Françoise B. Lacroix

Mammalian mad2 and bub1/bubR1 recognize distinct spindle-attachment and kinetochore-tension checkpoints

Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. Here we show that the arrest induced by nanomolar vinblastine correlates with loss of tension at the kinetochore, and that in response the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. We conclude that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively.

Multiple centrosomes arise from tetraploidy checkpoint failure and mitotic centrosome clusters in p53 and RB pocket protein-compromised cells

A high degree of aneuploidy characterizes the majority of human tumors. Aneuploid status can arise through mitotic or cleavage failure coupled with failure of tetraploid G1 checkpoint control, or through deregulation of centrosome number, thus altering the number of mitotic spindle poles. p53 and the RB pocket proteins are important to the control of G1 progression, and p53 has previously been suggested as important to the control of centrosome duplication. We demonstrate here that neither suppression of p53 nor of the RB pocket protein family directly generates altered centrosome numbers in any of several mammalian primary cell lines. Instead, amplification of centrosome number occurs in two steps. The first step is failure to arrest at a G1 tetraploidy checkpoint after failure to segregate the genome in mitosis, and the second step is clustering of centrosomes at a single spindle pole in subsequent tetraploid or aneuploid mitosis. The trigger for these events is mitotic or cleavage failure that is independent of p53 or RB status. Finally, we find that mouse embryo fibroblasts spontaneously enter tetraploid G1, explaining the previous demonstration of centrosome amplification by p53 abrogation alone in these cells.

Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)–dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.

Bacterial cell wall nanoimaging by autoblinking microscopy

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this ‘autoblinking’ phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.