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 endomemb rane system was weakly detected in live cells using the fluorescent me mbrane dye 3,3'-dihexyloxacarbocyanine iodide. The definitive identifi cation of endoplasmic reticulum required the development of a molecula r label, We expressed Giardial Bip in Escherichia coli and raised a po lyclonal antibody to the purified protein, In western blots, the antib ody was specific for Giardial Bip and did not react with human, monkey and rodent homologs, By immunofluorescence microscopy in methanol fix ed cells the antibody visualized tubular structures and other subcellu lar 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; an d (3) Bip presence in the nuclear envelope, Both the endoplasmic retic ulum and nuclear envelope were found either with or without a cleft re gion suggesting each may contain common specialized sub-regions, In st acked perinuclear membranes, which may represent either multilamellar endoplasmic reticulum or a Golgi apparatus, Bip labeling was restricte d to peripheral layers, also suggesting specialized sub-regions. Label ed 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 th at both the nucleus and endomembrane system co-evolved in a common anc estor of eukaryotic cells.