The microtubule-binding region of RECQL4 is required for chromosome alignment. The microtubule-binding region of RECQL4 is required for chromosome alignment. (A) Schematic representation of Xenopus RECQL4 and constructs used in add-back reactions. The position of the N-terminal Sld2-like domain, involved in DNA replication, the NLS, and the helicase domain are indicated. The Δ546–594 mutant lacks the NLS region. The K758M point mutant is a known helicase-defective RECQL4 version (Rossi et al, 2010). (B) RECQL4-depleted (ΔRECQL4) CSF extract was incubated with wild-type or Δ546–594 mRNAs for 90 min. Expression of the recombinant proteins were confirmed by Western blotting using Xenopus antibodies. The resulting extracts were used for microtubule (MT) binding assay. (C) Sperm was incubated in control (mock) or RECQL4-depleted (ΔRECQL4) CSF extracts supplemented with the indicated mRNAs for cycled spindle assembly in the presence of Alexa 488-labled tubulin. At the end of the reaction, samples were fixed and stained with DAPI for microscopy. Chromosome alignment was quantified analyzing all bipolar spindle structures identified. Columns show the average of three independent experiments and circles indicate individual data points. Scale bar, 20 μm. (D) Cycled spindles assembled as in (C) but with the helicase-defective K758M mutant. Depletion and add-back efficiency was analyzed at the end of the assay by Western blotting. Chromosome alignment was quantified. Columns show the average of two independent experiments and circles indicate individual data points. (E) Cycled spindles assembled as in (C) but supplemented with mRNA encoding for wild-type or different C-terminal RECQL4 truncations. Depletion and add-back efficiency was analyzed at the end of the assay by Western blotting. Chromosome alignment was also quantified. Columns show the average of at least two independent experiments and circles indicate individual data points. Hideki Yokoyama et al. LSA 2019;2:e201800120 © 2019 Yokoyama et al.