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Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane.

Zervas CG, Gregory SL, Brown NH - J. Cell Biol. (2001)

Bottom Line: Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B.ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters.Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK.

View Article: PubMed Central - PubMed

Affiliation: Wellcome/CRC Institute and Department of Anatomy, University of Cambridge, Cambridge CB2 1QR, United Kingdom.

ABSTRACT
Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

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ILK colocalizes with integrins during embryonic development. (a–e) Colocalization of ILK-GFP and PS integrins. ILK-GFP is visualized by GFP fluorescence (green) and the βPS subunit is detected with a monoclonal antibody (red), with colocalization appearing yellow. Anterior is left in this and all subsequent panels. (a) Dorsolateral view of an embryo at stage 13 during dorsal closure, where ILK-GFP and integrins are concentrated at the leading edges of epidermis and the edges of the amnioserosa (as). (b) Optical horizontal section of mid-stage 16 embryo focused on the internal organs to show the localization of ILK in pharyngeal muscles (pm) and visceral mesoderm (vm), with particularly strong expression of ILK seen here at the first midgut constriction (con), and at muscle attachment sites (ma). (c) Lateral view of a late stage 16 embryo showing ILK-GFP localization at muscle attachment sites. (d and e) Same embryo at higher magnification to show the tight colocalization of ILK with integrins (d) and the integrin staining alone (e). Bars, 20 μm.
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Figure 3: ILK colocalizes with integrins during embryonic development. (a–e) Colocalization of ILK-GFP and PS integrins. ILK-GFP is visualized by GFP fluorescence (green) and the βPS subunit is detected with a monoclonal antibody (red), with colocalization appearing yellow. Anterior is left in this and all subsequent panels. (a) Dorsolateral view of an embryo at stage 13 during dorsal closure, where ILK-GFP and integrins are concentrated at the leading edges of epidermis and the edges of the amnioserosa (as). (b) Optical horizontal section of mid-stage 16 embryo focused on the internal organs to show the localization of ILK in pharyngeal muscles (pm) and visceral mesoderm (vm), with particularly strong expression of ILK seen here at the first midgut constriction (con), and at muscle attachment sites (ma). (c) Lateral view of a late stage 16 embryo showing ILK-GFP localization at muscle attachment sites. (d and e) Same embryo at higher magnification to show the tight colocalization of ILK with integrins (d) and the integrin staining alone (e). Bars, 20 μm.

Mentions: Our finding that ilk mRNA is expressed mainly in mesodermal tissues was confirmed by the localization of ILK-GFP protein. However, we also found that low levels of ILK are distributed throughout the embryo. Some of these correspond to sites of integrin expression, such as at the leading edge of the epidermis and amnioserosa during dorsal closure (Fig. 3 a). Strong expression of ILK-GFP in the visceral mesoderm is first detected at stage 12, and it accumulates steadily during embryogenesis, following the level of mRNA expression. In mid–stage 16 embryos, ILK-GFP is particularly strong in the midgut constrictions and the pharyngeal muscles (Fig. 3 b). Low levels of ILK-GFP were found in the ventral nerve cord (not shown) and throughout the epidermis (Fig. 3, a and c). The most striking feature of ILK localization during embryogenesis is its tight localization at muscle attachment sites (Fig. 3 c), where PS integrins are strongly expressed (e; Bogaert et al. 1987). These data show that high levels of ILK-GFP are found at the places where integrins are found and the two proteins are tightly colocalized (Fig. 3 d). There is not strong expression of ILK at epidermal sites where wingless signaling through β-catenin is particularly active (Peifer et al. 1994), but low levels of ILK are detectable throughout the embryo, so this expression pattern does not exclude the possibility of the suggested interaction between ILK and β-catenin/T cell factor signaling (Novak et al. 1998) occurring in Drosophila.


Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane.

Zervas CG, Gregory SL, Brown NH - J. Cell Biol. (2001)

ILK colocalizes with integrins during embryonic development. (a–e) Colocalization of ILK-GFP and PS integrins. ILK-GFP is visualized by GFP fluorescence (green) and the βPS subunit is detected with a monoclonal antibody (red), with colocalization appearing yellow. Anterior is left in this and all subsequent panels. (a) Dorsolateral view of an embryo at stage 13 during dorsal closure, where ILK-GFP and integrins are concentrated at the leading edges of epidermis and the edges of the amnioserosa (as). (b) Optical horizontal section of mid-stage 16 embryo focused on the internal organs to show the localization of ILK in pharyngeal muscles (pm) and visceral mesoderm (vm), with particularly strong expression of ILK seen here at the first midgut constriction (con), and at muscle attachment sites (ma). (c) Lateral view of a late stage 16 embryo showing ILK-GFP localization at muscle attachment sites. (d and e) Same embryo at higher magnification to show the tight colocalization of ILK with integrins (d) and the integrin staining alone (e). Bars, 20 μm.
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Related In: Results  -  Collection

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Figure 3: ILK colocalizes with integrins during embryonic development. (a–e) Colocalization of ILK-GFP and PS integrins. ILK-GFP is visualized by GFP fluorescence (green) and the βPS subunit is detected with a monoclonal antibody (red), with colocalization appearing yellow. Anterior is left in this and all subsequent panels. (a) Dorsolateral view of an embryo at stage 13 during dorsal closure, where ILK-GFP and integrins are concentrated at the leading edges of epidermis and the edges of the amnioserosa (as). (b) Optical horizontal section of mid-stage 16 embryo focused on the internal organs to show the localization of ILK in pharyngeal muscles (pm) and visceral mesoderm (vm), with particularly strong expression of ILK seen here at the first midgut constriction (con), and at muscle attachment sites (ma). (c) Lateral view of a late stage 16 embryo showing ILK-GFP localization at muscle attachment sites. (d and e) Same embryo at higher magnification to show the tight colocalization of ILK with integrins (d) and the integrin staining alone (e). Bars, 20 μm.
Mentions: Our finding that ilk mRNA is expressed mainly in mesodermal tissues was confirmed by the localization of ILK-GFP protein. However, we also found that low levels of ILK are distributed throughout the embryo. Some of these correspond to sites of integrin expression, such as at the leading edge of the epidermis and amnioserosa during dorsal closure (Fig. 3 a). Strong expression of ILK-GFP in the visceral mesoderm is first detected at stage 12, and it accumulates steadily during embryogenesis, following the level of mRNA expression. In mid–stage 16 embryos, ILK-GFP is particularly strong in the midgut constrictions and the pharyngeal muscles (Fig. 3 b). Low levels of ILK-GFP were found in the ventral nerve cord (not shown) and throughout the epidermis (Fig. 3, a and c). The most striking feature of ILK localization during embryogenesis is its tight localization at muscle attachment sites (Fig. 3 c), where PS integrins are strongly expressed (e; Bogaert et al. 1987). These data show that high levels of ILK-GFP are found at the places where integrins are found and the two proteins are tightly colocalized (Fig. 3 d). There is not strong expression of ILK at epidermal sites where wingless signaling through β-catenin is particularly active (Peifer et al. 1994), but low levels of ILK are detectable throughout the embryo, so this expression pattern does not exclude the possibility of the suggested interaction between ILK and β-catenin/T cell factor signaling (Novak et al. 1998) occurring in Drosophila.

Bottom Line: Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B.ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters.Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK.

View Article: PubMed Central - PubMed

Affiliation: Wellcome/CRC Institute and Department of Anatomy, University of Cambridge, Cambridge CB2 1QR, United Kingdom.

ABSTRACT
Integrin-linked kinase (ILK) was identified by its interaction with the cytoplasmic tail of human beta1 integrin and previous data suggest that ILK is a component of diverse signaling pathways, including integrin, Wnt, and protein kinase B. Here we show that the absence of ILK function in Drosophila causes defects similar to loss of integrin adhesion, but not similar to loss of these signaling pathways. ILK mutations cause embryonic lethality and defects in muscle attachment, and clones of cells lacking ILK in the adult wing fail to adhere, forming wing blisters. Consistent with this, an ILK-green fluorescent protein fusion protein colocalizes with the position-specific integrins at sites of integrin function: muscle attachment sites and the basal junctions of the wing epithelium. Surprisingly, mutations in the kinase domain shown to inactivate the kinase activity of human ILK do not show any phenotype in Drosophila, suggesting a kinase-independent function for ILK. The muscle detachment in ILK mutants is associated with detachment of the actin filaments from the muscle ends, unlike integrin mutants, in which the primary defect is detachment of the plasma membrane from the extracellular matrix. Our data suggest that ILK is a component of the structure linking the cytoskeleton and the plasma membrane at sites of integrin-mediated adhesion.

Show MeSH
Related in: MedlinePlus