Here, we report the cloning and characterization of Drosophila
skeletor, one of the antigens recognized by the mAb2A (Johansen et al. 1999
). The skeletor locus is highly complex, with alternative splicing that gives rise to two mRNAs. The smaller mRNA codes for a cytoplasmic localized protein of 32 kD. The other and larger mRNA has bicistronic coding potential with the skeletor ORF localized downstream and the putative starting methionine followed by an inframe stop codon. At present, we do not know whether skeletor is translated from the indicated methionine and is able to read through the stop codon, or whether some type of frameshift mechanism or use of an alternative start codon is responsible for translation initiation. However, the detection of skeletor with multiple mono- and polyclonal antibodies, which were generated against separate domains of the protein and their identical immunocytochemical labeling of embryonic nuclei, demonstrate that, not withstanding the unconventional organization of the skeletor mRNA, the skeletor ORF is in fact translated. Skeletor is a novel protein of 81 kD, which, apart from a bipartite nuclear localization signal, is without functional domains identified in other proteins.
Labeling of embryonic nuclei in various stages of the cell cycle demonstrated that skeletor is associated with the chromosomes at interphase, but at prophase, it redistributes into a true fusiform spindle structure that precedes microtubule spindle formation, and that this spindle can exist independently of microtubules. Our interpretation of this dynamic redistribution is summarized and diagrammed in the model in . At interphase, skeletor is localized to the chromosomes in a staining pattern overlapping with that of Hoechst-labeled heterochromatin. However, during prophase, as the chromosomes start to condense, skeletor redistributes from the chromosomes and into a fibrous meshwork, which aligns to form a distinct spindle structure at late prophase. At this stage, nucleated microtubules have been accumulating around the centrosomes, but have not yet invaded the nuclear space as the nuclear lamina is still intact. During prophase, skeletor is also detected colocalizing with the nuclear envelope. At metaphase, the nuclear envelope has broken down at the poles and microtubule spindle fibers coalign with the skeletor-defined spindle, with the chromosomes positioned at the metaphase plate. Remnants of the nuclear lamina are still associated with the outer parts of the skeletor spindle. During anaphase, the skeletor spindle narrows, but remains stable and intact as the chromosomes segregate. At telophase, the chromosomes start to decondense and reassociate with skeletor where the two daughter nuclei are forming while skeletor still defines a spindle in the midregion. Microtubules associate with the skeletor spindle in this region, providing a basis for tubulin midbody formation and alignment.
Diagram of skeletor redistribution during mitosis. Skeletor is indicated in red, heterochromatin in blue, centrosomes and microtubules in green, and the nuclear lamina in yellow.
At present, we do not know whether skeletor itself forms the observed spindle or whether it associates with other macromolecules that provide the actual structural elements. However, since skeletor does not possess any known protein motifs with such a function, it is likely that the skeletor antibody-labeling pattern reflects the formation of a macromolecular complex constituted by several nuclear components. We further propose that this macromolecular complex, of which skeletor is a member, constitutes a spindle matrix. A spindle matrix has been hypothesized to provide a more or less stationary substrate that anchors motor molecules during force production and microtubule sliding (Pickett-Heaps et al. 1997
). Such a matrix would also have the properties of helping to organize and stabilize the microtubule spindle. Clearly, the formation of the skeletor-defined spindle before nuclear lamina breakdown suggests it may function as a guide for microtubule extension towards the metaphase plate. However, the fact that the skeletor-defined spindle matrix is aligned with the centrosomes implies the existence of a mechanism that coordinates this alignment with centrosome positioning across the nuclear membrane. The finding that the skeletor-defined spindle matrix maintains its fusiform spindle structure from end to end across the metaphase plate during anaphase when the chromosomes segregate makes it an ideal substrate for providing structural support stabilizing microtubules and for counterbalancing force production. The identification and characterization of skeletor is the first direct molecular evidence for the existence of a complete spindle matrix that forms within the nucleus. The NuMA (nuclear mitotic apparatus) protein, another proposed component of the spindle matrix, is located to the spindle poles at metaphase, assisting in stabilizing and focusing microtubules in the region near the centrosomes (Merdes et al. 1996
; Dionne et al. 1999
). CP60 and CP190, two proteins that shuttle between the centrosomes and the nucleus (Kellogg et al. 1995
; Oegema et al. 1995
), also localize in a fibrous pattern that persists into mitosis (Oegema et al. 1997
). The redistribution of CP190 from centrosomes into the nucleus that occurs during prophase could provide a mechanism by which the skeletor spindle becomes aligned with the centrosomes, despite the absence of microtubules in the nucleus. Skeletor is likely to play an important role in nuclear function as antibody perturbation experiments in Drosophila
embryos lead to nuclear fragmentation and reduced mitotic divisions. However, these experiments do not address whether this phenotype is a result of skeletor having a role in spindle assembly and/or function, or whether it may have additional roles in maintaining chromatin structure as its chromosomal localization during interphase may imply. The future isolation and characterization of mutants defective in skeletor in Drosophila
promises to resolve these questions and to provide further insights into the function of this protein and the spindle matrix.