Browsing by Author "Marsden, Mungo"

Now showing 1 - 3 of 3

Characterizing a role for α-parvin in Xenopus laevis Development
(University of Waterloo, 2021-09-20) Do, Richard; Marsden, Mungo
In metazoans dynamic regulation of cell adhesion is critical for the cell movements that characterize gastrulation. It is clear that the adhesive property of cells is modulated in a highly coordinated spatial and temporal fashion. In Xenopus, gastrulation has been extensively characterized at the cell and tissue level. The directed intercalation of cells generates the forces that drive tissue shape changes and rearrangements. In Xenopus, the adhesive properties of cells are mediated through the integrin and cadherin families of cell adhesion receptors. The integrins mediate bidirectional signaling across cell membranes. Integrin ligation recruits multi-protein complexes such as the ILK-PINCH-Parvin (IPP) complex to the cytoplasmic tail at sites of focal adhesion. α-parvin is a member of the IPP complex and has two functional calponin-homology (CH) domains. In the IPP complex, α-parvin primarily functions as a scaffolding molecule. However, the mechanistic role of α-parvin has yet to be described in Xenopus. I asked if α-parvin acts as an active modulator of integrin-mediated adhesion during Xenopus laevis gastrulation. I successfully isolated Xenopus α-parvin and performed a phylogenetic analysis on the sequence. α-parvin shares high sequence identity with α-parvin orthologs from other model organisms. The CH domains are also highly conserved. Xenopus α-parvin displays peak expression levels during gastrulation suggesting a role in the rearrangement of cells and tissues during gastrulation. To analyze function, I created GFP tagged α-parvin deletion constructs that isolate each domain. In Xenopus A6 cells, the full length α-parvin construct localizes to focal adhesions. The isolated CH1 domain is not recruited to focal adhesions, while the isolated CH2 domain is found in focal adhesions. This suggests that the CH2 domain of Xenopus α-parvin is responsible for recruitment to sites of integrin adhesion. In embryos, over-expression of full length α-parvin has no effect on development. When the CH1 domain is over expressed, blastopore closure is delayed and anterior–posterior axis extension is inhibited. These embryos develop axial protrusions that resemble a second axis. This would suggest that the CH1 domain is somehow influencing the β-catenin pathway that influences axial development. When the CH2 domain is over expressed, embryos show a dramatic delay and failure of blastopore closure. The anterior-posterior axis is truncated, and the blastocoel is retained. The embryos over-expressing the CH2 domain resemble those in which integrin-FN interactions are disrupted and it is likely that the CH2 domain is acting as a dominant negative to inhibit integrin adhesion. This thesis provides a preliminary analysis of α-parvin function in Xenopus laevis development.
Integrin α5β1 Function Is Regulated by XGIPC/kermit2 Mediated Endocytosis during Xenopus laevis Gastrulation
(Public Library of Science (PLOS), 2010) Spicer, Erin; Suckert, Catherine; Al-Attar, Hyder; Marsden, Mungo
During Xenopus gastrulation α5β1 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 α5 subunit and regulates the activity of α5β1 integrin. The interaction of kermit2 with α5β1 is essential for fibronectin (FN) matrix assembly during the early stages of gastrulation. We further demonstrate that kermit2 regulates α5β1 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 α5β1 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 α5β1 integrin through receptor endocytosis.
β‐Parvin Mediates Novel Integrin Signaling Pathways During Early Xenopus laevis Development.
(University of Waterloo, 2019-01-24) Knapp, Justin; Marsden, Mungo
Crosstalk between cell adhesion molecules is critical for the cell rearrangements that occur during early Xenopus laevis development. Cells primarily use integrin receptors to adhere to and interpret the extracellular environment, and cadherin receptors to modulate adhesion between cells. The Xenopus laevis gastrula provides a simple model to examine the molecular processes that regulate adhesion receptor crosstalk as epiboly and convergent extension rely on the modulation of integrin and cadherin adhesion. The adhesive crosstalk of integrin and cadherin is regulated in part by the binding of a shared pool of cytoplasmic molecules to the intracellular tail of these molecules. β-Parvin, a scaffolding molecule, has been described to accumulate at both cell-extracellular matrix (ECM) and cell-cell junctions. β-parvin consists of two calponin homology domains (CH1 and CH2). The CH1 domain was found to regulate the recruitment of β-parvin to cell-cell borders while the CH2 domain mediates the compartmentalization of β-parvin at sites of cell-ECM adhesion. Cell adhesion assays indicate that over-expression of the CH1 domain reduces cell adhesion to a cadherin substrate, whereas over-expression of the CH2 domain reduces cell adhesion to Fibronectin (FN). Furthermore, disruption of β-Parvin signaling inhibits cell intercalation behaviours that underlie epiboly and convergent extension. This suggests that β-parvin plays a role in mediating both integrin and cadherin adhesion. Co-immunoprecipitation analysis indicates that the CH2 domain of β-parvin regulates the association of β-parvin with the C-terminus of Xenopus laevis integrin-linked kinase (xILK). This interaction requires an outside/in signal originating from the ligation of integrin to FN. In addition, the recruitment of β-parvin to xILK is decreased in the presence of Activin A. This suggests that the activation state of integrin controls xILK- β-parvin interactions. The CH1 domain of β-parvin was found to regulate the binding of β-parvin to C-cadherin and β-catenin. GST-pull-down analysis indicates that β-catenin is not required for this interaction. Activin A increases the interaction between C-cadherin and β-Parvin. In addition, β-Parvin interacts with a T-cell factor (TCF)-selective pool of β-catenin known to translocate to the nucleus. Interestingly, the nuclear localization signal (NLS) in the N-terminus appears to regulate the exclusion of β-Parvin from the nucleus. Finally, this study also revealed the existence of a α-parvin homolog in Xenopus laevis. RT-PCR analysis indicates that α-parvin mRNA is expressed throughout early Xenopus laevis development. The present study has for the first time described the molecular mechanisms regulating the function of β-Parvin in integrin-cadherin receptor crosstalk during Xenopus laevis gastrulation. These findings also demonstrate the novel finding that β-parvin actively modulates cadherin mediated cell adhesion.