Quantitative Analysis of Cellular Traction Generation and Actomyosin Dynamics in a 3D Fibrin Matrix

Abstract

Substantial progress has been made in characterizing the mechanisms of tension sensing, adhesion, and traction in cells migrating on two-dimensional (2D), rigid surfaces. However, there are inherent differences in cellular architecture, signaling, and behavior when cells are embedded in the soft and confined environment of the extracellular matrix. Here we present a quantitative analysis of the dynamics of canonical actomyosin cytoskeletal proteins in primary dermal fibroblasts (HFFs) grown in a three-dimensional (3D) fibrin matrix, and correlate these dynamics to protrusion formation and traction generation during migration through the fibrin network. We observe that fibroblasts adopt an elongated cell morphology with adhesive protrusions that cyclically strain the surrounding fibrin matrix. We use GFP-tagged myosin regulatory light chain (MRLC1) and time-lapse confocal imaging to track myosin dynamics as cells explore the fibrin matrix. We observe highly dynamic alterations in MRLC1 localization coupled to both the extension and retraction of cellular protrusions. During protrusion extension, fibrin strain is often oriented bidirectionally several microns behind the protrusion tip and is transmitted via paxillin-dependent adhesions. ROCK inhibition by Y-27632 causes fibroblasts to develop extended protrusions despite a pronounced reduction in matrix deformation and adhesion maturation as judged by a dramatic reduction in matrix-associated paxillin plaques. Ongoing work characterizes the contribution of cytoskeletal contractility in regulating protrusion and adhesion dynamics.