Jason Stumpff

The Kinesin-8 Motor Kif18A Suppresses Kinetochore Movements to Control Mitotic Chromosome Alignment

During vertebrate cell division, chromosomes oscillate with periods of smooth motion interrupted by abrupt reversals in direction. These oscillations must be spatially constrained in order to align and segregate chromosomes with high fidelity, but the molecular mechanism for this activity is uncertain. We report here that the human kinesin-8 Kif18A has a primary role in the control of chromosome oscillations. Kif18A accumulates as a gradient on kinetochore microtubules in a manner dependent on its motor activity. Quantitative analyses of kinetochore movements reveal that Kif18A reduces the amplitude of preanaphase oscillations and slows poleward movement during anaphase. Thus, the microtubule-depolymerizing kinesin Kif18A has the unexpected function of suppressing chromosome movements. Based on these findings, we propose a molecular model in which Kif18A regulates kinetochore microtubule dynamics to control mitotic chromosome positioning.

Embryonic cleavage cycles: how is a mouse like a fly?

The evolutionary advent of uterine support of embryonic growth in mammals is relatively recent. Nonetheless, striking differences in the earliest steps of embryogenesis make it difficult to draw parallels even with other chordates. We suggest that use of fertilization as a reference point misaligns the earliest stages and masks parallels that are evident when development is aligned at conserved stages surrounding gastrulation. In externally deposited eggs from representatives of all the major phyla, gastrulation is preceded by specialized extremely rapid cleavage cell cycles. Mammals also exhibit remarkably fast cell cycles in close association with gastrulation, but instead of beginning development with these rapid cycles, the mammalian egg first devotes itself to the production of extraembryonic structures. Previous attempts to identify common features of cleavage cycles focused on post-fertilization divisions of the mammalian egg. We propose that comparison to the rapid peri-gastrulation cycles is more appropriate and suggest that these cycles are related by evolutionary descent to the early cleavage stages of embryos such as those of frog and fly. The deferral of events in mammalian embryogenesis might be due to an evolutionary shift in the timing of fertilization.

A Tethering Mechanism Controls the Processivity and Kinetochore-Microtubule Plus-End Enrichment of the Kinesin-8 Kif18A

Metaphase chromosome positioning depends on Kif18A, a kinesin-8 that accumulates at and suppresses the dynamics of K-MT plus ends. By engineering Kif18A mutants that suppress MT dynamics but fail to concentrate at K-MT plus ends, we identify a mechanism that allows Kif18A to accumulate at K-MT plus ends to a level required to suppress chromosome movements. Enrichment of Kif18A at K-MT plus ends depends on its C-terminal tail domain, while the ability of Kif18A to suppress MT growth is conferred by the N-terminal motor domain. The Kif18A tail contains a second MT-binding domain that diffuses along the MT lattice, suggesting that it tethers the motor to the MT track. Consistently, the tail enhances Kif18A processivity and is crucial for it to accumulate at K-MT plus ends. The heightened processivity of Kif18A, conferred by its tail domain, thus promotes concentration of Kif18A at K-MT plus ends, where it suppresses their dynamics to control chromosome movements.

Kif18A and chromokinesins confine centromere movements via microtubule growth suppression and spatial control of kinetochore tension.

Alignment of chromosomes at the metaphase plate is a signature of cell division in metazoan cells, yet the mechanisms controlling this process remain ambiguous. Here we use a combination of quantitative live-cell imaging and reconstituted dynamic microtubule assays to investigate the molecular control of mitotic centromere movements. We establish that Kif18A (kinesin-8) attenuates centromere movement by directly promoting microtubule pausing in a concentration-dependent manner. This activity provides the dominant mechanism for restricting centromere movement to the spindle midzone. Furthermore, polar ejection forces spatially confine chromosomes via position-dependent regulation of kinetochore tension and centromere switch rates. We demonstrate that polar ejection forces are antagonistically modulated by chromokinesins. These pushing forces depend on Kid (kinesin-10) activity and are antagonized by Kif4A (kinesin-4), which functions to directly suppress microtubule growth. These data support a model in which Kif18A and polar ejection forces synergistically promote centromere alignment via spatial control of kinetochore-microtubule dynamics.

Drosophila Wee1 Kinase Regulates Cdk1 and Mitotic Entry during Embryogenesis

Cyclin-dependent kinases (Cdks) are the central regulators of the cell division cycle. Inhibitors of Cdks ensure proper coordination of cell cycle events and help regulate cell proliferation in the context of tissues and organs. Wee1 homologs phosphorylate a conserved tyrosine to inhibit the mitotic cyclin-dependent kinase Cdk1. Loss of Wee1 function in fission or budding yeast causes premature entry into mitosis. The importance of metazoan Wee1 homologs for timing mitosis, however, has been demonstrated only in Xenopus egg extracts and via ectopic Cdk1 activation . Here, we report that Drosophila Wee1 (dWee1) regulates Cdk1 via phosphorylation of tyrosine 15 and times mitotic entry during the cortical nuclear cycles of syncytial blastoderm embryos, which lack gap phases. Loss of maternal dwee1 leads to premature entry into mitosis, mitotic spindle defects, chromosome condensation problems, and a Chk2-dependent block of subsequent development, and then embryonic lethality. These findings modify previous models about cell cycle regulation in syncytial embryos and demonstrate that Wee1 kinases can regulate mitotic entry in vivo during metazoan development even in cycles that lack a G2 phase.

Drosophila Wee1 Interacts with Members of the γTURC and Is Required for Proper Mitotic-Spindle Morphogenesis and Positioning

BACKGROUND Wee1 kinases delay entry into mitosis by phosphorylating and inactivating cyclin-dependent kinase 1 (Cdk1). Loss of this activity in many systems, including Drosophila, leads to premature mitotic entry. RESULTS We report here that Drosophila Wee1 (dwee1) mutant embryos show mitotic-spindle defects that include ectopic foci of microtubule organization, formation of multipolar spindles from adjacent centrosome pairs, and promiscuous interactions between neighboring spindles. Furthermore, centrosomes are displaced from the embryo cortex in dwee1 mutants. These defects are not observed to the same extent in embryos in which nuclei also enter mitosis prematurely as a result of a lack of checkpoint control or in embryos with elevated Cdk1 activity. dWee1 physically interacts with members of the gamma-tubulin ring complex (gammaTuRC), and gamma-tubulin is phosphorylated in a dwee1-dependent manner in embryo extracts. CONCLUSIONS Some of the abnormalities in dwee1 mutant embryos cannot be explained by premature entry into mitosis or bulk elevation of Cdk1 activity. Instead, dWee1 is also required for phosphorylation of gamma-tubulin, centrosome positioning, and mitotic-spindle integrity. We propose a model to account for these requirements.

Mitochondrial-targeted nitroxides disrupt mitochondrial architecture and inhibit expression of peroxiredoxin 3 and FOXM1 in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an intractable tumor of the peritoneal and pleural cavities primarily linked to exposure to asbestos. Recently, we described an interplay between mitochondrial‐derived oxidants and expression of FOXM1, a redox‐responsive transcription factor that has emerged as a promising therapeutic target in solid malignancies. Here we have investigated the effects of nitroxides targeted to mitochondria via triphenylphosphonium (TPP) moieties on mitochondrial oxidant production, expression of FOXM1 and peroxiredoxin 3 (PRX3), and cell viability in MM cells in culture. Both Mito‐carboxy‐proxyl (MCP) and Mito‐TEMPOL (MT) caused dose‐dependent increases in mitochondrial oxidant production that was accompanied by inhibition of expression of FOXM1 and PRX3 and loss of cell viability. At equivalent concentrations TPP, CP, and TEMPOL had no effect on these endpoints. Live cell ratiometric imaging with a redox‐responsive green fluorescent protein targeted to mitochondria (mito‐roGFP) showed that MCP and MT, but not CP, TEMPOL, or TPP, rapidly induced mitochondrial fragmentation and swelling, morphological transitions that were associated with diminished ATP levels and increased production of mitochondrial oxidants. Mdivi‐1, an inhibitor of mitochondrial fission, did not rescue mitochondria from fragmentation by MCP. Immunofluorescence microscopy experiments indicate a fraction of FOXM1 coexists in the cytoplasm with mitochondrial PRX3. Our results indicate that MCP and MT inhibit FOXM1 expression and MM tumor cell viability via perturbations in redox homeostasis caused by marked disruption of mitochondrial architecture, and suggest that both compounds, either alone or in combination with thiostrepton or other agents, may provide credible therapeutic options for the management of MM. J. Cell. Physiol. 228: 835–845, 2013.

Activating the DNA damage checkpoint in a developmental context

BACKGROUND Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, we examined the effect of X-rays on post-blastoderm embryos of Drosophila melanogaster. RESULTS In Drosophila, DNA damage was previously found to delay anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that included a G2 phase, we found that irradiation delayed the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurred normally after irradiation. The radiation-induced delay of mitosis accompanied the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drove a mitotic cyclin into the nucleus and overcame the delay of mitosis induced by irradiation. CONCLUSIONS Developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa.

Tyrosines in the Kinesin-5 Head Domain Are Necessary for Phosphorylation by Wee1 and for Mitotic Spindle Integrity

Mitotic spindle assembly and maintenance relies on kinesin-5 motors that act as bipolar homotetramers to crosslink microtubules. Kinesin-5 motors have been the subject of extensive structure-function analysis, but the regulation of their activity in the context of mitotic progression remains less well understood. We report here that Drosophila kinesin-5 (KLP61F) is regulated by Drosophila Wee1 (dWee1). Wee1 tyrosine kinases are known to regulate mitotic entry via inhibitory phosphorylation of Cdk1. Recently, we showed that dWee1 also plays a role in mitotic spindle positioning through gamma-tubulin and spindle fidelity through an unknown mechanism. Here, we investigated whether a KLP61F-dWee1 interaction could explain the latter role of dWee1. We found that dWee1 phosphorylates KLP61F in vitro on three tyrosines within the head domain, the catalytic region that mediates movement along microtubules. In vivo, KLP61F with tyrosine-->phenylalanine mutations fails to complement a klp61f mutant and dominantly induces spindle defects similar to ones seen in dwee1 mutants. We propose that phosphorylation of the KLP61F catalytic domain by dWee1 is important for the motors function. This study identifies a second substrate for a Wee1 kinase and provides evidence for phosphoregulation of a kinesin in the head domain.

Promiscuity Rules? The Dispensability of Cyclin E and Cdk2

The canonical view of the mammalian cell cycle arose from studies of cultured cells rather than mutant organisms. It depicts the many complexes of cyclin and Cdk (cyclin/Cdk) as fulfilling unique and essential steps that dictate the sequential order of cell cycle events. Recent analyses of knockout mice challenge this view. Cdk2 and cyclin E, long thought to be essential, are largely dispensable. Here, we discuss the phenotypes of these and other cyclin/Cdk mutants in genetically tractable metazoa (mouse, fly, and nematode) and explore possible reasons behind similarities and differences among experimental systems and cell types.