Ninein isoform contributions to intracellular processes and macrophage immune function.

Abstract

Ninein is a multifunctional protein, involved in microtubule (MT) organization and dynein/dynactin complex recruitment and activation. Several isoforms of ninein have been identified in various tissues, however, their relative contribution(s) are not clear. Here, we identify two ninein isoforms in mouse macrophages with distinct C-termini and disproportionate expression levels; a canonical ninein (ninein^CAN) isoform and ninein isoform 2 (ninein^ISO2). Analysis of ninein pre-mRNA exon-intron boundaries revealed that ninein^ISO2 transcript is likely generated by two alternative splicing site selection events predicted to result in a distinct 3D structure compared to ninein^CAN. We used selective and total protein knockdown experiments to assess intracellular and functional roles of ninein in macrophages. Live cell imaging analyses of macrophages implicated both isoforms in regulating cell proliferation. MT regrowth following nocodazole depolymerization showed that both isoforms contributed to MT nucleation and structural integrity of the centrosome, as cells lacking ninein^CAN or ninein^ISO2 contained multiple ectopic γ-tubulin foci. However, ninein^CAN, but not ninein^ISO2, was important for the separation of duplicated centrosomes during cell division. Despite a requirement of both ninein isoforms to recruit dynein/dynactin to the centrosome, only ninein^CAN was required for Golgi positioning and morphology, a dynein-dependent event. We additionally found that ninein^ISO2 was the primary isoform required for F-actin recruitment during internalization of IgG-opsonized particles. Our study indicates that alternative splicing promotes both redundant and differential activities for ninein in MT organization, organelle positioning and macrophage function.