Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila

Abstract

Mutations in the Drosophila gene greatwall cause improper chromosome condensation and delay cell cycle progression in larval neuroblasts. Chromosomes are highly undercondensed, particularly in the euchromatin, but nevertheless contain phosphorylated histone H3, condensin, and topoisomerase II. Cells take much longer to transit the period of chromosome condensation from late G2 through nuclear envelope breakdown. Mutant cells are also subsequently delayed at metaphase, due to spindle checkpoint activity. These mutant phenotypes are not caused by spindle aberrations, by global defects in chromosome replication, or by activation of a caffeine-sensitive checkpoint. The Greatwall proteins in insects and vertebrates are located in the nucleus and belong to the AGC family of serine/threonine protein kinases; the kinase domain of Greatwall is interrupted by a long stretch of unrelated amino acids.

Figure 1.

Chromosome condensation defects in gw l mutant brain cells. Wild-type (A and D), gwl ⁷¹⁶ mutant (B and E), and gwl ²⁹⁷⁰ mutant (C and F) brains were treated with colchicine, and the chromosomes were stained with either orcein (A–C) or Hoechst 33258 (D–F). Bar, 5 μm.

Figure 2.

Chromosomes from gwl mutants contain the condensin component Barren and phosphohistone H3. Wild-type (A–D) and gwl ²⁹⁷⁰mutant (E–H) neuroblasts stained for DNA (A and E), phosphohistone H3 (B and F), and Barren (C and G). (D and H) Merge of signals for DNA (blue), phosphohistone H3 (red), and Barren (green). Bar, 5 μm.

Figure 3.

Division of cultured neuroblasts in time-lapse microscopy. Time in h and min (h:min). (A) Wild-type neuroblasts. (B) gwl⁷¹⁶ mutant neuroblast. First and third rows, histone H2Av-GFP signal; second and fourth rows, concurrent DIC images. In the particular mutant neuroblast shown, chromosome condensation takes place at least in part, and the cells enter a defective anaphase with lagging chromatids. However, the time required to complete various stages of mitosis is much longer than in wild type. Bar, 5 μm.

Figure 4.

Depletion of Greatwall by RNAi. Treatment of Drosophila tissue culture cells with gwl dsRNA causes aberrant chromosome morphology reminiscent of that produced by depletion of condensin subunits. (A and B) Karyotypes of control colchicine-treated tissue culture cells in the absence of dsRNA. (C and D) RNAi with gwl dsRNA. (E) RNAi with dsRNA for Gluon (XCAP-C). (F) RNAi with dsRNA for Barren (XCAP-H). (G and H) Simultaneous RNAi with Greatwall and either Gluon (G) or Barren (H) dsRNAs. Bar, 5 μm.

Figure 5.

Structure of Greatwall proteins. Conserved domains of the D. melanogaster and human Greatwall proteins, compared with the related kinases Rim15p/Cek1p and IRE. The kinase domain in all these proteins (red) is split by the insertion of unrelated amino acids between kinase subdomains VII and VIII. Green and blue areas are less well-conserved regions found in all Greatwall kinases, but not in other kinases (except the short green stretch preceding the kinase domains in Rim15p/Cek1p and IRE). A small degree of homology is apparent between the inserts in insect and vertebrate Greatwall proteins (yellow). Asterisks indicate the lesions associated with mutant alleles: gwl ⁷¹⁶ = Gly66Ser; gwl ¹⁸⁰ = Gly69Glu; gwl ²⁹⁷⁰ = Gln102Ter; gwl ¹⁰²⁸ = Ser127Leu; gwl ⁸⁷³ = Ala728Val.