Influence of different freeze-fracture pretreatments on the fine structure of Physarum polycephalum. A freeze-fracture and freeze-substitution study

Abstract

The influence of different fixatives (glutaraldehyde, osmium, osmium/glutaraldehyde, and osmium/mercuric chloride) and freeze-protecting agents (glycerol and sucrose) on the fine-structural preservation of micro- and macroplasmodia of the acellular slime mold Physarum polycephalum was investigated in both freeze-substituted and freeze-fractured material. Glutaraldehyde fixation and subsequent infiltration with glycerol or sucrose caused severe destruction in the morphology of plasmodial strands and protoplasmic drops, whereas osmium- or osmium/mercuric chloride fixation prevented the formation of normal fracture planes running through the hydrophobic core of the plasma membranes. A short prefixation in a mixture of osmium/glutaraldehyde followed by postfixation in glutaraldehyde delivered the most satisfactory results in the preservation of the fine structure. For comparison, the distribution of integrated membrane particles (IMP) was analysed in freeze-fracture replicas of unfixed controls as well as glutaraldehyde- and osmium/glutaraldehyde-fixed specimens by evaluating the number of IMP per 1 micrometer 2 in two different plasmodial regions; in the peripheral plasmalemma and in the central plasmalemmal invaginations. In controls not receiving chemical pretreatment and in specimens fixed with osmium/glutaraldehyde, the central plasmalemmal invaginations showed a clearly reduced total amount of IMP (exoplasmic + protoplasmic fracture face: about 3100) as compared with the peripheral plasma membrane (about 3700). In addition both membrane systems were characterized by an asymmetrical distribution of IMP between the protoplasmic fracture face (PF) and the exoplasmic fracture face (EF): the PF:EF ratio (particle partition coefficient) in the peripheral plasma membrane is the same in controls and in osmium/glutaraldehyde-fixed specimens (2.4:1 and 2.5:1, respectively), whereas the PF:EF ratio in the central plasmalemmal invaginations is 1.5:1 in controls and 3.5:1 in fixed specimens. This shows that the membrane of the central plasmalemmal invaginations is more sensitive to chemical fixation than the peripheral plasmalemma. The results point to differences in the physiological properties and functions between the plasmalemma of the cell periphery and the plasmalemma of the invagination system.