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Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

Spicer E, Suckert C, Al-Attar H, Marsden M - PLoS ONE (2010)

Bottom Line: We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling.Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells.These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.

ABSTRACT
During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

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Kermit2 regulates internalization of antibody bound α5β1 integrin.Xenopus A6 cells were transfected with (A) GFP-tagged kermit2, or (B) GFP-tagged kermit2mut and the endocytosis of antibody labeled α5β1 was estimated from fluorescent intensity using the density slice function of Openlab. (C, D) Staining of internalized α5β1 with fluorescent anti-mouse antibody. Non-transfected cells in the same dish act as controls. Insets in C and D represent 25 µM2 ROI's used to estimate pixel densities. (A, C) Kermit2 transfected cells have 827.3±23.0 pixels/ROI as compared to control cell ROI pixel densities of 841.5±13.3 pixels/ROI. (B, D) In kermit2mut transfected cells the ROI pixel density is 530.8±51.2, while in non-transfected cells from the same dish have an average pixel density of 832.5±24.3 pixels/ROI. Pixel densities represent averages (±SD) from 10 individual cells from 4 separate transfections. Size marker  = 25 µM.
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pone-0010665-g008: Kermit2 regulates internalization of antibody bound α5β1 integrin.Xenopus A6 cells were transfected with (A) GFP-tagged kermit2, or (B) GFP-tagged kermit2mut and the endocytosis of antibody labeled α5β1 was estimated from fluorescent intensity using the density slice function of Openlab. (C, D) Staining of internalized α5β1 with fluorescent anti-mouse antibody. Non-transfected cells in the same dish act as controls. Insets in C and D represent 25 µM2 ROI's used to estimate pixel densities. (A, C) Kermit2 transfected cells have 827.3±23.0 pixels/ROI as compared to control cell ROI pixel densities of 841.5±13.3 pixels/ROI. (B, D) In kermit2mut transfected cells the ROI pixel density is 530.8±51.2, while in non-transfected cells from the same dish have an average pixel density of 832.5±24.3 pixels/ROI. Pixel densities represent averages (±SD) from 10 individual cells from 4 separate transfections. Size marker  = 25 µM.

Mentions: GIPC has a well-established role in the endocytosis of a number of transmembrane receptors. As integrin trafficking has been described as an essential component in cultured cell migration so we asked if kermit2 is involved in cell surface turnover of the α5β1 integrin during Xenopus embryonic cell migration. We initially approached this problem using a Xenopus tissue culture model. Xenopus kidney epithelial cells (A6 cells; ATCC # CCL 102) were transfected with GFP tagged kermit2 or kermit2mut. We obtain about 15–30% transfection rates and we have used un-transfected cells from the same plate as controls. We followed integrin α5β1 endocytosis by monitoring the internalization of the anti-α5β1 antibody P8D4 (Figure 8). In cells expressing the kermit2 construct significant quantities of α5β1 are found in cytoplasmic punctae (Figure 8A, C). In contrast cells expressing the kermit2mut construct show reduced levels of integrin endocytosis (Figure 8B, D). We quantified endocytosis using the measurements and density slice functions of Openlab on selected regions of interest (ROIs; Figure 8 insets). Images were collected at the same exposure and the same threshold values were used to quantify all images. Control and kermit2 expressing cells have similar rates of integrin endocytosis, while cells transfected with kermit2mut have fewer P8D4 positive vesicles than non-transfected cells in the same dish. This suggests that kermit2 plays a role in the endocytosis of integrin α5β1.


Integrin alpha5beta1 function is regulated by XGIPC/kermit2 mediated endocytosis during Xenopus laevis gastrulation.

Spicer E, Suckert C, Al-Attar H, Marsden M - PLoS ONE (2010)

Kermit2 regulates internalization of antibody bound α5β1 integrin.Xenopus A6 cells were transfected with (A) GFP-tagged kermit2, or (B) GFP-tagged kermit2mut and the endocytosis of antibody labeled α5β1 was estimated from fluorescent intensity using the density slice function of Openlab. (C, D) Staining of internalized α5β1 with fluorescent anti-mouse antibody. Non-transfected cells in the same dish act as controls. Insets in C and D represent 25 µM2 ROI's used to estimate pixel densities. (A, C) Kermit2 transfected cells have 827.3±23.0 pixels/ROI as compared to control cell ROI pixel densities of 841.5±13.3 pixels/ROI. (B, D) In kermit2mut transfected cells the ROI pixel density is 530.8±51.2, while in non-transfected cells from the same dish have an average pixel density of 832.5±24.3 pixels/ROI. Pixel densities represent averages (±SD) from 10 individual cells from 4 separate transfections. Size marker  = 25 µM.
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Related In: Results  -  Collection

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pone-0010665-g008: Kermit2 regulates internalization of antibody bound α5β1 integrin.Xenopus A6 cells were transfected with (A) GFP-tagged kermit2, or (B) GFP-tagged kermit2mut and the endocytosis of antibody labeled α5β1 was estimated from fluorescent intensity using the density slice function of Openlab. (C, D) Staining of internalized α5β1 with fluorescent anti-mouse antibody. Non-transfected cells in the same dish act as controls. Insets in C and D represent 25 µM2 ROI's used to estimate pixel densities. (A, C) Kermit2 transfected cells have 827.3±23.0 pixels/ROI as compared to control cell ROI pixel densities of 841.5±13.3 pixels/ROI. (B, D) In kermit2mut transfected cells the ROI pixel density is 530.8±51.2, while in non-transfected cells from the same dish have an average pixel density of 832.5±24.3 pixels/ROI. Pixel densities represent averages (±SD) from 10 individual cells from 4 separate transfections. Size marker  = 25 µM.
Mentions: GIPC has a well-established role in the endocytosis of a number of transmembrane receptors. As integrin trafficking has been described as an essential component in cultured cell migration so we asked if kermit2 is involved in cell surface turnover of the α5β1 integrin during Xenopus embryonic cell migration. We initially approached this problem using a Xenopus tissue culture model. Xenopus kidney epithelial cells (A6 cells; ATCC # CCL 102) were transfected with GFP tagged kermit2 or kermit2mut. We obtain about 15–30% transfection rates and we have used un-transfected cells from the same plate as controls. We followed integrin α5β1 endocytosis by monitoring the internalization of the anti-α5β1 antibody P8D4 (Figure 8). In cells expressing the kermit2 construct significant quantities of α5β1 are found in cytoplasmic punctae (Figure 8A, C). In contrast cells expressing the kermit2mut construct show reduced levels of integrin endocytosis (Figure 8B, D). We quantified endocytosis using the measurements and density slice functions of Openlab on selected regions of interest (ROIs; Figure 8 insets). Images were collected at the same exposure and the same threshold values were used to quantify all images. Control and kermit2 expressing cells have similar rates of integrin endocytosis, while cells transfected with kermit2mut have fewer P8D4 positive vesicles than non-transfected cells in the same dish. This suggests that kermit2 plays a role in the endocytosis of integrin α5β1.

Bottom Line: We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling.Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells.These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.

ABSTRACT
During Xenopus gastrulation alpha5beta1 integrin function is modulated in a temporally and spatially restricted manner, however, the regulatory mechanisms behind this regulation remain uncharacterized. Here we report that XGIPC/kermit2 binds to the cytoplasmic domain of the alpha5 subunit and regulates the activity of alpha5beta1 integrin. The interaction of kermit2 with alpha5beta1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates alpha5beta1 integrin endocytosis downstream of activin signaling. Inhibition of kermit2 function impairs cell migration but not adhesion to FN substrates indicating that integrin recycling is essential for mesoderm cell migration. Furthermore, we find that the alpha5beta1 integrin is colocalized with kermit2 and Rab 21 in embryonic and XTC cells. These data support a model where region specific mesoderm induction acts through kermit2 to regulate the temporally and spatially restricted changes in adhesive properties of the alpha5beta1 integrin through receptor endocytosis.

Show MeSH
Related in: MedlinePlus