Non-monotonic cellular responses to heterogeneity in talin protein expression-level

Abstract

Talin is a key cell-matrix adhesion component with a central role in regulating adhesion complex maturation, and thereby various cellular properties including adhesion and migration. However, knockdown studies have produced inconsistent findings regarding the functional influence of talin in these processes. Such discrepancies may reflect non-monotonic responses to talin expression-level variation that are not detectable via canonical "binary" comparisons of aggregated control versus knockdown cell populations. Here, we deployed an "analogue" approach to map talin influence across a continuous expression-level spectrum, which we extended with sub-maximal RNAi-mediated talin depletion. Applying correlative imaging to link live cell and fixed immunofluorescence data on a single cell basis, we related per cell talin levels to per cell measures quantitatively defining an array of cellular properties. This revealed both linear and non-linear correspondences between talin expression and cellular properties, including non-monotonic influences over cell shape, adhesion complex-F-actin association and adhesion localization. Furthermore, we demonstrate talin level-dependent changes in networks of correlations among adhesion/migration properties, particularly in relation to cell migration speed. Importantly, these correlation networks were strongly affected by talin expression heterogeneity within the natural range, implying that this endogenous variation has a broad, quantitatively detectable influence. Overall, we present an accessible analogue method that reveals complex dependencies on talin expression-level, thereby establishing a framework for considering non-linear and non-monotonic effects of protein expression-level heterogeneity in cellular systems.

Reticular adhesions: A new class of adhesion complex that mediates cell-matrix attachment during mitosis

“…Reticular ACs were more numerous than classical focal ACs at all sizes ( Fig.2A), increased in size more frequently (Fig.2B) and were generally localised further from the cell periphery (Fig.2C). There was no correlation between reticular AC size and integrin 5 clustering density, unlike the increased integrin density observed in larger focal ACs (Fig.2D) (Hernández-Varas et al, 2015;Kiss et al, 2015;Lock et al, 2014). This implies molecular-scale differences between the maturation of focal and reticular adhesions, with the latter being more homogenous.…”

“…Immunofluorescence labeling was performed either manually or using liquid-handling robotics (Freedom EVO, Tecan) to minimise experimental variability, as described previously (Kiss et al, 2015). In either case, standardised procedures were used except where otherwise stated.…”

“…Patch Morphology Analysis Dynamic software (Digital Cell Imaging Laboratories, Belgium) was used for analysis of static (fixed) and dynamic (live) cell imaging data, except where otherwise specified. Analysis strategy and parameterisation are as described previously (Hernández-Varas et al, 2015;Kiss et al, 2015;Kowalewski et al, 2015;Lock et al, 2014;Shafqat-Abbasi et al, 2016). Briefly, both cells and intracellular adhesion cohorts were segmented according to pixel intensity gradient analysis.…”

“…Cells were incubated for 48 h then plated for 3 h on 10 µg/ml VN. Fixation and permeabilisation conditions were tuned to retain cytoplasmic talin 2, as described previously (Kiss et al, 2015). Briefly, labelling was performed using liquid-handling robotics (Freedom EVO, Tecan) to reduce experimental variability.…”

Decision letter: An analysis toolbox to explore mesenchymal migration heterogeneity reveals adaptive switching between distinct modes

“…talin acts a critical intracellular regulator of integrin activation (Calderwood et al, 1999;Kiss et al, 2015;Moser et al, 2009;Tadokoro, 2003), and is also one of several CMAC proteins linking integrins to the F-actin cytoskeleton (Schwarz and Gardel, 2012). This enables cellular force application from F-actin, through CMACs, to the ECM, thereby driving membrane protrusion/retraction and cell translocation (Small and Resch, 2005).…”