Fracture—Permeation: A technique to assess cytoplasm compactness after glutaraldehyde‐fixation

Abstract

AbstractWe have developed a method—“fracture‐permeation”—to assess the compactness of the cytoplasm in glutaraldehyde‐fixed cells. Cells or tissues are fixed in glutaraldehyde, impregnated with glycerol, and frozen in liquid nitrogen. The cells are freeze‐fractured, thawed, deglycerinated, and immersed in concentrated solutions of globular proteins. In initial experiments, we used native ferritin (NF) to permeate model matrices made of bovine serum albumin (BSA). We show that permeation depends on the concentraion of proteins within the cross‐linked matrix: NF permeates matrices made from 10 or 15% (w/v) BSA solutions but do not permeate matrices made from solutions with 20% (w/v) protein or more. With freeze‐fractured cells, ferritin molecules were unable to permeate the cross‐linked cytoplasm of fungal zoospores and cysts, used here as examples of resting cells. In human lymphocytes from peripheral blood, permeation of ferritin was limited or absent, but it became massive in cells activated by phytohemagglutinin. Massive permeation of ferritin was also observed within the cytoplasmic matrix of other active cells (fungal sporangia, germinating cysts). In the examined resting cells, glutaraldehyde crosslinks the proteins into a dense matrix that effectively excludes ferritin (diameter 12 nm). These findings cannot be explained by models that envisage all cytoplasmic proteins congregated into a single‐phase microtrabecular lattice with the nature and dimensions proposed by Porter and co‐workers. We conclude that compactness of the cytoplasm matrix depends on the physiological state of the cell: It varies through differentiation and is related to the degree of cellular activity.