Analysis of rab10 localization in sea urchin embryonic cells by three-dimensional reconstruction.

Abstract

Rabs are a subfamily of ras-like GTPases required for membrane traffic in eukaryotic cells. In this report we describe the analysis of a rab10 GTPase expressed during sea urchin development. Protein distance measurements suggest that rab10 is less evolutionarily conserved than rabs 1, 2, and 3, particularly in the hypervariable C-terminus responsible for membrane targeting. Immunoblots and immunofluorescent stainings show that rab10 protein (rab10p) is expressed during all stages of sea urchin early development and in all embryonic cell types. Iterative deconvolutions of immunofluorescently stained embryos reveal that rab10p is localized to an extensive tubular network. Rab10p is not exclusively localized to the endoplasmic reticulum, as identified by anti-calsequestrin immunofluorescence. Double-labeling experiments with anti-rab10 antisera and wheat germ agglutinin, a trans-Golgi and trans-Golgi network (TGN) marker, demonstrate that rab10p is not localized to the trans-Golgi/TGN. Three-dimensional reconstructions of immunofluorescently labeled sea urchin embryonic cells show that tubules with greater concentrations of rab10p are closely apposed to trans-Golgi/TGN in a cis orientation-suggesting localization of rab10p to the cis-Golgi network. In mammalian cell lines, Rab10 has been localized to the trans-Golgi/trans-Golgi network (Y.-T. Chen et al., 1993, Proc. Natl. Acad. Sci. USA 90, 6508-6512). The localization of rab10 may not have been evolutionarily conserved between echinoderms and mammals because of the high rate of change in the hypervariable domain.