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Many proteins act in multiple pathways which complicates phenotypic analysis. Xenopus egg extracts reconstitute complex reactions in vitro, and this can be used to develop assays that isolate a single function of a multifunctional protein. We have applied this system to study regulators of cytoplasmic dynein (dynein), which has numerous roles in the cell including trafficking, nuclear migration, and mitotic spindle formation. Here we describe a functional assay to specifically study the regulation of spindle pole self-organization by dynein and summarize an experimental approach that was used to perform a structure-function analysis of its regulator Ndel1. The approaches presented here can be generalized to isolate a single function of other multifunctional proteins.

Ndel1 has been implicated in a variety of dynein-related processes, but its specific function is unclear. Here we describe an experimental approach to evaluate a role of Ndel1 in dynein-dependent microtubule self-organization using Ran-mediated asters in meiotic Xenopus egg extracts. We demonstrate that extracts depleted of Ndel1 are unable to form asters and that this defect can be rescued by the addition of recombinant N-terminal coiled-coil domain of Ndel1. Ndel1-dependent microtubule self-organization requires an interaction between Ndel1 and dynein, which is mediated by the dimerization fragment of the coiled-coil. Full rescue by the coiled-coil domain requires LIS1 binding, and increasing LIS1 concentration partly rescues aster formation, suggesting that Ndel1 is a recruitment factor for LIS1. The interactions between Ndel1 and its binding partners are positively regulated by phosphorylation of the unstructured C terminus. Together, our results provide important insights into how Ndel1 acts as a regulated scaffold to temporally and spatially regulate dynein.

Tetraspanin CD9 is the only protein of the oocyte membrane (oolemma) known to be required for the fusion of gametes during fertilization in the mouse. Using electron microscopy and immunostaining we examined the differences in localization of CD9 between ovulated oocytes, zygotes and parthenogenetically activated eggs (parthenogenotes). Changes in ultrastructure of oolemma, which take place in oocytes after fertilization or artificial activation, were also assessed. We demonstrated that after fertilization the level of CD9 present on microvilli of zygote was two times lower than its level on the oolemma of the oocyte. In addition, we showed that the distribution of microvilli is less uniform in the zygotes than in the unfertilized oocytes. We propose that the changes of microvilli distribution and their CD9 content are responsible for the development of the oocyte membrane block to sperm penetration.