Midzone microtubules of mammalian cells play an essential role in the induction of cell cleavage, serving as a platform for a number of proteins that play a part in cytokinesis. We demonstrate that PRC1, a mitotic spindle-associated Cdk substrate that is essential to cell cleavage, is a microtubule binding and bundling protein both in vivo and in vitro. Overexpression of PRC1 extensively bundles interphase microtubules, but does not affect early mitotic spindle organization. PRC1 contains two Cdk phosphorylation motifs, and phosphorylation is possibly important to mitotic suppression of bundling, as a Cdk phosphorylation-null mutant causes extensive bundling of the prometaphase spindle. Complete suppression of PRC1 by siRNA causes failure of microtubule interdigitation between half spindles and the absence of a spindle midzone. Truncation mutants demonstrate that the NH2-terminal region of PRC1, rich in α-helical sequence, is important for localization to the cleavage furrow and to the center of the midbody, whereas the central region, with the highest sequence homology between species, is required for microtubule binding and bundling activity. We conclude that PRC1 is a microtubule-associated protein required to maintain the spindle midzone, and that distinct functions are associated with modular elements of the primary sequence.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor‐based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol‐4,5‐bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol‐4‐phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide‐PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol‐3,4,5‐trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X‐linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.

A "spindle assembly" checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21 WAF1 and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity. INTRODUCTIONAneuploidy is common among tumor cells and frequently follows after an intermediate tetraploid state (Shackney et al., 1989;Galipeau et al., 1996). The presence of aneuploidy in tumors is correlated with metastatic progression and poor prognosis (Sandberg, 1977;Rabinovitch et al., 1989). A carefully studied progression toward tumor status in Barrett's esophagus cells has shown that the precancerous state is characterized by loss of p53 function, followed by tetraploidy, and then aneuploidy (Galipeau et al., 1996). Subversion of the capacity of the cell to evade the consequences of tetraploidization, which inevitably leads to aneuploidy, may be a common intermediate in the multistep process by which tumor cells arise in situ.Tetraploidy can arise by exit of a cell from mitosis following failures of spindle assembly, of chromosome segregation, or of cytokinesis . Mammalian cells have a checkpoint that maintains cdc2-cyclin B activity and induces mitotic arrest in the presence of inhibitors of microtubule assembly (Kung et al., 1990;Andreassen and Margolis, 1994). This mechanism can logically be expected to ensure the assembly of a functioning mitotic spindle before exit from mitosis. But in actuality, cells have a widely varied capacity to arrest in mitosis in the presence of microtubule inhibitors (Kung et al., 1990;Schimke et al., 1991;Cahill et al., 1998), and many nontransformed cells undergo only a transient mitotic arrest, and then exit mitosis without chromosome segregation and become tetraploid (Minn et al., 1996;Lanni and Jacks, 1998). However, a p53-dependent backup mechanism induces G1 arrest in cell...

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