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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 is required for coupling the actin cytoskeleton to the plasma membrane. Visualization of the plasma membrane (green) and actin filaments (red) of the muscles in embryos that are wild type (a), lack ILK [ilk1/Df(3L)Pc-14d] (b), or lack the PS2 integrin (if B4/Y) (c). Lateral views are shown of the ventral longitudinal muscles of embryos at stage 17 (a and b) or 16 (c). The detachment of actin from the plasma membrane in the absence of ILK is shown by an arrow in b, and the detachment of the muscle plasma membrane is shown by dashed arrows in b and c. The plasma membrane is labeled with Src-GFP (green) and the filamentous actin with rhodamine-labeled phalloidin (red). Bar, 10 μm.
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Figure 8: ILK is required for coupling the actin cytoskeleton to the plasma membrane. Visualization of the plasma membrane (green) and actin filaments (red) of the muscles in embryos that are wild type (a), lack ILK [ilk1/Df(3L)Pc-14d] (b), or lack the PS2 integrin (if B4/Y) (c). Lateral views are shown of the ventral longitudinal muscles of embryos at stage 17 (a and b) or 16 (c). The detachment of actin from the plasma membrane in the absence of ILK is shown by an arrow in b, and the detachment of the muscle plasma membrane is shown by dashed arrows in b and c. The plasma membrane is labeled with Src-GFP (green) and the filamentous actin with rhodamine-labeled phalloidin (red). Bar, 10 μm.

Mentions: There are a number of possible roles that ILK could have in the generation of the normal actin configuration within the muscles. It could be required for the extracellular adhesion of the muscle to the ECM, for example by modifying integrin adhesion to the ECM, or it could be required for the link between the actin filaments and the intracellular face of the muscle membrane. To distinguish between these two possibilities, we marked the plasma membrane of muscle cells so that we could examine the membrane attachment to the ECM as well as the actin filaments in the muscle cells. We used GFP fused to the NH2-terminal myristylation signal of Src kinase to mark the membrane (Kaltschmidt et al. 2000). In wild-type embryos, the actin filaments extend to the very ends of the muscles so that the signal from rhodamine-phalloidin and Src-GFP overlap (Fig. 8 a). By contrast, in the ilk1/Df(3L)Pc-14d mutant embryos, we see muscles where the actin has detached from the plasma membrane and the membrane remains attached at its normal position adjacent to the ECM (Fig. 8 b). This is visible because the filamentous actin retracts to one end of the muscle, presumably due to the contraction of the actin/myosin fibers. We also see muscles where both the plasma membrane and actin has retracted, although they are still separate. This detachment of the membrane from the ECM-containing attachment site is seen in muscles lacking PS integrin function, but in that case the actin filaments are still anchored to the membrane (Fig. 8 c). Thus, both ILK and integrins are required for the ECM–cytoskeletal link, but the point at which breakage occurs differs: in the absence of integrins, the membrane pulls away from the ECM as a severe, early defect; whereas, in the absence of ILK, the cytoskeleton also pulls away from the membrane as a later defect after the integrins have been localized and bound ECM (Fig. 9). This phenotype allows a clear distinction between the two possible roles for ILK in muscle attachment: ILK is not required for the stage 16 adhesion of integrins to the ECM, but instead is required later to maintain the link between the contractile actin filaments and the plasma membrane at the ends of the muscles.


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 is required for coupling the actin cytoskeleton to the plasma membrane. Visualization of the plasma membrane (green) and actin filaments (red) of the muscles in embryos that are wild type (a), lack ILK [ilk1/Df(3L)Pc-14d] (b), or lack the PS2 integrin (if B4/Y) (c). Lateral views are shown of the ventral longitudinal muscles of embryos at stage 17 (a and b) or 16 (c). The detachment of actin from the plasma membrane in the absence of ILK is shown by an arrow in b, and the detachment of the muscle plasma membrane is shown by dashed arrows in b and c. The plasma membrane is labeled with Src-GFP (green) and the filamentous actin with rhodamine-labeled phalloidin (red). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 8: ILK is required for coupling the actin cytoskeleton to the plasma membrane. Visualization of the plasma membrane (green) and actin filaments (red) of the muscles in embryos that are wild type (a), lack ILK [ilk1/Df(3L)Pc-14d] (b), or lack the PS2 integrin (if B4/Y) (c). Lateral views are shown of the ventral longitudinal muscles of embryos at stage 17 (a and b) or 16 (c). The detachment of actin from the plasma membrane in the absence of ILK is shown by an arrow in b, and the detachment of the muscle plasma membrane is shown by dashed arrows in b and c. The plasma membrane is labeled with Src-GFP (green) and the filamentous actin with rhodamine-labeled phalloidin (red). Bar, 10 μm.
Mentions: There are a number of possible roles that ILK could have in the generation of the normal actin configuration within the muscles. It could be required for the extracellular adhesion of the muscle to the ECM, for example by modifying integrin adhesion to the ECM, or it could be required for the link between the actin filaments and the intracellular face of the muscle membrane. To distinguish between these two possibilities, we marked the plasma membrane of muscle cells so that we could examine the membrane attachment to the ECM as well as the actin filaments in the muscle cells. We used GFP fused to the NH2-terminal myristylation signal of Src kinase to mark the membrane (Kaltschmidt et al. 2000). In wild-type embryos, the actin filaments extend to the very ends of the muscles so that the signal from rhodamine-phalloidin and Src-GFP overlap (Fig. 8 a). By contrast, in the ilk1/Df(3L)Pc-14d mutant embryos, we see muscles where the actin has detached from the plasma membrane and the membrane remains attached at its normal position adjacent to the ECM (Fig. 8 b). This is visible because the filamentous actin retracts to one end of the muscle, presumably due to the contraction of the actin/myosin fibers. We also see muscles where both the plasma membrane and actin has retracted, although they are still separate. This detachment of the membrane from the ECM-containing attachment site is seen in muscles lacking PS integrin function, but in that case the actin filaments are still anchored to the membrane (Fig. 8 c). Thus, both ILK and integrins are required for the ECM–cytoskeletal link, but the point at which breakage occurs differs: in the absence of integrins, the membrane pulls away from the ECM as a severe, early defect; whereas, in the absence of ILK, the cytoskeleton also pulls away from the membrane as a later defect after the integrins have been localized and bound ECM (Fig. 9). This phenotype allows a clear distinction between the two possible roles for ILK in muscle attachment: ILK is not required for the stage 16 adhesion of integrins to the ECM, but instead is required later to maintain the link between the contractile actin filaments and the plasma membrane at the ends of the muscles.

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