The cytoskeleton of murine leukemia virus (MuLV)‐infected mouse fibroblasts as observed under varying conditions of formaldehyde fixation

Abstract

Mouse fibroblasts chronically infected with Moloney murine leukemia virus (MuLV) were fixed using variable amounts of formaldehyde, then examined by indirect immunofluorescence light microscopy. Several antisera were employed to detect both external and internal antigens associated with the cells, eg, MuLV gp70, tubulin, vimentin, and actin. Our results indicate that the cell membranes could be partially permeabilized to IgG molecules directed against the three cytoskeletal antigens only after 3.7%, but not 1 %, formaldehyde treatment. Complete permeabilization was achieved by subsequent acetone treatment of cells after 3.7 % formaldehyde fixation. In such cells, normal-appearing cytoskeletal networks of microtubules and intermediate filaments were observed. Stress fibers were also seen; however, they appeared less numerous and thinner than those of uninfected mouse fibroblasts. Further, a significant amounts of F-actin fluorescence was localized in granules in the cytoplasm of infected cells. Similar observations were made using JLS-V9 mouse cells chronically infected with 334C virus, another MuLV. These results taken together suggest that subtle differences exist in the organization of actin within MuLV-infected and uninfected mouse fibroblasts.

Probes for Fluorescent Visualization of Specific Cellular Organelles

“…It functions both statically as the structural platform of the cell and dynamically as a means to facilitate organelle and cellular migration. It is essential for processes, such as muscle contraction, chromosome segregation, cytokinesis, endocytosis, intracellular vesicular transport, and migration of viruses through the cell [21,[58][59][60][61]. Furthermore, it can function to form specialized structures for synaptogenesis and neurite outgrowth, for movement of the cell (lamellipodia, filopodia and podosomes) or for virus and bacterial egress from infected cells [21,[58][59][60][62][63][64].…”

“…It is essential for processes, such as muscle contraction, chromosome segregation, cytokinesis, endocytosis, intracellular vesicular transport, and migration of viruses through the cell [21,[58][59][60][61]. Furthermore, it can function to form specialized structures for synaptogenesis and neurite outgrowth, for movement of the cell (lamellipodia, filopodia and podosomes) or for virus and bacterial egress from infected cells [21,[58][59][60][62][63][64]. The cell cytoskeleton contains three main types of filaments: microfilaments, intermediate filaments (neurofilaments in neurons), and microtubules.…”

“…Alcohol and acetone based organic solvents remove lipids and dehydrate cells, thereby permeabilizing the plasma membrane, while also precipitating proteins onto the remaining cellular architecture. By contrast, aldehyde-based fixatives crosslink macromolecules, including proteins, and are excellent at preserving cell morphology and architecture making them a reliable choice for visualization of cellular organelles [60,82]. Formaldehyde type fixatives are most commonly utilized for these types of studies, with the most common being formaldehyde, paraformaldehyde (a dry powder polymer of formaldehyde), and formalin (a saturated 37-40% formaldehyde liquid that normally contains up to 15% methanol to prevent repolymerization to paraformaldehyde).…”