JCS044818 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Identification of the kinetochore-binding domain of Mast. (A) Schematic drawing of the Mast protein with the C-terminus located at the right side. The grey box represents the microtubule-binding domain (MBD) (S.G. et al, unpublished results). The solid bars below represent fragments of Mast expressed in S2 cells and used to define the kinetochore-targeting region. (B) S2 cells transfected with the different truncated proteins fused to EGFP, incubated with colchicine and immunostained with the centromere marker Cid. (C) Alignment of Mast-Kt2 amino acid sequence to homologues from different species. Residues that are identical among the sequences have a black background, and those that are similar among the sequences have a grey background. Scale bar: 5 µm.

• Supplemental Figure S2 -

  Fig. S2. Functional analysis of EGFP-Mast transgenes. To determine the ability of EGFP-Mast to complement mutations in the endogenous mast gene we expressed this transgene using a UAS promoters either in neuroblasts or in the whole fly and analyzed individual cell phenotype or quantified the mitotic phenotypes across the whole tissue. (A) Brain from a control individual expressing EGFP-Mast on a wild type background and (D,G) higher magnification of the region highlighted in A. Note that cells have regular size and no polyploidy cells are observed. (B) Brain from a mast⁴/mast⁴: UAS-EGFP-Mast individual but which does not contain either GAL4 driver and therefore does not express the EGFP-Mast trangene and (E,H) higher magnification of the region highlighted in B. Note that the mast⁴ mutation causes the formation of large polyploidy cells throughout the tissue. (C) Brain from a mast⁴/mast⁴: UAS-EGFP-Mast carrying the MZ1061 neuroblast specific driver showing expression of EGFP-Mast (green) in clearly defined areas of the brain. Note that all cells that expresses EGFP-Mast show normal ploidy and no signs the mast⁴ mitotic phenotype. (J) Quantification of the percentage of polyploid/monopolar spindles present in third instar larval neuroblasts from W1118 (control), mast⁴ mutants (w; UAS-EGFP-Mast/Cyo; mast⁴/mast⁴) or flies mast⁴ expressing ubiquitously EGFP-Mast (w; UAS-EGFP-Mast/Act-Gal4; mast⁴/mast⁴). All mitotic cells from five different brains for each strain were quantified.

• Supplemental Figure S3 -

  Fig. S3. Western blots of cell extracts after depletion of Mast alone or Mast+Dynein and Mast+ZW10. Level of protein depletion after 120 hours of treatment is indicated on the table below. Tubulin was used as loading control.

• Supplemental Figure S4 -

  Fig. S4. Zw10 localization in cells lacking both Mast and dynein. S2 cells labelled for γ-tubulin (red), KLP10A (green) and Cid (blue) showing KLP10A localization in control cells and cells lacking both Mast and Dynein. Scale bar: 5 µm.

• Movie 1 -

  Movie 1. Embryos expressing EGFP-Mast. Laser-confocal time-lapse imaging of syncytial embryos at stage 13expressing EGFP-Mast. Images were acquired every 10 seconds.

• Movie 2 -

  Movie 2. Embryos expressing EGFP-Mast-Kt2. Laser-confocal time-lapse imaging of syncytial embryos at stage 12expressing EGFP-Mast-Kt2. Images were acquired every 10 seconds.

• Movie 3 -

  Movie 3. Embryos expressing EGFP-Mast-Kt2 and mRFP-Cid. Laser-confocal time-lapse imaging of syncytial embryos at stage 11expressing EGFP-Mast-Kt2 (green) and mRFP-Cid (red). Images were acquired every 10 seconds.

• Movie 4 -

  Movie 4. Embryos expressing EGFP-Mast. Laser-confocal time-lapse imaging of syncytial embryos at stages12 expressing EGFP-Mast. Images were acquired every 10 seconds.