TIMP-1 promotes hypermigration of Toxoplasma-infected primary dendritic cells via CD63–ITGB1–FAK signaling

Abstract

The same GAPDH loading control blots were used in Fig. 4A,B without an explanation. The correct legend is shown below clarifying that results are from a single experiment. Fig. 4. Phosphorylation of FAK and SRC upon challenge with Toxoplasma and inhibition of hypermotility by gene silencing of Ptk2 (FAK) or by antagonism of FAK-SRC-PI3K. (A,B) Western blot analysis of total and phosphorylated protein expression of FAK (A) and SRC (B) in DCs unchallenged or challenged with T. gondii tachyzoites for 1 h. Graphs show total protein (FAK and SRC) and phosphorylated protein (p-397: FAK and p-416: SRC), relative to unchallenged DCs, normalized to GAPDH expression. ADU, arbitrary densitometry unit. n=4 biological replicates from independent blots. Representative blots from one experiment are shown, with same loading control (GAPDH) for total FAK/p-397 and total SRC/p-416, respectively.

Dysregulation of focal adhesion kinase upon Toxoplasma gondii infection facilitates parasite translocation across polarised primary brain endothelial cell monolayers

“…Impact of focal adhesion kinase (FAK) inhibition on cellular barrier integrity and transmigration of Toxoplasma gondii(a) Transcellular electrical resistance (TCER) of Caco2 and murine brain endothelial cell (MBEC) monolayers upon treatment with FAK inhibitor 12.5 μM). In contrast, challenge with MOI-equivalent or higher doses of T. gondii lysate (or LPS) did not reduce pFAK levels.This indicated an effect mediated by live intracellular tachyzoites and is well in line with recent reports of FAK dysregulation in toxoplasma-infected DCs and monocytic cells(Cook, Ueno, & Lodoen, 2018;Olafsson, Ross, Varas-Godoy, & Barragan, 2019). (b) Permeability to FITC-dextran (3 kDa) in same setup as in (a).…”

“…Although that study did not address the impact of infection on polarisation or barrier integrity, it is plausible that similar mechanisms explain the dephosphorylation of FAK upon T. gondii infection. Indeed, β1-integrin regulation and motility in toxoplasma-infected DCs and monocytes was recently linked to FAK dysregulation (Cook et al, 2018;Olafsson et al, 2019). Additionally, because redistribution of β1-integrins is observed upon T. gondii infection in DCs (Weidner et al, 2013), it is also possible that FAK dysregulation impacts on β1-integrin signalling and focal adhesion reorganisation in endothelial and epithelial cells and that the resulting increased vascular or epithelial permeability facilitates passage of T. gondii (Izawa et al, 2017).…”

“…Additionally, because redistribution of β1-integrins is observed upon T. gondii infection in DCs (Weidner et al, 2013), it is also possible that FAK dysregulation impacts on β1-integrin signalling and focal adhesion reorganisation in endothelial and epithelial cells and that the resulting increased vascular or epithelial permeability facilitates passage of T. gondii (Izawa et al, 2017). Indeed, β1-integrin regulation and motility in toxoplasma-infected DCs and monocytes was recently linked to FAK dysregulation (Cook et al, 2018;Olafsson et al, 2019).…”

Convergent Met and voltage-gated Ca2+channel signaling on Ras-Erk MAPK drives migratory activation of dendritic cells parasitized byToxoplasma gondii

“…We recently reported the implication of the non-receptor tyrosine kinase Fak in DC hypermotility (Olafsson et al, 2019). However, receptor tyrosine kinases (RTKs) also play central roles in cell migration and metastasis (Lemmon and Schlessinger, 2010).…”

“…Hypermigratory parasitized DCs are characterized by high-velocity amoeboid locomotion (termed hypermotility), cytoskeletal reorganization with loss of podosomes, decreased pericellular proteolysis and redistribution of integrins (Kanatani et al, 2015; Olafsson et al, 2018; Weidner et al, 2013). Hypermigration is triggered by non-canonical GABA A receptor-mediated (Fuks et al, 2012) activation of VGCCs, subtype Ca V 1.3 (Kanatani et al, 2017) and Timp-1-mediated Itgb1-dependent activation of focal adhesion kinase (Fak) (Olafsson et al, 2019). However, the signaling elements downstream of VGCC and Itgb1-Fak signaling, and their respective contribution to the hypermigratory phenotype of DCs, have remained unknown.…”