CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle

A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-β and subsequently suppresses TGF-β signalings

AbstractWe previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-β1 (TGF-β1)–stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-β1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-β1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-β receptors (TβRI and TβRII) in either cell types nor altered the specific binding of 125I TGF-β1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TβRI and TβRII present on the same cells exclusively form aggregates in TGF-β1-stimulated cells; (e) co-treatment of TGF-β1–stimulated cells with bik suppresses TGF-β1–induced complex formation of TβRI and TβRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-β1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-β1–elicited signaling cascades in cells possibly by abrogating the coupling between TβRI and TβRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression