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A novel mutation in β integrin reveals an integrin-mediated interaction between the extracellular matrix and cki-1/p27KIP1.

Kihira S, Yu EJ, Cunningham J, Cram EJ, Lee M - PLoS ONE (2012)

Bottom Line: RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization.These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell.Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Baylor University, Waco, Texas, United States of America.

ABSTRACT
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.

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CKI-1::GFP is localized to the nucleus and nucleolus in pat-3 transgenic rescued animals.CKI-1::GFP transgenic animals were examined using fluorescence microscopy. Panels A and B show mid-body regions of pat-3(+) and pat-3(sp) rescued animals, respectively. Arrows indicate the nuclei of hypodermal cells at early adult stages. Panels C and D depict a hypodermal nucleus in pat-3(+) and pat-3(sp) worms, respectively. CKI-1::GFP appeared to be nucleolar (arrow heads) in pat-3(+), while the CKI-1::GFP appeared clumped in pat-3(sp) nuclei (arrows). Scale bar = 50 µm.
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pone-0042425-g002: CKI-1::GFP is localized to the nucleus and nucleolus in pat-3 transgenic rescued animals.CKI-1::GFP transgenic animals were examined using fluorescence microscopy. Panels A and B show mid-body regions of pat-3(+) and pat-3(sp) rescued animals, respectively. Arrows indicate the nuclei of hypodermal cells at early adult stages. Panels C and D depict a hypodermal nucleus in pat-3(+) and pat-3(sp) worms, respectively. CKI-1::GFP appeared to be nucleolar (arrow heads) in pat-3(+), while the CKI-1::GFP appeared clumped in pat-3(sp) nuclei (arrows). Scale bar = 50 µm.

Mentions: Studies of the mammalian β1B and β1C integrins in mammalian cells revealed that expression of these integrins suppresses cell adhesion and proliferation [5] and upregulates the expression of p27KIP1[39]. To investigate a potential linkage between PAT-3 and CKI-1, the C. elegans homolog of mammalian p27KIP1[26], [29], [40], we created rescued transgenic lines containing pat-3(+) or pat-3(sp) genomic DNA by co-injecting with DNA encoding a CKI-1::GFP fusion protein [26], [34]. As previously described [26], [29], CKI-1::GFP is expressed in hypodermal cells of late L4 and young adult animals. Nuclear expression is observed within an ER meshwork, typical of the hypodermal syncytium (hyp 7) (Figure 2A and 2B) [41]. In pat-3(+) rescued animals, the appearance of CKI-1::GFP is a distinct fluorescent spot within a round green nucleus, suggesting nuclear and nucleolar localization (Figures 2A and 2C). To substantiate our interpretation that CKI-1::GFP localizes to the nucleolus, the ncl-1 gene, disruption of which results in enlarged nucleoli, was depleted using RNAi [42], [43]. In the pat-3(+) background, ncl-1 (RNAi) significantly increased the size of the CKI-1::GFP spot. ImageJ analysis showed that the size of the green spot was increased by 2.4 fold when compared to that of control RNAi animals (Figure S1, Table S1). Therefore, we conclude that the observed spots on the nuclei are likely to represent nucleolar localization.


A novel mutation in β integrin reveals an integrin-mediated interaction between the extracellular matrix and cki-1/p27KIP1.

Kihira S, Yu EJ, Cunningham J, Cram EJ, Lee M - PLoS ONE (2012)

CKI-1::GFP is localized to the nucleus and nucleolus in pat-3 transgenic rescued animals.CKI-1::GFP transgenic animals were examined using fluorescence microscopy. Panels A and B show mid-body regions of pat-3(+) and pat-3(sp) rescued animals, respectively. Arrows indicate the nuclei of hypodermal cells at early adult stages. Panels C and D depict a hypodermal nucleus in pat-3(+) and pat-3(sp) worms, respectively. CKI-1::GFP appeared to be nucleolar (arrow heads) in pat-3(+), while the CKI-1::GFP appeared clumped in pat-3(sp) nuclei (arrows). Scale bar = 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412830&req=5

pone-0042425-g002: CKI-1::GFP is localized to the nucleus and nucleolus in pat-3 transgenic rescued animals.CKI-1::GFP transgenic animals were examined using fluorescence microscopy. Panels A and B show mid-body regions of pat-3(+) and pat-3(sp) rescued animals, respectively. Arrows indicate the nuclei of hypodermal cells at early adult stages. Panels C and D depict a hypodermal nucleus in pat-3(+) and pat-3(sp) worms, respectively. CKI-1::GFP appeared to be nucleolar (arrow heads) in pat-3(+), while the CKI-1::GFP appeared clumped in pat-3(sp) nuclei (arrows). Scale bar = 50 µm.
Mentions: Studies of the mammalian β1B and β1C integrins in mammalian cells revealed that expression of these integrins suppresses cell adhesion and proliferation [5] and upregulates the expression of p27KIP1[39]. To investigate a potential linkage between PAT-3 and CKI-1, the C. elegans homolog of mammalian p27KIP1[26], [29], [40], we created rescued transgenic lines containing pat-3(+) or pat-3(sp) genomic DNA by co-injecting with DNA encoding a CKI-1::GFP fusion protein [26], [34]. As previously described [26], [29], CKI-1::GFP is expressed in hypodermal cells of late L4 and young adult animals. Nuclear expression is observed within an ER meshwork, typical of the hypodermal syncytium (hyp 7) (Figure 2A and 2B) [41]. In pat-3(+) rescued animals, the appearance of CKI-1::GFP is a distinct fluorescent spot within a round green nucleus, suggesting nuclear and nucleolar localization (Figures 2A and 2C). To substantiate our interpretation that CKI-1::GFP localizes to the nucleolus, the ncl-1 gene, disruption of which results in enlarged nucleoli, was depleted using RNAi [42], [43]. In the pat-3(+) background, ncl-1 (RNAi) significantly increased the size of the CKI-1::GFP spot. ImageJ analysis showed that the size of the green spot was increased by 2.4 fold when compared to that of control RNAi animals (Figure S1, Table S1). Therefore, we conclude that the observed spots on the nuclei are likely to represent nucleolar localization.

Bottom Line: RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization.These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell.Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Baylor University, Waco, Texas, United States of America.

ABSTRACT
The cell-extracellular matrix (ECM) interaction plays an essential role in maintaining tissue shapes and regulates cell behaviors such as cell adhesion, differentiation and proliferation. The mechanism by which the ECM influences the cell cycle in vivo is poorly understood. Here we demonstrate that the β integrin PAT-3 regulates the localization and expression of CKI-1, a C. elegans homologue of the cyclin dependent kinase inhibitor p27(KIP1). In nematodes expressing wild type PAT-3, CKI-1::GFP localizes primarily to nucleoli in hypodermal cells, whereas in animals expressing mutant pat-3 with a defective splice junction, CKI-1::GFP appears clumped and disorganized in nucleoplasm. RNAi analysis links cell adhesion genes to the regulation of CKI-1. RNAi of unc-52/perlecan, ina-1/α integrin, pat-4/ILK, and unc-97/PINCH resulted in abnormal CKI-1::GFP localization. Additional RNAi experiments revealed that the SCF E3 ubiquitin-ligase complex genes, skpt-1/SKP2, cul-1/CUL1 and lin-23/F-box, are required for the proper localization and expression of CKI-1, suggesting that integrin signaling and SCF E3 ligase work together to regulate the cellular distribution of CKI-1. These data also suggest that integrin plays a major role in maintaining proper CKI-1/p27(KIP1) levels in the cell. Perturbed integrin signaling may lead to the inhibition of SCF ligase activity, mislocalization and elevation of CKI-1/p27(KIP1). These results suggest that adhesion signaling is crucial for cell cycle regulation in vivo.

Show MeSH
Related in: MedlinePlus