Mechanisms of Nuclear, Spindle and Mitotic Chromosome Scaling

Abstract

Cell size varies widely among different organisms as well as within the same organism in different tissue types and during development, placing variable functional demands on internal structures. A fundamental question is how essential subcellular components such as the nucleus, mitotic spindle and chromosomes are regulated to accommodate cell size differences. Xenopus frogs offer two physiological contexts in which we can investigate this question. First, we can compare Xenopus laevis to the smaller, related species Xenopus tropicalis, which lays smaller eggs and has proportionally smaller cells throughout development. Second, we can compare different stages of Xenopus laevis embryogenesis, as the ∼1 millimeter diameter egg rapidly cleaves to form smaller blastomeres, which by the 15th division are reduced to 40 microns across. A unique aspect of our approach is to prepare cytoplasmic extracts from eggs and embryos that recapitulate organelle scaling in vitro, which we can use to identify molecular differences that underlie size changes. We identified two factors, importin α and Ntf2, whose levels alter nuclear import and are largely responsible for the difference in nuclear size between the two frog species. With respect to spindle size regulation, based on predictions from a 2-D meiotic spindle simulation, we identified katanin-dependent MT severing as an activity reduced in X. laevis compared X. tropicalis. Interestingly, X. tropicalis lacks an inhibitory Aurora B phosphorylation site in the p60 catalytic subunit of katanin found in X. laevis at Ser131, which is largely responsible for the difference in spindle length. Mitotic chromosomes must also decrease in size to permit their proper segregation in smaller cells. We have established an in vitro system that recapitulates chromosome scaling during development, and are now poised to elucidate the underlying molecular mechanisms.