Figure 6. Complex phenotypes arising in top-ranked translocations discovered by the active screen.
EGFP-tagged Fatty acid 2-hydroxylase (Fa2h, clone number 23) expressed in NIH-3T3 cells exhibits a broad range of localization patterns in two top-ranked treatments, cycloheximide (drug 4), and Econazole (drug 16). (A) Image features calculated from confocal images (60X) of two treatments (orange squares and green triangles, respectively) as well as the vehicle treatment (purple dots) are reasonably well classified by logistic regression. The resulting 2D projection of the 173-dimensional feature space transforms the distribution of each treatment into a 2D Gaussian (95% confidence intervals as colored ellipses). Consistent with the active screen results, drug treatments are distinguishable from vehicle and from each other. Fa2h exhibits an unusual spread of secretory-associated, or subnuclear (but not nucleolar), and sometimes both localizations in single cells. (B) In order to visually assess the distributional differences between treatments, we can extend the usual visual vocabulary of coarse localization phenotypes (e.g. 'Golgi,' 'ER' and the like) by decomposing the feature vectors of each single-cell image in terms of a fixed set of single-cell images. Each image can be expressed as an additive combination; for example, a cell exhibiting both a subnuclear and small vesicular localization can be linearly approximated by adding together feature vectors of a subnuclear and a separate small vesicular cell. Subtractive cases (B, bottom row) are false colored with red instead of green for EGFP signal. (C) Archetypal cells (top row), chosen by minimax clustering (Bien and Tibshirani, 2011), can be approximately added in different weighted combinations to reveal nuanced differences in condition-dependent localization (see Materials and methods). For each treatment (pair of rows), two cells (leftmost column) and their additive description in terms of the archetypes are displayed. White arrows highlight dim subnuclear signal where visually subtle. Overall, Econazole appears to have the effect of generally enhancing post perinuclear/ER secretory structure localization, whereas cycloheximide generally suppresses ER localization in favor of presumably later secretory vesicular localization.