The Cellular Content of Non-Erythroid Spectrins and Ankyrins is Modulated by External Forces

Abstract

Cells are exposed to mechanical forces in the form of stretch, shear stress, and hydro-static pressure and they transduce those forces into physical as well as biochemical responses. An important, mechanical cell-component that also takes part in this process of mechano-transduction is the spectrin based membrane skeleton. In the context of non-erythroid cells this structure has seen very little attention so far, while its counterpart in erythroid cells, which lack any other significant mechanical structure, has been extensively studied. For example, it was investigated that the erythroid α-spectrin has an E2/E3 ubiquitination site that allows it to either ubiquitinate itself or spectrin associated Ankyrin. Interestingly, the corresponding domain in non-erythroid α-Spectrins is both conserved and is located at a mechano-sensitive site within a tri-helical spectrin repeat. Thus, we looked at the change in non-erythroid Spectrins' and Ankyrins', suspected targets' contents in NIH-3T3 Fibroblasts and multipotent-10T1/2 cells as they are exposed to stretch magnitudes of 20% at a frequency of 1Hz. Using a combination of fluorescence microscopy, quantitative gel analysis and immunoprecipitation, we find that spectrin content drastically decreases by up to 80%, while ankyrin content increases by up to 30% in the two cell types when they are exposed to bi-axial stretch. Technically, we were also able to establish that quantitative fluorescence microscopy on immunostained fixed cells is as suitable to measure relative changes in protein content as immunoprecipitated Coomassie blue stained protein gels. Finally, our results from co-immunoprecipitation and Western blotting indeed suggest that the cause of these changes in protein content is the poly-ubiqutination activity of spectrin on itself as well as ankyrin. Since both proteins act also as scaffolds for membrane associated proteins, these force induced changes should also impact membrane organization and structure.