Quantitative Phase Imaging of Spreading Fibroblasts Identifies the Role of Focal Adhesion Kinase in the Stabilization of the Cell Rear.

Olga Ramaniuk,Zuzana Klímová,Tomáš Groušl,Tomáš Vomastek

doi:10.3390/biom10081089

Abstract

Cells attaching to the extracellular matrix spontaneously acquire front–rear polarity. This self-organization process comprises spatial activation of polarity signaling networks and the establishment of a protruding cell front and a non-protruding cell rear. Cell polarization also involves the reorganization of cell mass, notably the nucleus that is positioned at the cell rear. It remains unclear, however, how these processes are regulated. Here, using coherence-controlled holographic microscopy (CCHM) for non-invasive live-cell quantitative phase imaging (QPI), we examined the role of the focal adhesion kinase (FAK) and its interacting partner Rack1 in dry mass distribution in spreading Rat2 fibroblasts. We found that FAK-depleted cells adopt an elongated, bipolar phenotype with a high central body mass that gradually decreases toward the ends of the elongated processes. Further characterization of spreading cells showed that FAK-depleted cells are incapable of forming a stable rear; rather, they form two distally positioned protruding regions. Continuous protrusions at opposite sides results in an elongated cell shape. In contrast, Rack1-depleted cells are round and large with the cell mass sharply dropping from the nuclear area towards the basal side. We propose that FAK and Rack1 act differently yet coordinately to establish front–rear polarity in spreading cells.

Highlights

The directional migration of mesenchymal cells is an adhesion-dependent process characterized by a repetition of the cell’s front protrusion, rear retraction, and cell body forward translocation [1]
The phenotypes of focal adhesion kinase (FAK)- and Rack1-depleted cells are different as Rack1-depleted cells spread with radial symmetry without establishing the bipolar phenotype. These results suggest that FAK and Rack1 differently regulate the development of the cell rear and the establishment of front–rear polarity in spreading cells
We investigated cell dry mass distribution in cells treated with siRNA targeting the FAK or Rack1 mRNAs

Summary

Introduction

The directional migration of mesenchymal cells is an adhesion-dependent process characterized by a repetition of the cell’s front protrusion, rear retraction, and cell body forward translocation [1]. Efficient migration requires the establishment and maintenance of front–rear polarity that allows a migrating cell to differently regulate the processes at leading and trailing edges. Rho-family GTPase Rac is the central activator of actin polymerization; Cdc and RhoA are activated in this region and participate in the regulation of actin polymerization and cell edge dynamics [3,4,5]. Membrane protrusions are coupled to the extracellular matrix (ECM) through small dot-like integrin mediated adhesions [2,7,8]. These stationary yet dynamic adhesions stabilize the protrusions and are essential to transform part of the polymerization mechanical force into a protrusion [9]. The protruding leading edge and adhesions serve as the sites of the formation of dorsal stress fibers, transverse actin arcs, and perinuclear actin fibers [10,11,12]

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Conclusion