IQGAP1 interacts with numerous binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 plays a role in cell-matrix interactions and actin cytoskeleton dynamics in membrane ruffling and lamellipodium protrusion. Phosphorylation in the CT domain regulates intramolecular interaction and IQGAP1 cellular activity. In a recent study, we discovered that IQGAP1 surprisingly localizes to actively retracting edges, instead of protruding areas, in B16F10 mouse melanoma cells and some other cells types. In these current studies we examined localization of IQGAP1 mutants to retracting versus protruding areas in phorbol ester-stimulated B16F10 cells. Cells were co-transfected with GFP-IQGAP1 full length (GFP-IQGAP1-FL), as an internal control, and one of five Myc-tagged IQGAP1 constructs (FL, CA, ΔCHD, ΔGRD or ΔCT). The cotransfected cells were plated onto laminin for 30 minutes, stained with anti-Myc and anti-WAVE2 antibodies, and normalized fluorescence measurements were made in retracting and protruding areas. Retracting cell areas were defined as GFP-IQGAP1-FL positive and WAVE2 negative, while protruding cell areas were defined as GFP-IQGAP1-FL negative and WAVE2 positive. In retracting areas there were large decreases in both ΔGRD and ΔCT localization, a slight decrease in ΔCHD localization, and normal localization of the CA mutant. In areas of cell protrusion there were large increases in both ΔGRD and ΔCT localization, and normal localization of ΔCHD and CA mutants. These results indicate that two domains, GRD and CT, are essential for normal localization of IQGAP1 to retracting cell areas. Furthermore, our results suggest a model in which IQGAP1 in the areas of cell retraction is in the open, phosphorylated, conformation. Additionally we investigated the knockdown of IQGAP1 in B16F10 cells by means of actin images. Cells were exposed to the lentil virus which contained short hairpin Ribonucleic acid (shRNA) that would silence IQGAP1. Two controls were used in the experiment, untransfected B16F10 cells and B16F10 cells which were exposed to the lentil virus without any shRNA. We found that the knockdown cells were in general much more compact and that they did not polarize. Surface stiffness was investigated in the effect it would have on B16F10 cells. Polyacrylamide gels were made and cross-linked using sulfo-SANPAH. Laminin was added to the cross-linked gels and B16F10 cells were placed on top of the laminin coated hydrogels. Investigation of the cells by means of actin images revealed that surface stiffness had an effect on cell morphology. The 1 kPa surfaces did not allow for spreading of the cells, while the surfaces greater than 100 kPa exhibited normal cell behavior.
|Commitee:||Shaw, Michael, Witt, Kenneth|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 54/03M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Cellular biology, Biochemistry|
|Keywords:||Cell motility, Cell polarity, Iqgap1|
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