ABSTRACT
To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244-538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination.
Subject(s)
CDC2 Protein Kinase/metabolism , Calmodulin-Binding Proteins/metabolism , Cell Division , Cell Membrane/physiology , Actins/metabolism , Amino Acid Sequence , Animals , CHO Cells , Calmodulin-Binding Proteins/genetics , Cell Size , Cricetinae , Fibroblasts , Gene Expression , Humans , Mutagenesis, Site-Directed , Phosphorylation , Point Mutation , Time Factors , Tropomyosin/analysis , Tropomyosin/isolation & purificationABSTRACT
Two hybridoma clones, CMYL3 and CMYL30, were generated by immunizing Balb/c mice with excysted oocysts of Cryptosporidium muris. Both clones secreted monoclonal antibodies against an oocyst-wall antigen with apparent molecular mass of 250 kDa (called CM250) from C. muris and C. parvum. The epitope appeared to be periodate-sensitive, suggesting the involvement of a carbohydrate moiety. Immunofluorescence and confocal microscopy on purified oocysts and infected mouse tissues revealed staining confined to the oocyst wall of both Cryptosporidium species. Immunogold labeling further revealed the presence of the CM250 antigen in electron-dense vesicles and cytoplasm of developing macrogametocytes, and ultimately localized to the oocyst wall of mature oocysts. Both antibodies cross-reacted with C. serpentis oocysts but did not recognize the other enteropathogenic protozoans Giardia muris, Eimeria falciformis and E. nischulz. These antibodies may be valuable tools for the analysis of oocyst-wall formation in Cryptosporidium and characterization of the common antigen.
