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Comparative Study
. 2005 Dec;171(4):1823-35.
doi: 10.1534/genetics.105.047464. Epub 2005 Sep 2.

The HhH2/NDD domain of the Drosophila Nod chromokinesin-like protein is required for binding to chromosomes in the oocyte nucleus

Wei Cui  1 , R Scott Hawley
Affiliations
Comparative Study

The HhH2/NDD domain of the Drosophila Nod chromokinesin-like protein is required for binding to chromosomes in the oocyte nucleus

Wei Cui et al. Genetics. 2005 Dec.

Abstract

Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9-10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (Cui et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes.

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Figures

Figure 1.
Figure 1.
The structure of the Drosophila Nod chromokinesin-like protein. (A) A schematic comparison of the structure of the full-length Nod protein. The motor-like domain of Nod falls entirely within the first 318 amino acids at the N terminus. Shaded regions indicate the HMGN domains, the putative D-box cyclin destruction domain (see discussion), and the two HhH motifs. Note that the HhH(2)/NDD domain, as described by Doherty et al. (1996), is composed of two HhH motifs, both of which are encompassed by regions of predicted α-helical structure. (B) Sequence composition of numerous proteins containing the HhH(2)/NDD domain (adapted from Doherty et al. 1996). Listed below the Nod sequence are three of the nine sequences used by Doherty et al. (1996) to define the HhH(2)/NDD domain. The last two sequences are from the chromokinesins XKid and HKid (Funabiki and Murray 2000; Tokai et al. 1996). Cylinder represents α-helix and arrows represent β-strands. (C) The structure of existing mutants in the Nod HhH(2)/NDD domain (Rasooly et al. 1994; Zwick et al. 1999). Underlining denotes the mutations existing in nod alleles, * denotes the breakpoint in nodb17, and ** denotes that the remaining 20 amino acids are altered by a frameshift mutation in nodb1. (D) Mutants in the HhH(2)/NDD domain produced by site-directed in vitro mutagenesis. * denotes the positions of the mutated amino acids, and underlining denotes the mutations.
Figure 2.
Figure 2.
Wild-type or mutant Nod-GFP localization stage 7 nod + oocytes. Oocytes expressing wild-type or mutant UAS-Nod-GFP were stained with DAPI (blue) and anti-GFP antibody (GFP) and analyzed by deconvolution microscopy. Arrowhead indicates the location of oocyte nucleus. Bar, 10 μm.
Figure 3.
Figure 3.
NodNDD1,2-GFP localizes within the oocyte nuclei envelope but not on the oocyte nuclei. Oocytes expressing NodNDD1,2-GFP were stained with DAPI (blue) and anti-GFP antibody (GFP) and analyzed by deconvolution microscopy. Arrowhead indicates the location of oocyte nucleus. Bar, 10 μm.
Figure 4.
Figure 4.
Localization of wild-type or mutant Nod-GFP in stage 13–14 nod+/nod+ oocytes. Wild-type or mutant UAS-Nod-GFP expressing oocytes were stained with DAPI (blue), anti-GFP antibody (green), and anti-tubulin antibody (red) and analyzed by deconvolution microscopy. Bar, 10 μm.
Figure 5.
Figure 5.
Localization of wild-type or mutant Nod-GFP in stage 7 nod−/nod− oocytes. FM7nodb27/y noda oocytes carrying either wild-type or mutant UAS-Nod-GFP expressing oocytes were stained with DAPI (blue) and anti-GFP antibody (GFP) and analyzed by deconvolution microscopy. Arrowhead indicates the location of oocyte nucleus. Bar, 10 μm.
Figure 6.
Figure 6.
Localization of wild-type or mutant Nod-GFP in stage 13–14 nod oocytes. FM7nodb27/y noda;pol/pol oocytes carrying wild-type or mutant UAS-Nod-GFP construct were stained with DAPI (blue), anti-GFP antibody (green), and anti-tubulin antibody (red) and analyzed by deconvolution microscopy. Bar, 10 μm.
Figure 7.
Figure 7.
Expression of wild-type or mutant Nod-GFP construct in stage 9 nod+/nod+ oocytes. Wild-type or mutant UASp:Nod-GFP expressing oocytes were stained with anti-GFP antibody and analyzed by deconvolution microscopy. Bar, 20 μm.
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