Authors

Joseph H. R. HetmanskiID*, Matthew C. JonesID, Fatima Chunara, Jean-Marc SchwartzID, Patrick T. CaswellID*

Affiliations

Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester, United Kingdom

Topic

Cell Biology, Time-Lapse Microscopy

Abstract

Cell migration in 3D microenvironments is a complex process which depends on the coordinated activity of leading edge protrusive force and rear retraction in a push-pull mechanism. While the potentiation of protrusions has been widely studied, the precise signalling and mechanical events that lead to retraction of the cell rear are much less well understood, particularly in physiological 3D extra-cellular matrix (ECM). We previously discovered that rear retraction in fast moving cells is a highly dynamic process involving the precise spatiotemporal interplay of mechanosensing by caveolae and signalling through RhoA. To further interrogate the dynamics of rear retraction, we have adopted three distinct mathematical modelling approaches here based on (i) Boolean logic, (ii) deterministic kinetic ordinary differential equations (ODEs) and (iii) stochastic simulations. The aims of this multi-faceted approach are twofold: firstly to derive new biological insight into cell rear dynamics via generation of testable hypotheses and predictions; and secondly to compare and contrast the distinct modelling approaches when used to describe the same, relatively under-studied system. Overall, our modelling approaches complement each other, suggesting that such a multi-faceted approach is more informative than methods based on a single modelling technique to interrogate biological systems. Whilst Boolean logic was not able to fully recapitulate the complexity of rear retraction signalling, an ODE model could make plausible population level predictions. Stochastic simulations added a further level of complexity by accurately mimicking previous experimental findings and acting as a single cell simulator. Our approach highlighted the unanticipated role for CDK1 in rear retraction, a prediction we confirmed experimentally. Moreover, our models led to a novel prediction regarding the potential existence of a ‘set point’ in local stiffness gradients that promotes polarisation and rapid rear retraction.

DOI: https://dx.doi.org/10.1371%2Fjournal.pcbi.1008213

Extract

All cells were seeded sparsely in cell derived matrix (CDM, [51]) and imaged for 16h using on an Eclipse Ti inverted microscope (Nikon) with a 20x/ 0.45 SPlan Fluar objective and the Nikon filter sets for Brightfield and a pE-300 LED (CoolLED) fluorescent light source with imaging software NIS Elements AR.46.00.0.

Product Associated Features

A 16 hour time-lapse assay was performed to analyse cell migration following CDK1 siRNA and inhibitor treatment. The pE-300 Series is optimised for live cell time-lapse applications, where irradiance can be tightly controlled to reduce photobleaching and photodamage even over longer protocols.

Product Type

pE-300white, pE-300lite, pE-300ultra

Journal

PLoS Comput. Biol.

Year of Publication

2021

Country of Publication

UK