ABSTRACT
Summary The atomic force microscope (AFM) has provided nanoscale analyses of surfaces of cells that exhibit strong adhesive and cell spreading properties. However, it is frequently reported that prior fixation is required for reliable imaging of cells with lower adhesive properties. In the present study, the AFM is used to assess the effects of fixation by glutaraldehyde on the elastic modulus of a human rhabdomyosarcoma transfectant cell line RDX2C2. Our results show a sharp increase in the elastic modulus for even mild fixation (0.5% glutaraldehyde for 60 s), accompanied by a dramatic improvement in imaging reproducibility. An even larger increase is seen in NIH-3T3 mouse fibroblasts, although in that case fixation is not typically necessary for successful imaging. In addition, our results suggest that treatment with glutaraldehyde restricts the content of the resulting images to features nearer to the cell surface.
Subject(s)
Microscopy, Atomic Force , Tissue Fixation/methods , Animals , Elasticity , Fibroblasts/ultrastructure , Glutaral , Humans , Image Enhancement , Mesoderm/cytology , Mesoderm/ultrastructure , MiceABSTRACT
In a previous study, we show that stimulation of chemotaxis in rat pheochromocytoma PC12 cells by nerve growth factor (NGF) and epidermal growth factor (EGF) requires activation of the RAS-ERK signaling pathway. In this study, we compared the threshold levels of ERK activation required for EGF and NGF-stimulated chemotaxis in PC12 cells. The threshold ERK activity required for NGF to stimulate chemotaxis was approximately 30% lower than that for EGF. PD98059 treatment inhibited EGF stimulation of growth and chemotaxis; however, stimulation of chemotaxis required an EGF concentration approximately 10 times higher than for stimulation of PC12 cell growth. Thus, ERK-dependent cellular functions can be differentially elicited by the concentration of EGF. Also, treatment of PC12 cells with the PI3-K inhibitor LY294002 reduced ERK activation by NGF; thus, higher NGF concentrations were required to initiate chemotaxis and to achieve the same maximal chemotactic response seen in untreated PC12 cells. Therefore, the threshold NGF concentration to stimulate chemotaxis could be adjusted by the crosstalk between the ERK and PI3-K pathways, and the contributions of PI3-K and ERK to signal chemotaxis varied with the concentrations of NGF used. In comparison, LY294002 treatment had no effect on ERK activation by EGF, but the chemotactic response was reduced at all the concentrations of EGF tested indicating that NGF and EGF differed in the utilization of ERK and PI3-K to signal chemotaxis in PC12 cells.
Subject(s)
Chemotaxis/drug effects , Epidermal Growth Factor/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Nerve Growth Factor/pharmacology , Adrenal Gland Neoplasms/enzymology , Adrenal Gland Neoplasms/pathology , Animals , Cell Movement/drug effects , Cell Proliferation/drug effects , Chromones/pharmacology , Differential Threshold , Enzyme Activation , Enzyme Inhibitors/pharmacology , Flavonoids , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/drug effects , Morpholines/pharmacology , PC12 Cells , Pheochromocytoma/enzymology , Pheochromocytoma/pathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Rats , Signal TransductionABSTRACT
The ability of cells to undergo shape changes is essential for diverse cellular functions including cell growth, differentiation, and movement. The present study examines how an integration of the function of alpha2beta1 integrin with that of the receptor for epidermal growth factor (EGFR) modulates EGF-stimulated morphological changes in human rhabdomyosarcoma RD transfectant cells. Upon EGF stimulation, RD transfectant cells that lacked alpha2beta1 integrin expression (RDpF) underwent contraction; in contrast, expression of alpha2beta1 on RD cells (RDX2C2) resulted in transient cell spreading. Integrin alpha2 cytoplasmic domain played a critical role in the observed alpha2beta1-mediated conversion from a cell rounding to a cell spreading phenotype. Thus, the expression of an alpha2 cytoplasmic domain deletion variant (X2C0) or a chimeric alpha2beta1 containing the cytoplasmic domain of alpha4 (X2C4) or alpha5 (X2C5), instead of alpha2, failed to mediate spreading upon EGF stimulation. Using dominant negative (DN) mutants of RhoGTPases, results revealed that RhoA activation was required for both EGF-stimulated responses of cell rounding and spreading, Cdc42 functioned in the re-spreading of cells after undergoing EGF-stimulated contraction, and Rac1 was required in alpha2beta1-mediated RD cell spreading. Therefore, alpha2beta1 integrin function can switch the Rho GTPase-dependent cell shape changes in RD cells from an EGF-stimulated cell contraction to a spreading morphology. Together, results show that integrin alpha2 cytoplasmic domain plays an indispensable role in the ability of integrin alpha2beta1 to modulate EGF stimulation of Rho-GTPase-dependent morphological changes in RD cells.
