Freeze-fracture analysis of plasma membranes of isolated astrocytes from rat brain

Abstract

Plasma membranes of mature rat astrocytes separated by differential centrifugation have been reported to be intact, based on electron microscopic examination of thin plastic sections. However, the effects of the separation procedure on the internal structure of the plasma membranes are unknown. The degree of membrane integrity is of concern to us since our goal is the separation of astrocytic plasma membranes and characterization of the specific intramembranous particle groups called assemblies. We have taken advantage of the astrocyte membrane-marker, the assembly, in order to monitor, by freeze-fracture, the identity of the separated astrocytes and the integrity of their cell membrane. Since some processes of an astrocyte contain assemblies whereas other processes of the same cell do not, it was also necessary to determine if processes with assemblies were separated by this technique. Astrocytic cell membranes were also examined to determine if trypsinization or the mechanical disruption steps of the separation affected the intramembranous particles. Freeze-fracture of the plasma membranes revealed that the particles were rearranged resulting in patches of clumped intramembranous particles and areas of bare membrane. The assemblies were rearranged rather than lost from the membrane since they could be identified among the clumped particles. More astrocytic plasma membranes contained non-clumped, normally distributed particles in the trypsin treated fractions. The non-trypsinized fractions had more damaged astrocytes with aggregated intramembranous particles and much more cellular debris. We interpret the findings for the non-trypsinized astrocytes as due to greater mechanical stress placed on the cells during tissue disruption. Trypsin treatment lessens this stress, thereby, tending to preserve the normal distribution of intramembranous particles.