epidermal-growth-factor and tetramethylrhodamine-isothiocyanate

Cell migration is critically important for the repair of chronic wounds, which cost billions of dollars each year to treat and can lead to serious complications, including amputation and death. Growth factors, including epidermal growth factor (EGF) and insulin-like growth factor-1 (IGF-1), are known to be deficient in chronic wounds; unfortunately, traditional delivery of soluble growth factors to wounds is expensive and complicated by their degradation. We have previously shown that directed and accelerated keratinocyte migration could be achieved by creating immobilized gradients of EGF. In this work, we have optimized EGF gradients for cell migration, synthesized and characterized gradient patterns of IGF-1, and tested for migration synergy upon combination of EGF and IGF-1 patterns. An optimal EGF concentration and pattern was identified, resulting in migration that was almost 10-fold that achieved on unpatterned controls. Immobilization of IGF-1 gradients also accelerated and directed keratinocyte migration (p < 0.05), however, no difference in migration was found across various IGF-1 concentrations or gradient patterns. Although combining EGF with IGF-1 patterns did not accelerate migration beyond levels achieved using EGF alone, these methods can be applied to create other types of multi-component gradients that may ultimately be utilized to create bioactive wound dressings.

Topics: Azides; Cell Line, Transformed; Cell Movement; Cross-Linking Reagents; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Fluorescent Dyes; Humans; Indoles; Insulin-Like Growth Factor I; Keratinocytes; Recombinant Proteins; Reproducibility of Results; Rhodamines; Succinimides; Wound Healing

Epidermal growth factor (EGF) exerts tropic effects on salivary epithelial cells. We examined EGF-mediated signaling pathways in the salivary epithelial cells of patients with Sjögren's syndrome (SS). We compared the immunohistochemical expression of EGF receptor (EGF-R), phosphatidylinositol 3-kinase (PI3K), Akt and nuclear factor kappa B (NF-kappaB) in the labial salivary glands of SS patients (n = 6) with those of control subjects (n = 2). EGF-mediated signaling pathways were further studied in vitro (n = 3) using primary salivary epithelial cells; NF-kappaB p65 nuclear translocation and Akt phosphorylation were examined by immunofluorescence and western blotting, respectively. The phosphorylation of EGF-R and Akt, and the nuclear expression of NF-kappaB p65, were increased in situ in the salivary epithelial cells of SS patients compared with those of control subjects. Epidermal growth factor induced rapid EGF-R phosphorylation and NF-kappaB p65 nuclear translocation in primary salivary epithelial cells in vitro. However, EGF also induced late Akt phosphorylation (after 12 h). Chemical inhibition of PI3K-Akt by LY294002/wortmannin did not affect EGF-mediated NF-kappaB p65 nuclear translocation; and NF-kappaB inhibition by Bay 11-7082 did not suppress Akt phosphorylation. Our data suggest that EGF stimulates both the PI3K-Akt pathway and NF-kappaB via distinct mechanisms, promoting tropic effects in SS salivary epithelial cells.

Topics: Biopsy; Case-Control Studies; Cells, Cultured; Enzyme Activation; Epidermal Growth Factor; Epithelial Cells; ErbB Receptors; Female; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Humans; Immunohistochemistry; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Recombinant Proteins; Rhodamines; Salivary Glands, Minor; Sjogren's Syndrome

The classical Arp2/3-mediated dendritic network defines the cytoskeleton at the leading edge of crawling cells, and it is generally assumed that Arp2/3-mediated actin polymerization generates the force necessary to extend lamellipods. Our previous work suggested that successful lamellipod extension required not only free barbed ends for actin polymerization but also a proper ultrastructural organization of the cytoskeleton. To further explore the structural role of the Arp2/3 complex-mediated networks in lamellipod morphology and function, we performed a detailed analysis of the ultrastructure of the Arp2/3-mediated networks, using the WA domains of Scar and WASp to generate mislocalised Arp2/3 networks in vivo, and to reconstruct de novo Arp2/3-mediated actin nucleation and polymerization on extracted cytoskeletons. We present here evidence that spatially unrestricted Arp2/3-mediated networks are intrinsically three-dimensional and multilayered by nature and, as such, cannot sustain significant polarized extension. Furthermore, such networks polymerize only at preferred locations in extracted cells, corresponding to pre-existing Arp2/3 networks, suggesting that the specific molecular organization of the actin cytoskeleton, in terms of structure and/or biochemical composition, dictates the location of Arp2/3 complex-mediated actin polymerization. We propose that successful lamellipod extension depends not only on localized actin polymerization mediated through local signalling, but also on spatial restriction of the Arp2/3 complex-mediated nucleation of actin polymerization, both in terms of location within the cell and ultrastructural organization of the resulting network.

Topics: Actin-Related Protein 2-3 Complex; Actins; Animals; Blood Platelets; Cell Line, Tumor; Cytoskeleton; Epidermal Growth Factor; Epithelial Cells; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Fluorescent Dyes; Glutathione Transferase; Humans; Mammary Neoplasms, Experimental; Microinjections; Muscle, Skeletal; Polymers; Protein Structure, Tertiary; Pseudopodia; Rabbits; Rats; Recombinant Fusion Proteins; Rhodamines; Wiskott-Aldrich Syndrome Protein

Fluorescence Correlation Spectroscopy is a powerful method to analyze densities and diffusive behavior of molecules in membranes, but effects of photodegradation can easily be overlooked.. Based on experimental photophysical parameters, calculations were performed to analyze the consequences of photobleaching in fluorescence correlation spectroscopy (FCS) cell surface experiments, covering a range of standard measurement conditions.. Cumulative effects of photobleaching can be prominent, although an absolute majority of the fluorescent molecules would pass the laser excitation beam without being photo-bleached. Given a distribution of molecules on a cell surface with different diffusive properties, the fraction of molecules that is actually analyzed depends strongly on the excitation intensities and measurement times, as well as on the size of the reservoir of freely diffusing molecules. Both the slower and the faster diffusing molecules can be disfavored.. Apart from quantifying photobleaching effects, the calculations suggest that the effects can be used to extract additional information, for instance about the size of the reservoirs of free diffusion. By certain choices of measurement conditions, it may be possible to more specifically analyze certain species within a population, based on their different diffusive properties, different areas of free diffusion, or different kinetics of possible transient binding.

Topics: Animals; Biophysical Phenomena; Biophysics; Cell Line, Tumor; Cell Membrane; Diffusion; Epidermal Growth Factor; Fluorescent Dyes; Mathematics; Photobleaching; Rats; Rhodamines; Spectrometry, Fluorescence; Time Factors; Urinary Bladder Neoplasms