At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.

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