Systematic functional profiling of low-density lipoprotein receptor (<i>LDLR</i>) alleles.

<p><b>(A)</b><i>LDLR</i> missense variants were functionally characterized by monitoring cellular uptake of fluorescently-labeled LDL (DiI-LDL; <i>red</i>) into cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004855#sec004" target="_blank">Methods</a>). Shown are automatically acquired images of HeLa-Kyoto cells transiently expressing siRNA-resistant full-length human wildtype LDLR linked to EGFP (LDLR’-GFP), empty GFP-control plasmid, or two FH mutants known to inhibit transport (p.G549D; FH class-2) or endocytosis (p.Y828C; FH class-4) of the LDLR protein. Arrows denote GFP-positive cells. Note the localization of FH mutants to different subcellular compartments. Bars = 15μm. <b>(B)</b> Graphs depict relative signal intensities of total DiI-LDL in endosome-like subcellular compartments (total LDL signal; y-axis, in arbitrary units) plotted against total cellular GFP expression (x-axis, in arbitrary units) for wildtype LDLR (LDLR’-GFP, upper panel) and indicated FH mutants. Each graph depicts results from a single experimental replica upon either overexpression of the respective cDNA-construct (left graphs) or complementation settings (i.e., siRNA knockdown of endogenous LDLR followed by reconstitution with indicated LDLR-GFP constructs; right graphs). Each dot represents one individual cell. Dashed vertical bars separate cells classified as GFP-negative (left from bar) from cells defined as GFP-positive. Dashed horizontal lines in complementation setting indicate mean total LDL signal in control siRNA-treated cells expressing endogenous LDLR. Cells where total LDL signal fell above this threshold (indicating over-compensation by LDLR’-GFP expression) were not respected for quantifications (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004855#sec004" target="_blank">Methods</a>). <b>(C)</b> LDLR activity was measured with five phenotypic parameters: total LDL signal in endosome-like compartments, LDL concentration, number (seg. number) and area (seg. area) of subcellular DiI-positive endosome-like structures, and cellular GFP-expression. The heatmap represents means from all experimental replicas per variant under the overexpression setting. Red reflects reduced, blue increased signal relative to wildtype LDLR’-GFP. Phenotypes meeting statistical criteria as described in Methods are framed in orange. Bar graph depicts total LDL signal ±SD normalized to wildtype LDLR’-GFP.