Abstract
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.
Highlights
Mad1 is a key component of the mitotic checkpoint, the mechanism that delays anaphase onset until all chromosomes have properly attached to the spindle
We considered the possibility that MINTs might correspond to some kind of unusual redeployment of nuclear pore complex (NPC) within spermatocyte nucleoplasm and might contain other nucleoporins besides Mtor/Tpr
This study presents evidence that: (i) Mad1 is present in the nucleoplasm of both mitotic and postmitotic cells in Drosophila; (ii) Mad1 associates with at least three proteins, Mtor/ Tpr, Ulp1 and Raf2, with known or suspected roles in chromatin packaging; (iii) Mad1 helps organize a prominent nucleoplasmic structure in spermatocytes containing these three proteins; and (iv) Mad1 can influence chromatin conformation, and gene expression, in the imaginal eye and leg tissues during development
Summary
Mad is a key component of the mitotic checkpoint, the mechanism that delays anaphase onset until all chromosomes have properly attached to the spindle. Mad and Mad are usually described as localizing to the nuclear envelope (NE) [2,3,4], by binding the nucleoporin Tpr, a component of the inner basket of the nuclear pore complex (NPC), [5,6,7,8]. This interaction is conserved in yeast, plants and metazoans. NPC-bound Mad is reported to be a source of anaphase inhibitor prior to the assembly of functional kinetochores [7]
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