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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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Expression of kermit2 mRNA rescues morpholino knock down of kermit2.(A–D) Ventral view of stage 12 embryos. (E–H) Late gastrula stage BCR stained for FN. (A) Stage 12 control embryos gastrulate normally and (E) assemble a dense FN matrix. Embryos injected with 10 ng of Kermit2 morpholino (MO; B) gastrulate normally, and (F) exhibit a small decrease in FN matrix assembly. (C) Embryos injected with kermit2 inhibiting morpholino and kermit2 mRNA (MO+kermit2) exhibit a small delay in blastopore closure and (G) a partial rescue of FN matrix assembly. (D) Embryos injected with the kermit2 morpholino and the dominant negative kermit2 construct (MO+kermit2mut) exhibit delayed blastopore closure and (H) significant reduction in FN matrix assembly. (A–D) size marker  = 200 µm. (E–H) size marker  = 30 µm.
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pone-0010665-g005: Expression of kermit2 mRNA rescues morpholino knock down of kermit2.(A–D) Ventral view of stage 12 embryos. (E–H) Late gastrula stage BCR stained for FN. (A) Stage 12 control embryos gastrulate normally and (E) assemble a dense FN matrix. Embryos injected with 10 ng of Kermit2 morpholino (MO; B) gastrulate normally, and (F) exhibit a small decrease in FN matrix assembly. (C) Embryos injected with kermit2 inhibiting morpholino and kermit2 mRNA (MO+kermit2) exhibit a small delay in blastopore closure and (G) a partial rescue of FN matrix assembly. (D) Embryos injected with the kermit2 morpholino and the dominant negative kermit2 construct (MO+kermit2mut) exhibit delayed blastopore closure and (H) significant reduction in FN matrix assembly. (A–D) size marker  = 200 µm. (E–H) size marker  = 30 µm.

Mentions: We used morpholino knockdowns to further elucidate the role kermit2 plays in early development. A 5 mismatch control morpholino (CO) has minor effects on FN assembly that are not significant enough to block gastrulation (Figure 4B, E). The kermit2 morpholino (MO) inhibits FN assembly and blocks blastopore closure (Figure 4C, F). Western blots indicate GIPC is a maternal protein and expressed throughout all stages of early development (Figure 4G). The inhibiting morpholino reduces the amount of kermit2 protein, particularly in the post gastrula stages. In our hands injection of increasing amounts of morpholino resulted in no further decrease in kermit2 protein levels (Figure 4H) and increasing amounts of the control morpholino resulted in non-specific inhibition (not shown). Injection of kermit2 mRNA in the presence of the inhibiting morpholino rescues blastopore closure and FN matrix assembly (Figure 5C, G). Embryos co-injected with the morpholino and the dominant negative kermit2mut construct cannot close their blastopores and have very little FN on the BCR (Figure 5D, H). Similar to the results we obtained with the dominant negative construct, the defects in gastrulation are not due to disruptions of mesodermal genes or molecules involved in FN assembly (Supplemental material S 2). In our hands the dominant negative kermit2mut construct has a more consistent phenotype than the morpholino and we used this construct in most of our experiments.


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)

Expression of kermit2 mRNA rescues morpholino knock down of kermit2.(A–D) Ventral view of stage 12 embryos. (E–H) Late gastrula stage BCR stained for FN. (A) Stage 12 control embryos gastrulate normally and (E) assemble a dense FN matrix. Embryos injected with 10 ng of Kermit2 morpholino (MO; B) gastrulate normally, and (F) exhibit a small decrease in FN matrix assembly. (C) Embryos injected with kermit2 inhibiting morpholino and kermit2 mRNA (MO+kermit2) exhibit a small delay in blastopore closure and (G) a partial rescue of FN matrix assembly. (D) Embryos injected with the kermit2 morpholino and the dominant negative kermit2 construct (MO+kermit2mut) exhibit delayed blastopore closure and (H) significant reduction in FN matrix assembly. (A–D) size marker  = 200 µm. (E–H) size marker  = 30 µm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2871791&req=5

pone-0010665-g005: Expression of kermit2 mRNA rescues morpholino knock down of kermit2.(A–D) Ventral view of stage 12 embryos. (E–H) Late gastrula stage BCR stained for FN. (A) Stage 12 control embryos gastrulate normally and (E) assemble a dense FN matrix. Embryos injected with 10 ng of Kermit2 morpholino (MO; B) gastrulate normally, and (F) exhibit a small decrease in FN matrix assembly. (C) Embryos injected with kermit2 inhibiting morpholino and kermit2 mRNA (MO+kermit2) exhibit a small delay in blastopore closure and (G) a partial rescue of FN matrix assembly. (D) Embryos injected with the kermit2 morpholino and the dominant negative kermit2 construct (MO+kermit2mut) exhibit delayed blastopore closure and (H) significant reduction in FN matrix assembly. (A–D) size marker  = 200 µm. (E–H) size marker  = 30 µm.
Mentions: We used morpholino knockdowns to further elucidate the role kermit2 plays in early development. A 5 mismatch control morpholino (CO) has minor effects on FN assembly that are not significant enough to block gastrulation (Figure 4B, E). The kermit2 morpholino (MO) inhibits FN assembly and blocks blastopore closure (Figure 4C, F). Western blots indicate GIPC is a maternal protein and expressed throughout all stages of early development (Figure 4G). The inhibiting morpholino reduces the amount of kermit2 protein, particularly in the post gastrula stages. In our hands injection of increasing amounts of morpholino resulted in no further decrease in kermit2 protein levels (Figure 4H) and increasing amounts of the control morpholino resulted in non-specific inhibition (not shown). Injection of kermit2 mRNA in the presence of the inhibiting morpholino rescues blastopore closure and FN matrix assembly (Figure 5C, G). Embryos co-injected with the morpholino and the dominant negative kermit2mut construct cannot close their blastopores and have very little FN on the BCR (Figure 5D, H). Similar to the results we obtained with the dominant negative construct, the defects in gastrulation are not due to disruptions of mesodermal genes or molecules involved in FN assembly (Supplemental material S 2). In our hands the dominant negative kermit2mut construct has a more consistent phenotype than the morpholino and we used this construct in most of our experiments.

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