Abstract 5112: To be or not to be a protrusion: Unraveling the determinants of protrusion formation

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Abstract The shape of cells is largely determined by the arrangement of complexes integrating cytoskeletal structures with focal adhesions (FAs) and contacts (FCs). The transformed phenotype of the cells could be identified mathematically by a computerized morphometric assay. The assay is implemented by analyzing the shape of cells’ interference contours. Previous work established a database of shape variable value which was applied to classify cell features. By deconstructing cell shape, we discovered that filopodia, i.e. thin, flat, tapering projections from the cell edge (indexed by factor #4), and knob-shaped, triangular, or strap-shaped features (indexed by factor #7) were decreased and increased, respectively, in transformed cells. Factor #4 represents a feature that accounts for approximately 15% of the phenotype specifying a cancer cell, and here we explore the hypothesis that filopodia may be subtracted by elaborating the larger protrusions. The objective of this work was to model the determinants of the latter protrusions and test different models to gain insights into this feature. Although ruffling is the main mechanism for motility in cells, there was no correlation between #7 values and ruffle number or ruffling frequency. One model that stable contacts were stationed at the cell edge and the actin protrusion force pushed the membrane out parallel to the line of contacts, is supported by some data. First, data implicated PAK (p21-activated kinase)-PIX (PAK-interacting exchange factor) complex in the dynamics of FCs formation and turnover. Consistent with a role for PAK, studies done by expressing GFP-paxillin in the cells, inducing the formation of neurite-like protrusions, and then mapping the location of the bright spots and streaks (paxillin-containing scaffolds), showed that the range of angles formed between FCs and cell edge was lower in protrusions than in other portions of the edge. However, it was unlikely that actin-myosin interactions exerted outward force on the FCs, as ATPase inhibitor blebbistatin and myosin light chain kinase inhibitor SPC 16524 had no effect on the protrusions. A second model held that the protrusions were anchored by actin integration into FCs in the interior of the cell, e.g. at the boundary between the lamellum and lamellipodium. The size and shape of each such site, as well as its orientation and the x, y coordinates of its centroid were read out in MetaMorph software from digital images. A statistical analysis with these variables, testing for correlations with the factor #7 feature values (representing the neurite-like protrusions), showed that the smaller, more compact FCs were positively correlated with factor # 7. This indicated that widely-distributed, smaller contacts might be important for formation of neurite-like features, which could be consistent with either model. These two models were not mutually exclusive and so the necessity for both mechanisms is being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5112.