SUNDAY, DECEMBER 4- Late Abstracts 1 - Molecular Biology of the ...

SUNDAY
additive effectiveness with proven or suggested regenerative agents. WS1 human skin
fibroblasts are seeded at a density of 105 cells/well on a 6-well culture plate. Using a 4mm
sterile biopsy punch, three wound areas are created in each well at confluency. WS1 cell
wounds are exposed to 10-fold serial dilutions of calcium hydroxide, avobenzene, or ZnO in
appropriate solvents for 24 hours in control wells, or before cells are exposed to UVA. Effective
concentrations are selected from standard dose response curves for the various regenerative
reagents and are used in combination with the vitamin D3 and E isomers. During the test
periods, wound areas are digitally recorded beginning at day 0, for every 12 hours over a four
day period. Measurements of the radius (rw - radius of the minimal diameter of the wound), the
wound area (Aw) are taken using Motic 2.0 software and compared to day 0 (r0 and A0).
Triplicate data sets are statistically analyzed using a one-way ANOVA with Tukey’s post hoc
tests. Effective concentrations of calcium/ D3 and cholcalciferol alone are seen at 10-4 mg/ml.
Vitamin E isomers did improve cell proliferation and overall wound healing. The most effective
doses were 10-7 mg/ml for alpha-tocopherol and 10-8 mg/ml for delta-tocotrienol, showing
complete wound healing between 48 and 60 hours. It is suggested that agents with greater
antioxidant activities may shorten healing times, reduce the incidence of wound infection and, in
the most serious wound cases, lower the mortality rate in burn patients.
2062
Arp 2/3 Plays a Crucial Role in Focal Adhesion Initiation at the Leading Edge.
R. J. Vasquez 1 , K. Sayegh 2 , J. Stricker 3 , Y. Beckham 3 , M. Gardel 3 ; 1 Department of Pediatrics,
Section of Hematology, Oncology and Stem Cell Transplantation, University of Chicago,
Chicago, IL, 2 University of Chicago, Chicago, IL, 3 Department of Physics, University of Chicago,
Chicago, IL
The lamellipodia is a dense, Arp-2/3 mediated actin network at the leading edge of migrating
cells that generates forces sufficient to generate sheet-like protrusions of the cell edge. In
addition to this force generating role, the initiation of focal adhesions also occurs within the
lamellipodia. However, the role of lamellipodial actin in focal adhesion initiation is not well
understood. We used a recently identified inhibitor of the ARP 2/3 complex, CK 869, to
investigate the role of the lamellipodia in leading edge protrusion and focal adhesion formation
in cell motility and adhesion in two cell types- MCF10A cells (a human breast epithelial cell line)
and U20S cells (a human osteosarcoma cell line). We find that treatment with CK 869 disrupts
or inhibits the formation of an organized lamellipod, as demonstrated by phalloidin staining and
immunofluoresce with cortactin, a protein enriched in the lamellipod. Motility assays of single
cells and scratch assays with MCF-10A cells demonstrate that treatment with CK-869 reduced
the number of motile cells and reduced the motility rate, and in the case of the scratch assay,
greatly increased the time to closure of the cell sheet. Despite these effects on cell migration,
protrusions of the cell edge persist, suggesting that ARP 2/3 mediated actin assembly is not the
only mechanism through which to generate sheet-like membrane protrusions. We hypothesized
that these membrane protrusions might differ from lamellipodial protrusions in their ability to
form new focal adhesions. Immuno-staining of the focal adhesion proteins paxillin and vinculin
demonstrated that the ARP 2/3 inhibited cells had fewer but larger peripheral adhesions and
fewer central focal adhesions. Live cell imaging with GFP-paxillin in U2OS cells revealed that
the protrusions formed in the presence of the ARP-inhibitor revealed reduced focal adhesion
assembly. Collectively, these results indicate that ARP 2/3 is not necessary for sheet-like
protrusions of the cell edge but plays a crucial role in focal adhesion formation.