Quantitation of Compositional Changes in the Non-Erythroid Membrane Skeleton due to External Forces

Abstract

The spectrin based membrane skeleton plays important roles in the mechanical and biological functions of living cells. Unlike the membrane skeleton of red blood cells, its non-erythroid counterpart has seen very little attention. Yet, it is assumed that also in non-erythrocytes the membrane skeleton is mostly composed of heterotetramers of α-II and β-II spectrin. Because of α-spectrins’ potential role as mechanotransducers, we investigate the effect of mechanical stimulation on the spectrin-ankyrin B membrane skeleton of NIH 3T3 fibroblasts and murine C3H 10T½ cells. Using quantitative fluorescence microscopy and biochemical techniques, the absolute changes in cellular spectrin and ankyrin B content were determined. Before stimulation the spectrins and ankyrin B make up more than 10% of cytosolic and membrane proteins. Thus, they constitute a much more significant part of the non-erythroid cytoskeleton in these cells than previously assumed. Interestingly, both cell types contain all four major isoforms of spectrin. However, while α-II spectrin is the dominant α spectrin in 3T3, in 10T½ cells it is the ‘erythroid’ α-I spectrin. Also, at least half of the α spectrins do not have β-spectrin counterparts and thus cannot be part of the classical heterotetramere structure. Mechanical stimulation decreases the overall spectrin amount by up to 60% with α-spectrins exhibiting the largest loss. In contrast, the number of ankyrin B copies increases by almost 30%. Furthermore, the fraction of polyubiquitinated spectrin actually increases, which in part could account for the reduction in α-spectrins and is in line with their proposed mechanically enhanced self-ubiquitination activity.