Examination of the effects of selected factors on the talin-actin interaction

Schmidt, Jean
Major Professor
Richard M. Robson
Committee Member
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Biochemistry, Biophysics and Molecular Biology

Talin is an adhesion plaque protein believed to be involved in linking actin filaments to cell membranes. In these studies I examined the effects of selected factors on the ability of talin and its 190 kDa proteolytic fragment to interact with actin. A modified method for purifying talin from avian smooth muscle was devised. Cosedimentation assays showed that increasing amounts of talin bound to actin filaments as pH was decreased from 7.4 to 6.4. Talin's ability to increase viscosity of F-actin solutions also was increased with decreasing pH within this range. Electron microscope observations supported a pH-dependent, actin filament crosslinking activity for talin. The ability of talin to crosslink actin filaments was markedly decreased with increasing ionic strength, regardless of pH. Increasing the molar ratio of talin to actin enhanced the interaction. Viscometry studies showed that increasing temperature from 4∘ to 37∘C increased the ability of talin to interact with actin. Talin crosslinked actin filaments into networks and bundles in times as short as 5 min at pH 6.4 and low ionic strength. Talin also bound to and crosslinked preformed actin filaments to the same extent as it did when filaments were formed in its presence. The effects of pH and ionic strength on the ability of talin to bind to actin were shown to be reversible;The 190 kDa talin fragment did not bind actin strongly under conditions that favored the interaction between intact talin and actin (pH 6.4, low ionic strength). However, when the pH was lowered to 6.0, the 190 kDa fragment crosslinked actin filaments into networks and bundles. The effects of the adhesion plaque proteins filamin, vinculin, [alpha]-actinin, and tropomyosin on the ability of talin to cosediment with actin filaments were also studied. Filamin and tropomyosin could reduce the amount of talin that cosedimented with actin, but did not eliminate the talin-actin interaction. Vinculin and [alpha]-actinin did not measurably affect the ability of talin to cosediment with actin. Overall, results from these experiments support a role for talin in the organization and attachment of actin filaments at adhesion plaques.