Early events in cell spreading as a model for quantitative analysis of biomechanical events

Abstract

In this review, we focus on the early events in the process of fibroblast spreading on fibronectin matrices of different rigidities. We present a focused position piece that illustrates the many different tests that a cell makes of its environment before it establishes mature matrix adhesions. When a fibroblast is placed on fibronectin-coated glass surfaces at 37°C, it typically spreads and polarizes within 20-40 min primarily through αvβ3 integrin binding to fibronectin. In that short period, the cell goes through three major phases that involve binding, integrin activation, spreading, and mechanical testing of the surface. The advantage of using the model system of cell spreading from the unattached state is that it is highly reproducible and the stages that the cell undergoes can thus be studied in a highly quantitative manner, in both space and time. The mechanical and biochemical parameters that matter in this example are often surprising because of both the large number of tests that occur and the precision of the tests. We discuss our current understanding of those tests, the decision tree that is involved in this process, and an extension to the behavior of the cells at longer time periods when mature adhesions develop. Because many other matrices and integrins are involved in cell-matrix adhesion, this model system gives us a limited view of a subset of cellular behaviors that can occur. However, by defining one cellular process at a molecular level, we know more of what to expect when defining other processes. Because each cellular process will involve some different proteins, a molecular understanding of multiple functions operating within a given cell can lead to strategies to selectively block a function.

Figure 1

Early steps of fibroblast spreading on fibronectin substrates. A fibroblast cell goes through several distinct phases when plated on a fibronectin surface. Attachment is followed by the formation of initial integrin clusters, best observed on supported lipid bilayer, using the mobile Arg-Gly-Asp (RGD) peptide as the integrin ligand (P0; figure modified from Yu et al. (27) (top panel) and Iskratsch et al. (28) (bottom panel). Cluster formation is accompanied by binding of the actin assembly protein FHOD1 and actin assembly (see kymograph). Myosin II then contracts the clusters (not shown), activating fast actin polymerization and cell spreading. As the cell spreads out, the membrane folds and blebbs flatten out and feed into the expanding membrane surface (P1, epifluorescence images of the membrane dye FM1-43; images modified from Gauthier et al. (38). Additionally, activation of exocytosis increases the availability of phospholipids. A decrease in the pool of available lipids leads to a sharp rise in membrane tension and a switch to the subsequent protrusion-retraction phase (P2). During the P2 phase, the cell tests its environment with local contractions (see the force map on PDMS pillar arrays from Ghassemi et al. (34)) during the periodic protrusion-retractions (kymograph of the spreading cell edge from Giannone et al. (3)). To see this figure in color, go online.