Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip.

Giardia lamblia trophozoites contain a complex endomembrane system as demonstrated by fluorescence and cryoelectron microscopy. The endomembrane system was weakly detected in live cells using the fluorescent membrane dye 3,3'-dihexyloxacarbocyanine iodide. The definitive identification of endoplasmic reticulum required the development of a molecular label. We expressed Giardial Bip in Escherichia coli and raised a polyclonal antibody to the purified protein. In western blots, the antibody was specific for Giardial Bip and did not react with human, monkey and rodent homologs. By immunofluorescence microscopy in methanol fixed cells the antibody visualized tubular structures and other subcellular components that required characterization by electron microscopy. Using cryotechniques we directly demonstrate the presence of a complex endomembrane system at the ultrastructural level. In conjunction with Bip immunogold labeling of cryosections we identify: (1) endoplasmic reticulum cisternae and tubules; (2) stacked perinuclear membranes; and (3) Bip presence in the nuclear envelope. Both the endoplasmic reticulum and nuclear envelope were found either with or without a cleft region suggesting each may contain common specialized sub-regions. In stacked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricted to peripheral layers, also suggesting specialized sub-regions. Labeled endomembrane systems could be observed associated with microtubule structures, including axonemes and the adhesive disk. The presence of an extensive endomembrane system in Giardia lamblia, which represents one of the earliest diverging eukaryotic species, supports the view that both the nucleus and endomembrane system co-evolved in a common ancestor of eukaryotic cells.