We hypothesized that the actin cytoskeleton might be responsible for the differential uptake between tissue culture plastic and polyacrylamide gels. Actin stress fibers are affected by substrate stiffness. Actin stress fibers are also regulated by cell-cell contact. Cells on soft substrates do not typically exhibit stress fibers, however, when cells on soft substrates are in cell-cell contact, stress fibers reappear. The actin cytoskeleton is critical in caveolae-mediated endocytosis, and is necessary for the closure and initial uptake of caveolar vesicles. Other investigators have shown that increased density of organized stress fibers impedes clathrin-mediated endocytosis. Furthermore, stress fibers are not prominent in cells in vivo. However, our data suggests that YARA uptake is independent of the state of actin polymerization since neither LPA nor cytochalasin D affected YARA uptake. Microtubules are also important in TAK-875 biological activity endosome trafficking. In this study microtubules were shown to be important in YARA uptake or 870281-82-6 trafficking since nocodazole treatment significantly enhanced YARA uptake. Microtubules are confirmed to affect endosome trafficking including recycling to the plasma membrane; thus, it is likely that disruption of microtubules does not increase the rate of endocytosis of YARA, but delays recycling of YARA to the membrane. The end result of microtubule disruption and delayed recycling is accumulation of YARA within the cells. Uptake appears to be independent of actin polymerization, while accumulation of YARA within the cell is dependent upon microtubule polymerization. Understanding how substrate stiffness affects intracellular uptake has broad implications in the design of drug screening platforms, both in screening potential drugs for evidence of efficacy and for understanding how uptake might differ in cells within a diseased state. Several different disease states are characterized by changes in tissue rigidity due to inflammation, fibrosis, calcification, or other biochemical changes within the tissue. Understanding whether a drug is influenced by tissue rigidity can help physicians choose therapies that may be more effective for the patient, depending on the stage of the disease. Pancreatic cancer is the fourt
