Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures.

Abstract

The intracellular precipitation of nonerythrocyte spectrin was achieved by the microinjection into cells of either a monoclonal antibody (IgM) directed against the .alpha. chain of nonerythrocyte spectrin or an affinity-purified polyclonal antibody raised against bovine brain spectrin (fodrin). This antibody-induced precipitation of spectrin was observed in fibroblastic and epithelial cell types, including embryonic bovine tracheal fibroblasts, a bovine kidney epithelial cell line (MDBK), Hela cells, gerbil fibroma cells and fibroblast lines of human and mouse origins. The precipitation of the spectrin was specific and 2 proteins with a similar distribution to the nonerythrocyte spectrin were not induced to co-precipitate in the spectrin aggregates. Comparing the 2 types of antibody microinjected, the affinity-purified polyclonal antibody resulted in more compact aggregates of spectrin and these were more frequently aligned with microfilament bundles. The rate at which the spectrin aggregates were cleared into presumptive lysosomes varied with different cell types: in some such as the bovine kidney epithelial cells, this appeared complete within 3 h after microinjection, whereas in some of the fibroblasts the spectrin aggregates were prominent in the cytoplasm at 24 and even 48 h after microinjection. Microfilament bundles appeared unaffected by the aggregation of spectrin. The integrity of the actin microfilament bundles does not require nonerythrocyte spectrin and that most probably these structures are linked at their termini to the membrane through proteins other than nonerythrocyte spectrin. No effect of the intracellular spectrin precipitation was observed on cell shape or on the distribution of coated vesicles or microtubules. The aggregation of the nonerythrocyte spectrin did affect the distribution of the vimentin type of intermediate filaments in most of the cell types studied. These filaments became more distorted and condensed, but generally did not collapse around the nucleus as occurs following microtubule disruption induced by colchicine treatment. The clumped intermediate filaments were frequently seen to coincide with regions of aggregated spectrin. This aggregation of intermediate filaments was not induced by microinjection of irrelevant antibodies, nor was it induced by the monoclonal antibody against spectrin in cells with which it did not cross-react. There may exist some association (either direct or indirect) between the vimentin type of intermediate filaments and nonerythrocyte spectrin and that possibly the nonerythrocyte spectrin is involved in linking intermediate filaments to the plasma membrane. In contrast to the situation in erythrocytes, the data do not support a major role for a nonerythrocyte spectrin network as the structural basis of cytoskeletal organization underlying the plasma membrane in fibroblasts. Instead, nonerythrocyte spectrin appears to be just one element in a far more complex submembranous meshwork than that found in erythrocytes.