Pattern of lipids associated with cytoskeletal protein prepared as Triton X 100 insoluble residues in some single cell types.

Abstract

The cytoskeletal proteins from erythrocytes, lymphoid cells, unstimulated and stimulated platelets, HeLa cells, and Ehrlich ascites cells were prepared as Triton X 100 insoluble residues. The pellet was extracted using the Bligh-Dyer procedure. After separation of the lipids by thin-layer chromatography, phospholipids and neutral lipids were estimated and the lipid pattern was compared with the lipid composition of the total cell. The percentage of the lipids associated with the Triton X 100 insoluble pellet ranged between 10 and 50 depending on the lipid and the cell type. Despite of the heterogenous protein composition of the residue in the different cells involving microfilaments and intermediate filaments together with associated proteins and minor components, in all cells sphingomyeline (Sph) and free fatty acids (FA) could be found in outstanding contents. In HeLa cells we found beside the high proportion of Sph a different species pattern of diacyl-, alkylacyl-, and alkenylacyl classes of endogenous diacylglycerol (DG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The discussion involved the data from literature showing lipid associations with all 3 classes of cytoskeletal filaments: microtubules, intermediate filaments, and microfilaments. These results were obtained by histological observation, by in vitro binding studies between cytoskeletal proteins and purified lipids, and--as we have practised--by lipid analysis after extraction of the more or less purified cytoskeleton. Artefacts could not be excluded, but the different lipid pattern in the total cell compared with the cytoskeletal let us assume that the results can not be explained by coprecipitation of micelles or organelle remnants with the Triton X 100 insoluble residue alone. An in vivo association of lipids, mainly of Sph, with F-actin and/or associated proteins might be concluded.