to the nucleus, whereas Groucho5 lacks a nuclear localization signal and in both nuclear and cytoplasmic localization (Figure 3.2.2).
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Figure 3.2.1 Sox2 subcellular localiziation
COS7 cells were transfected with Sox2 by electroporation and seeded on poly-D-lysine coated coverslips. After 24 hours, the cells were immunostained using anti-Sox2 antibody. DAPI staining identified the nucleus of the cells. Sox2 was detected in the nucleus.
Figure 3.2.2 Groucho protein subcellular localization
COS7 cells were transfected with Myc-Groucho3 or Myc-Groucho5 by electorporation and seeded on poly-D-lysine coated coverslips. After 24 hours, the cells were immunostained using anti-Myc antibody. Groucho3 was detected in the nucleus and 10% of the Groucho3 stained cells exhibited nuclear bodies which did not co-localize with the nucleolus. Groucho5 was detected in the cytoplasm and nucleus, with a brighter staining in the nucleus than the cytoplasm. (N: Nucleolus; NB: Nuclear body)
71 According to the previous studies in our lab, the Sox3 and Groucho5 interaction was demonstrated using a nuclear translocation assay (PhD thesis, Zulfiqar Laghari, 2010). Here, the same experiment was carried out to investigate the interaction of Sox2 with Groucho5. As a positive control, COS7 cells were transfected with Sox3 and a Myc-tagged Groucho5 either alone or together and immunostaining was used to detect their subcellular localization (Figure 3.2.3). Sox3 localized to the nucleus and, also expressed alone, Myc- tagged Groucho5 was broadly distributed in cytoplasm and nucleus. When co- expressing both Sox3 and Groucho5, Groucho5 was mainly relocated to the nucleus together with Sox3 which implied an interaction with Sox3. These results agree with the previous studies in our lab.
Figure 3.2.3 Sox3 translocates Groucho5 to the nucleus
COS7 cells were transfected with Sox3 or Myc-Groucho5 by electorporation and seeded on poly-D-lysine coated coverslips. After 24 hours, the cells were immunostained using anti- Sox3 and anti-Myc antibody. When expressed alone, Sox3 was detected in the nucleus while Myc-Groucho5 was detected in the cytoplasm and nucleus, with a brighter staining in the nucleus than the cytoplasm. Co-expressing Sox3 with Groucho5 caused Groucho5 to translocates into nucleus.
72 A similar analysis was carried out for Sox2 and Groucho5 interaction. When transfected alone, Groucho5 was distributed throughout the cells but staining was more intense in the nuclei (Figure 3.2.4.A). When both Sox2 and Groucho5 were overexpressed in COS7 cells, the nuclear Groucho5 was still detected whereas the cytoplasmic Groucho5 was significantly reduced (Figure 3.2.4.B; p <0.001). This nuclear restricted localization of Groucho5 represents co-localization with Sox2. This co-localization of Sox2 and Groucho5 implied the interaction between two proteins. Quantification of these data demonstrated a distinct increase in the nucleus of cells that had nuclear only Groucho5 (Figure 3.2.4.C).
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Figure 3.2.4 Sox2 translocates cytoplasmic Grouch5 to the nucleus
COS7 cells were transfected with Sox2, Myc-Groucho3 or both by electorporation and seeded on poly-D-lysine coated coverslips. After 24 hours, the cells were immunostained using anti-Sox2, anti-Myc or both antibodies. (A.) When co-transfected with Sox2, Groucho5 was detected in the nucleus, but no Groucho5 was detected in the cytoplasm. (B.) Quantification of cytoplasmic Groucho5. The unsaturated Groucho5 immunostaining picture were analysed by ImageJ software. The pixel number of cytoplasm was calculated as whole cell pixel number minus the nucleus pixel number. 15 cell pictures were analysed per set. (C) Quantification of Groucho5 nuclear localization. 100 Groucho5-expressing cells were counted. The number of cells express no cytoplasmic Groucho5 were counted and the percentage was calculated.
A.
B.
74 In order to test the interaction between Sox2 with Groucho, a construct encoding Sox2 and/or Myc-tagged Groucho3 was introduced into cells either alone or together and the subcellular localization of the proteins produced was detected using immunostaining. When cells were transfected with Myc- Groucho3 construct alone, Myc-Groucho3 protein inclusion bodies were seen, which did not co-localize with the nucleolus. When both Sox2 and the Myc- Groucho3 were introduced into COS7 cells simultaneously, the inclusion bodies of Groucho3 stained brighter in the periphery of than the centre and Sox2 now also formed inclusion bodies which co-localized with Groucho3 (Figure 3.2.5.A). This subcellular localization change of both Sox2 and Groucho3 when co-expressed in the cell implied interaction between the two proteins. To further quantify the frequency of the phenomena, the cells with or without Groucho3 nuclear bodies were counted in cells transfected with Groucho3 alone or with both Groucho3 and Sox2 co-expressed. The results in presented in Figure 3.2.5.B as percentage of the dots formed in the nucleus. When Groucho3 was expressed alone, 1% of cells had Groucho3 inclusion bodies with bright peripheral staining. When Groucho3 was co-transfected with Sox2, approximately 16% of Groucho3 positive nuclear bodies were colocalized with Sox2 and exhibited bright peripheral staining.
To sum up, the subcellular localization change of Grouchos after introducing Sox2 into cells suggests some kind of connection between Sox2 and Grouchos
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Figure 3.2.5 Sox2 co-localized in the nucleus with Groucho3
COS7 cells were transfected with either Sox2, Myc-Groucho3 or both constructs by electroporation and seeded on poly-D-lysine coated coverslips. After 24 hours, the cells were immunostained using anti-Sox2, anti-Myc or both antibodies. (A) When co- transfected with Groucho3, Sox2 formed inclusion bodies which colocalized with Groucho3. Groucho3 was detected in the nucleus and the inclusion bodies of Groucho3 only stained in the periphery inclusion bodies with bright edge were counted. The number of cells expressing Groucho3 was counted and the percentage of inclusion bodies with bright edge was calculated (first column). The number of cells co-localized Sox2 and Groucho3 in inclusion bodies was counted and the percentage was calculated (second column).
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A.
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