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Cas, Fak and Pyk2 function in diverse signaling cascades to promote Yersinia uptake

Pamela J. Bruce-Staskal1, Cheryl L. Weidow, Jennifer J. Gibson2,* and Amy H. Bouton1,{ddagger}

1 Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, VA 22908-0734, USA
2 Department of Health Evaluation Sciences, University of Virginia Health System, Charlottesville, VA 22908-0734, USA
* Present address: Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center, Lebanon, NH 03756



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  		Fig. 1. Fak-/- MEFs are deficient for Y. pseudotuberculosis uptake. (A) The percentage of internalized Y. pseudotuberculosis observed in infected Fak+/+, Fak-/- and Fak-/- MEFs expressing ectopic Fak is presented as the average of eight experiments. Error bars indicate standard deviation from the mean. (B) A representative immunoblot showing Fak expression from lysates derived from Fak+/+, Fak-/-, and Fak-/- MEFs reconstituted to express Fak. (C) Overlay of fluorescence images (400x magnification) of infected Fak-/- MEFs, including one Fak-transfected cell (blue). Intracellular Y. pseudotuberculosis appear red and extracellular Y. pseudotuberculosis appear yellow.

 


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  		Fig. 2. The absence of functional Fak and Cas leads to similar morphological defects in Yersinia uptake. Representative transmission electron micrographs of Fak+/+ MEFs (A), Fak-/- MEFs (B), HeLa/RK5 (C) or HeLa/Cas-{Delta}YXXP (D) cells infected with YP17/pVector.

 


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  		Fig. 3. Cas promotes Fak-independent Y. pseudotuberculosis uptake. (A) Fak-/- MEFs were transfected with the designated amounts of a plasmid encoding Cas. Uptake levels for each condition were compared by ANOVA to the level observed for mock-transfected cells. The data presented were obtained from 11 independent experiments. Uptake of YP17/pVector into mock-transfected cells averaged 35%. Error bars represent limits at a 95% confidence level. (B) Representative Cas immunoblot showing Cas expression in lysates from Fak-/- MEFs transfected with the designated amounts of DNA Cas.

 


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  		Fig. 4. Y. pseudotuberculosis induces Pyk2 autophosphorylation. Representative immunoblots containing lysates from Fak-/- MEFs (lanes 1-4) or J774A.1 macrophages (lanes 5-8) following infection with the indicated strains of Y. pseudotuberculosis. Lysates were immunoblotted for phospho-Y402 (upper panel) or Pyk2 (lower panel).

 




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