Partial purification and characterization of the intercellular bridge from cultured mouse cells

Publisher Website
Google Scholar
Abstract
At the completion of mitosis, the 2 daughter cells are connected by a channel of membrane-bound cytoplasm, the intercellular bridge. This structure contains parallel arrays of spindle microtubules which are associated, at the bridge midline, with a metallophilic band called the midbody. In an effort to characterize midbody components, intercellular bridges were partially purified. The purification consisted of brief sonication of telophase populations of mouse L929 (neoplastic fibroblast) cells in order to shear intercellular bridges from daughter cells, digestion of chromatin by an excess of micrococcal nuclease, and differential centrifugation to enrich for intercellular bridges. EM of these preparations substantiated the identity of the bulk of material as intercellular bridges. After solubilization with sodium dodecyl sulfate, the protein components of these preparations were iodinated with Na125I and separated by 2-dimensional gel electrophoresis. From these analyses, the major polypeptide components of intercellular bridges appear to be tubulin, varying amounts of plasma membrane proteins and a polypeptide with an apparent MW of 42,000. Time-course studies reveal that this polypeptide is first detectable in a pelletable form approximately 30 min after cells are released from metaphase block, reaches maximal spot intensity in late telophase and is no longer detectable in G1 populations. The 42,000 dalton polypeptide is probably a component of the midbody.