Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics

Mingyue Jin,Takaki Miyata,Tomoyasu Shinoda,Daiju Kitagawa,Bruno Reversade,Ken’Ya Furuta,Takuo Yasunaga,Shigeru Matsumura,Takayuki Torisawa,Oz Pomp,Thong Teck Tan,Kazuhiro Oiwa,Shinji Hirotsune,Shiori Toba

doi:10.1038/srep39902

Abstract

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Highlights

Lysate used for this immunoprecipitation assay. (c) glutathione S-transferase (GST) pull-down assay with 0.2 μM GST-NuMA-FLAG
Mutations in KATNB1 (p80), which encodes the non-catalytic regulatory p80 subunit of katanin[14,15,16,17], have been shown to cause severe microlissencephaly[18,19]. These findings highlight the critical functions of KATNB1, WDR62 and NDE1 during neurogenesis and neuronal migration which suggest the existence of a common pathophysiological pathway responsible for microcephaly and lissencephaly
We demonstrate that p80 is essential for the proper regulation of MT dynamics at the centrosome/spindle pole in combination with cytoplasmic dynein and NuMA

Summary

Discussion

The current study combined in vitro and in vivo strategies to provide evidence that p80 interacts with NuMA and cytoplasmic dynein to regulate and maintain proper MT organization into asters. Two NuMA isoforms, which are likely to be expressed at low levels, were found to be clustered at the centrosomal region in interphase CHOP cells[43] These findings suggest the possibility that, similar to p80, cytoplasmic NuMA may participate in neuronal migration. Our findings that a p80-NuMA-dynein-MT complex is sufficient to form asters in vitro, whereas p80 mutations from patients were unable to elicit aster formation, indicate that severe brain abnormalities in patients may be, in part, a result of the loss of the ability of p80 to regulate MT dynamics These findings provide novel molecular and cellular insights into the potential pathogenesis of severe microlissencephaly

Methods

Author Contributions

Findings

Additional Information