EFFECT OF MECHANICAL LOADING ON CYTOSKELETAL STRUCTURE

Participants: J.D. Miller, M.R. Moalli, S.A. Goldstein

Keywords: mechanotransduction, cytoskeleton, focal adhesion

Introduction

It is well known that tissues and their component cells respond to mechanical forces. Bone, for example, remodels to better support an applied load. Cells in culture respond to fluid shear and to stretch by induction of cell division and by activation of a number of well-defined signal transduction pathways. The process by which cells sense forces in their environment and then respond appropriately is known as mechanotransduction, i.e.- the conversion of an inherently mechanical signal into a biological one. The integrin family of trans-membrane proteins may play a role in passing the signal from the outside milieu to the interior of the cell because they bind to the extracellular matrix through their extracellular domain as well as to the cytoskeleton via their cytoplasmic domain. The cytoskeletal connection is mediated by a group of proteins known as a focal adhesion complex, which bind to both integrin as well as to actin microfillaments. Force could then be transmitted from the extracellular matrix, through integrin and the focal adhesion complex/cytoskeleton, to the cytoplasm. If so, cells may respond to mechanical load by modulating focal adhesions and/or the cytoskeleton. Our long term goals are to evaluate these pathways in cells loaded by fluid shear, hydrostatic and biaxial substrate strain. These experiments will also be conducted with in-vivo models to test for contrasts of response of the cells with and without a normal extracellular matrix. This specific project was designed to begin to establish our paradigm and assays and focused on hydrostatic loading.

Materials and Methods

Cell isolation and culture

Established fibroblast cell line Rat-2 was grown in Dulbecco’s Modified Eagle’s Medium+10% fetal calf serum.

Rat calvarial cells were isolated by centrifugation following collagenase digestion of newborn rat skulls. Cells were plated in alpha-Minimal Essential Medium+10% fetal calf serum and assayed directly without passaging them.

Cell hydrostatic pressure loading

Cells to be loaded were plated onto LabTek 8 well glass slides (Nunc) and grown at 37degrees C for 12-48 hours. Loading was performed in a sealed chamber to which a defined hydrostatic pressure could be applied. The chamber was placed in a tissue culture incubator at 37 degrees and was pre-equilibrated with the incubator atmosphere prior to sealing. Cyclic loading from 0-2 psi at 1 Hz was carried out for 30 minutes.

Immunofluoresence

Following loading, cells were rinsed in phosphate buffered saline at pH-7.4 (PBS) and then fixed in 3.7% paraformaldehyde/PBS for 15 minutes at room temperature followed by a PBS wash. Cells were then permeabilized in 0.4% Triton X-100/PBS for 15 minutes at room temperature. After a PBS wash, blocking was performed in 1% bovine serum albumin/PBS for 20 minutes at room temperature. Mouse monoclonal antibodies directed against paxillin, src, focal adhesion kinase (Upstate Biotechnology) and rho A (Santa Cruz Biotechnology, Inc.) proteins were diluted to 10 micrograms/milliliter in 1% bovine serum albumin/PBS and incubated with the cells for 12 hours at 4 degrees C. Following a PBS wash, cells were incubated with Alexa flourescently labeled goat anti-mouse IgG antibody (Molecular Probes) at 10 micrograms/milliliter for 4 hours at room temperature. After washing with PBS, slides were mounted in Vectashield shield anti-fade compound (Vector Laboratories, Inc.) and examined using a Leitz Aristoplan fluorescence microscope. Images were recorded with a Sony digital camera for subsequent processing.

Progress

Focal adhesion proteins FAK and paxillin as well as the associated proteins src and rho have been studied +/- hydrostatic loading in both the rat fibroblast cell line, Rat-2, and in rat calvarial osteoblasts. The study will now be extended to include cytoskeletal proteins as well as alternate loading regimens.

Results

No change in focal adhesion pattern, intensity or number has been noted in response to loading. No change in subcellular localization of src or rho has been noted in response to loading. This was not unexpected, since related studies in the literature found similar results in gross cellular responses to hydrostatic load. This study does verify our system and establishes one of several methods for loading cells that will be established in the next year.