Association of glycoconjugates with the cytoskeletal framework.

Abstract

The association of glycoconjugates with the cytoskeletal framework was examined in detergent-extracted cells. Sparse cultures offibroblasts that assemble only minimal amounts of extracellular matrix were extracted under mild conditions with Triton X-100 which remove most of the lipids and soluble cellular proteins. The detergent-resistant framework retains lectin binding sites in the nucleus, in the perinuclear area occupied by the rough endoplasmic reticulumGolgi system of the intact cell, and in a network throughout the cytoskeletal framework. Fluorescent-antibody staining with antibody against collagen type I and fibronectin reveals extensive perinuclear staining of the remnant rough endoplasmic reticulum-Golgi system. In contrast, only sporadic staining of the pericellular area is obtained with these antibodies, in sparse cultures of whole cells. Lectin binding sites were detected in the nucleus and are attributed to chromatin-associated glycoconjugates. They can be removed by DNase under conditions that preserve the cytoplasmic lectin binding sites and the nuclear matrix. The results suggest a high degree of integration of the membrane residues of the cytoplasmic elements and the nuclear matrix with the skeletal framework and indicate a possible role for the glycoconjulgates in this structural integration.The study of subcellular structure and its organization indicates the existence of highly interconnected filamentous networks in the cytoplasm of eucaryotic cells. These include the major filaments, i.e., microfilaments, intermediate filaments, and microtubules, which are also termed "cytoskeleton." A more detailed insight into the organization of the cytoplasmic networks is afforded by using a high-voltage electron microscope to examine critical point-dried whole cells (8,22,23). These studies reveal a dense, highly interconnected three-dimensional network, termed "microtrabeculae" by Wolosewick and Porter (22,23) as Triton X-100, and several laboratories have used these "Triton skeletons" for biochemical and morphological studies (4, 6, 7, 9, 10, 14, 18, 21). This extraction removes most of the lipids and the soluble cellular proteins, leaving intact the structural elements, and reveals aspects of cell structure such as a lamina that forms the outer boundary of the extracted cell (4). The term "skeletal framework" was suggested to include the entire structure obtained after detergent extraction of the intact cell. It consists of the cytoplasmic filaments, the plasma lamina, and possibly the nuclear matrix (6,10). This report is a study of the association of glycoconjugates with the skeletal framework.The results suggest that glycoconjugates and the proteinaceous membrane moieties of the rough endoplasmic reticulum (RER)-Golgi system are intimately associated with the skeletal framework. The nucleus also contains glycoconjugates which can be removed by dissolution of the chromatin. The extraction of chromatin leaves the cytoskeletal lectin binding sites intact together with a nuclear matrix. These studies...

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