The Nurse cell-parasite complex is a unique feature of infection with Trichinella spiralis. It arises from a portion of myocyte and is induced by the invading worm during its first 14 days of life as an intracellular parasite. The Nurse cell is characterized by the presence of abundant smooth membranes throughout its cytoplasm, dysfunctional mitochondria, 40+ enlarged nuclei, mostly found at either end of the cytoplasm, and an outer acellular capsule composed mainly of collagen. In addition, the Nurse cell is surrounded by a circulatory rete that is also induced during infection. Nothing is known as to any of the molecular signals from the parasite that coordinate all the changes necessary for the formation of this unusual host cell. We have described a secreted protein specific to the infectious L1 larva, 43 kDa (apparent mw), that is located in all enlarged nuclei of the Nurse cell. We have cloned and sequenced the cDNA encoding this protein and, using antibodies against a fusion protein consisting of the last 95 aa have identified it in the nucleoplasm, thus clarifying which protein is in the nuclei of the Nurse cell. We now propose to further characterize the association of this protein with the Nurse cell by determining its pattern of differential expression employing Northern analysis and in situ hybridization. Its precise nuclear localization will also be determined using co-focal microscopy and immunoelectron microscopy. We will relate these findings to two other Nurse cell-specific processes-namely, over-expression of collagen during capsule formation and the acquisition of the circulatory rete, by determining their patterns of differential expression using similar methods. All of these studies will employ synchronized infections in mouse muscle. Functional motifs of the 43 kDa molecule identified by computer- assisted structural analysis (eg. HLH, Cxxx box, and the ras exon 1 phosphate binding site) will be investigated by domain exchange, deletion and mutation. An in vitro system employing myoblasts and fibroblasts will be explored for their potential in assisting with investigating functional motifs in which several versions of the 43 kDa protein will be transfected into them.
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