An alternative splice isoform of mouse CDK5RAP2 induced cytoplasmic microtubule nucleation

Akari Nakamura; Mami Ikeda; Seina Kusayanagi; Kensuke Hayashi

doi:10.1016/j.ibneur.2022.09.004

An alternative splice isoform of mouse CDK5RAP2 induced cytoplasmic microtubule nucleation

IBRO Neurosci Rep. 2022 Sep 15:13:264-273. doi: 10.1016/j.ibneur.2022.09.004. eCollection 2022 Dec.

Authors

Akari Nakamura¹, Mami Ikeda¹, Seina Kusayanagi¹, Kensuke Hayashi¹

Affiliation

¹ Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, Tokyo, Japan.

Abstract

The centrosome lacks microtubule (MT)-nucleation activity in differentiated neurons. We have previously demonstrated that MTs were nucleated at the cytoplasm of mouse neurons. They are supposed to serve seeds for MTs required for dendrite growth. However, the factors that activate the cytoplasmic γ-tubulin ring complex (γTuRC) are unknown. Here we report an alternative splicing isoform of cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CKD5RAP2) as a candidate for the cytoplasmic γTuRC activator. This isoform lacked exon 17 and was expressed predominantly in the brain and testis. The expression was transient during the development of cortical neurons, which period coincided with the period we reported cytoplasmic MT nucleation. This isoform resulted in a frameshift and generated truncated protein without a centrosomal localization signal. When this isoform was expressed in cells, it localized diffusely in the cytoplasm. It was co-immunoprecipitated with γ-tubulin and MOZART2, suggesting that it can activate cytosolic γTuRCs. After cold-nocodazole depolymerization of MTs and subsequent washout, we observed numerous short MTs in the cytoplasm of cells transfected with the cDNA of this isoform. The isoform-overexpressing cells exhibited an increased amount of MTs and a decreased ratio of acetylated tubulin, suggesting that MT generation and turnover were enhanced by the isoform. Our data suggest the possibility that alternative splicing of CDK5RAP2 induces cytoplasmic nucleation of MTs in developing neurons.

Keywords: CKD5RAP2, cyclin-dependent kinase 5 regulatory subunit-associated protein 2; CM1, centrosomin motif 1; Centrosome; DMEM, Dulbecco’s Modified Eagle’s Medium; Dendrite growth; FBS, fetal bovine serum; HBSS, Hanks' Balanced Salt Solution; IB, immunoblotting; IP, immunoprecipitation; MT, microtubule; MZT2; MZT2, MOZART2; Microcephaly; NSD, nonsense-mediated mRNA decay; Neuron differentiation; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; γTuRC; γTuRC, γ-tubulin ring complex.