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  • In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2.

In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2.

Structure (London, England : 1993) (2022-09-24)
Robert Fagiewicz, Corinne Crucifix, Torben Klos, Célia Deville, Bruno Kieffer, Yves Nominé, Johan Busselez, Paola Rossolillo, Helgo Schmidt
ABSTRACT

Cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles, and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis, interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here, we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2. We also describe pH-dependent conformational changes of BicD2 using cryoelectron microscopy (cryo-EM), template-free structure predictions, and biophysical tools. Our results will help define the biochemical parameters for the in vitro reconstitution of higher-order BicD2 protein complexes.

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Sf9 Insect Cells - Novagen, Serum-free, adapted Sf9 cells