Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozen/freeze-substituted protoplasts of white spruce (Picea glauca)

Abstract

An ultrastructural study of endocytosis has been made for the first time in protoplasts of a gymnosperm, white spruce (Picea glauca), fixed by high-pressure freezing and freeze substitution. Protoplasts derived from the WS1 line of suspension-cultured embryogenic white spruce were labelled with cationized ferritin, a non-specific marker of the plasma membrane. The timing of cationized ferritin uptake and its subcellular distribution were determined by fixing protoplasts at various intervals after labelling. To address concerns about using chemical fixation to study the membrane-bound transport of cationized ferritin, protoplasts were fixed both by conventional glutaraldehyde fixation and by rapid freezing in a Balzers high-pressure freezing apparatus (followed by freeze substitution). Cationized ferritin appeared rapidly in coated pits and coated vesicles after labelling. Later it was present in uncoated vesicles, and in Golgi bodies, trans-Golgi membranes and partially coated reticula, then subsequently in multivesicular bodies, which may ultimately fuse with and deliver their contents to lytic vacuoles. The results show that the time course and pathway of cationized ferritin uptake in the gymnosperm white spruce is very similar to the time course and pathway elucidated for cationized ferritin uptake in the angiosperm soybean. High-pressure freezing yielded much better preservation of intracellular membranes and organelles, although plasma membranes appeared ruffled. Protoplasts fixed by both methods possessed numerous smooth vesicles in the cortex and smooth invaginations of the plasma membrane. These became labelled with cationized ferritin, but apparently did not contribute directly to the internalization of cationized ferritin, except via the formation of coated pits and vesicles from their surfaces.