['To determine whether integrin-linked kinase (ILK) controls the organization of the actin cytoskeleton in the trabecular meshwork (TM) by regulating integrin co-signaling.', 'The cell binding domain and the Heparin II (Hep II) domain of fibronectin were used to activate α5β1 and α4β1 integrin signaling, respectively, in differentiated human TM (HTM) cells. The role of ILK was determined using either ILK small interfering RNA (siRNA) to knockout ILK expression or the ILK inhibitors, KP392 and QLT0267. The knockdown of ILK expression was verified by Western blot analysis. The presence of actin stress fibers and focal adhesions was determined by labeling cultures with phalloidin and anti-talin or ILK antibodies, respectively.', 'Cell spreading in differentiated HTM cells required ILK, since ILK siRNA and the ILK inhibitors significantly reduced cell spreading, actin polymerization, and the localization of talin and ILK in focal adhesions (FAs). Both cell spreading and the localization of talin and ILK to FAs in differentiated HTM cells could be rescued by inducing α4β1 integrin signaling with a recombinant Hep II domain of fibronectin, even though α4β1 integrins were not found in FAs. In the absence of ILK inhibition, the Hep II domain had minimal effect on α5β1 integrin-mediated spreading.'] Does integrin-linked kinase regulate integrin signaling in human trabecular meshwork cells?