Mad1 influences interphase nucleoplasm organization and chromatin regulation in Drosophila.

The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.

A mitotic role for Mad1 beyond the spindle checkpoint.

Unattached kinetochores generate an anaphase inhibitor, through the spindle assembly checkpoint (SAC), that allows cells more time to establish proper kinetochore-microtubule (K-MT) linkages and thus avoid aneuploidy. Mad1 is the receptor for Mad2 at kinetochores, where it catalyzes the formation of Mad2-Cdc20 complexes, an essential part of the anaphase inhibitor, but whether it has any other mitotic function is unknown. We have generated a mad1-null mutation in Drosophila. This mutant is SAC defective and Mad2 is no longer localized to either nuclear envelope or kinetochores, but it displays normal basal mitotic timing. Unlike mad2 mutants, which have relatively normal mitoses, mad1 anaphases show high frequencies of lagging chromatids, at least some of which are caused by persistent merotelic linkages. A transgene expressing GFP-Mad1 rescues both the SAC and the anaphase defects. In an attempt to separate the SAC function from the mitotic function, we made a mad1 transgene with a mutated Mad2-binding domain. Surprisingly, this transgene failed to complement the anaphase phenotype. Thus, Mad1 has activity promoting proper K-MT attachments in addition to its checkpoint function. This activity does not require the presence of Mad2, but it does depend in some unknown way on key residues in the Mad2-binding domain of Mad1.

Separating the spindle, checkpoint, and timer functions of BubR1.

BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture and/or dynamics but is relatively dispensable for SAC function. In contrast, bubR1-KEN flies have normal spindles but no SAC. Nevertheless, mitotic timing is normal as long as Mad2 is present. Thus, the SAC, timer, and spindle functions of BubR1 are substantially separable. Timing is shorter in bubR1-KEN mad2 double mutants, yet in these flies, lacking both critical SAC components, chromosomes still segregate accurately, reconfirming that in Drosophila, reliable mitosis does not need the SAC.

Flies without a spindle checkpoint.

Mad2 has a key role in the spindle-assembly checkpoint (SAC) - the mechanism delaying anaphase onset until all chromosomes correctly attach to the spindle. Here, we show that unlike every other reported case of SAC inactivation in metazoans, mad2-null Drosophila are viable and fertile, and their cells almost always divide correctly despite having no SAC and an accelerated 'clock', which is caused by premature degradation of cyclin B. Mitosis in Drosophila does not need the SAC because correct chromosome attachment is achieved very rapidly, before even the cell lacking Mad2 can initiate anaphase. Experimentally reducing spindle-assembly efficiency renders the cells Mad2-dependent. In fact, the robustness of the SAC may generally mask minor mitotic defects of mutations affecting spindle function. The reported lethality of other Drosophila SAC mutations may be explained by their multifunctionality, and thus the 'checkpoint' phenotypes previously ascribed to these mutations should be considered the consequence of eliminating both the checkpoint and a second mitotic function.