Filament organization revealed in platinum replicas of freeze-dried cytoskeletons.

TLDR

Freeze‑dried cytoskeletons resemble the microtrabeculae seen in whole cells, but without fixation artifacts, enabling high‑resolution study of filament organization across cytoplasmic regions and developmental stages. The study aims to describe the spatial organization of actin and intermediate filaments in different regions of fully spread mouse fibroblasts. Rapid freezing, freeze‑drying, platinum rotary replication, and TEM imaging were used to visualize the cytoskeleton. The technique resolves individual filaments, distinguishing actin, microtubules, and intermediate filaments by surface substructure, with identification confirmed by antibody labeling and extraction, and preserves the three‑dimensional cytoskeletal architecture.

Abstract

This report presents the appearance of rapidly frozen, freeze-dried cytoskeletons that have been rotary replicated with platinum and viewed in the transmission electron microscope. The resolution of this method is sufficient to visualize individual filaments in the cytoskeleton and to discriminate among actin, microtubules, and intermediate filaments solely by their surface substructure. This identification has been confirmed by specific decoration with antibodies and selective extraction of individual filament types, and correlated with light microscope immunocytochemistry and gel electrophoresis patterns. The freeze-drying preserves a remarkable degree of three-dimensionality in the organization of these cytoskeletons. They look strikingly similar to the meshwork of strands or "microtrabeculae" seen in the cytoplasm of whole cells by high voltage electron microscopy, in that the filaments form a lattice of the same configutation and with the same proportions of open area as the microtrabeculae seen in whole cells. The major differences between these two views of the structural elements of the cytoplasmic matrix can be attributed to the effects of aldehyde fixation and dehydration. Freeze-dried cytoskeletons thus provide an opportunity to study--at high resolution and in the absence of problems caused by chemical fixation--the detailed organization of filaments in different regions of the cytoplasm and at different stages of cell development. In this report the pattern of actin and intermediate filament organization in various regions of fully spread mouse fibroblasts is described.