High-resolution image of HeLa cells expressing YFP-paxillin.

Image courtesy of Dr Benjamin Geiger, Weizmann Institute of Science, Rehovot, Israel

Focal adhesions (FAs) are highly dynamic transmembrane multiprotein complexes that undergo continuous assembly and disassembly during cell migration and attachment to the extracellular matrix (ECM). In the Journal of Cell Biology, Benjamin Geiger and colleagues now report a high-throughput RNA interference (RNAi) screen aimed at understanding the molecular pathways that regulate FA assembly.

The authors screened three human small interfering RNA (siRNA) libraries, jointly selected with Joan Brugge’s laboratory: one targeting 576 kinase-encoding genes, one targeting 192 phosphatase-encoding genes, and a custom library, designed to target 312 genes known or predicted to affect migration or adhesion (MAR library). HeLa cells expressing yellow fluorescent protein (YFP)-paxillin were selected for their uniform FA morphology and distribution and used as a reporter cell line. Upon transfection, the effects of siRNA treatment on different FA features were assessed using a high-resolution automatic microscope and quantitative image analysis.

Four main categories of phenotypic features were examined: FA morphology, including area, length and intensity, and the percentage of small dot-like FAs; FA distribution, including abundance and peripheral location; cell coverage, which reflects cell number and spreading; and cell shape.

Screening multiple parameters had the advantage of providing information on how different FA features might be connected to one another. The authors identified different siRNA clusters, where each cluster was characterised by a 'phenotypic signature' that represents the overall effect of the particular siRNAs. For example, when screening for altered morphological features, siRNAs were divided in eight clusters. Interestingly, four of these clusters had similar effects although at varying degrees - they combined reduced FA area, intensity and FA length with a higher number of dot-like FAs. However, the other clusters either uncoupled these features or had no effect on FA morphology. When studying FA distribution and cell shape, nine siRNA clusters were identified. The authors then calculated the correlation between pairs of features to determine which FA and cell features might be co-regulated by the targeted genes.

This approach led Benjamin Geiger and colleagues to propose a model where FA growth is regulated by a common molecular pathway that includes signalling molecules, proteases and factors that affect actin dynamics. In addition, some morphological features are controlled independently as suggested by their ‘uncoupling’ in certain siRNA clusters.

Independent testing of 86 siRNAs with stronger phenotypes using siRNAs modified to reduce off-target effects yielded 44 validated siRNAs, 22 of which were not previously associated with adhesion and migration.

Together this work provides the first dataset of genes that regulate FA formation and implies previously unknown relationships between various gene functions and FA features.

Author: Kim Baumann