title: Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia.
abstract: Controlled secretion of a protective extracellular matrix is required for transmission of the infective stage of a large number of protozoan and metazoan parasites. Differentiating trophozoites of the highly minimized protozoan parasite Giardia lamblia secrete the proteinaceous portion of the cyst wall material (CWM) consisting of three paralogous cyst wall proteins (CWP1-3) via organelles termed encystation-specific vesicles (ESVs). Phylogenetic and molecular data indicate that Diplomonads have lost a classical Golgi during reductive evolution. However, neogenesis of ESVs in encysting Giardia trophozoites transiently provides basic Golgi functions by accumulating presorted CWM exported from the ER for maturation. Based on this "minimal Golgi" hypothesis we predicted maturation of ESVs to a trans Golgi-like stage, which would manifest as a sorting event before regulated secretion of the CWM. Here we show that proteolytic processing of pro-CWP2 in maturing ESVs coincides with partitioning of CWM into two fractions, which are sorted and secreted sequentially with different kinetics. This novel sorting function leads to rapid assembly of a structurally defined outer cyst wall, followed by slow secretion of the remaining components. Using live cell microscopy we find direct evidence for condensed core formation in maturing ESVs. Core formation suggests that a mechanism controlled by phase transitions of the CWM from fluid to condensed and back likely drives CWM partitioning and makes sorting and sequential secretion possible. Blocking of CWP2 processing by a protease inhibitor leads to mis-sorting of a CWP2 reporter. Nevertheless, partitioning and sequential secretion of two portions of the CWM are unaffected in these cells. Although these cysts have a normal appearance they are not water resistant and therefore not infective. Our findings suggest that sequential assembly is a basic architectural principle of protective wall formation and requires minimal Golgi sorting functions.
authors.affiliation: Institute of Parasitology, University of Zürich, Zürich, Switzerland.
authors.forename: Christian
authors.initials: C
authors.lastname: Konrad
authors.forename: Cornelia
authors.initials: C
authors.lastname: Spycher
authors.forename: Adrian B
authors.initials: AB
authors.lastname: Hehl
mesh_terms: D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D016829:Giardia lamblia / Q000378:metabolism* / Q000648:ultrastructure*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D021381:Protein Transport / Q000502:physiology; D015800:Protozoan Proteins / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins
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