The Drosophila midblastula transition (MBT), a major event in embryogenesis, remodels and slows the cell cycle. In the pre-MBT cycles, all genomic regions replicate simultaneously in rapid S phases that alternate with mitosis, skipping gap phases. At the MBT, down-regulation of Cdc25 phosphatase and the resulting inhibitory phosphorylation of the mitotic kinase Cdk1 create a G2 pause in interphase 14. However, an earlier change in interphase 14 is the prolongation of S phase. While the signals modifying S phase are unknown, the onset of late replication-where replication of constitutively heterochromatic satellite sequences is delayed-extends S-phase 14. We injected Cdc25 mRNA to bypass the developmentally programmed down-regulation of Cdc25 at the MBT. Introduction of either Cdc25 isoform (String or Twine) or enhanced Cdk1 activity triggered premature replication of late-replicating sequences, even after their specification, and thereby shortened S phase. Reciprocally, reduction of Cdk1 activity by knockdown of mitotic cyclins extended pre-MBT S phase. These findings suggest that high Cdc25 and Cdk1 contribute to the speed of the rapid, pre-MBT S phases and that down-regulation of these activities plays a broader role in MBT-associated changes than was previously suspected.

Metazoans start embryogenesis with a relatively naïve genome. The transcriptionally inert, late-replicating heterochromatic regions, including the constitutive heterochromatin on repetitive sequences near centromeres and telomeres, need to be re-established during development. To explore the events initiating heterochromatin formation and examine their temporal control, sequence specificity, and immediate regulatory consequence, we established a live imaging approach that enabled visualization of steps in heterochromatin emergence on specific satellite sequences during the mid-blastula transition (MBT) in Drosophila. Unexpectedly, only a subset of satellite sequences, including the 359-base-pair (bp) repeat sequence, recruited HP1a at the MBT. The recruitment of HP1a to the 359-bp repeat was dependent on HP1a's chromoshadow domain but not its chromodomain and was guided by maternally provided signals. HP1a recruitment to the 359-bp repeat was required for its programmed shift to later replication, and ectopic recruitment of HP1a was sufficient to delay replication timing of a different repeat. Our results reveal that emergence of constitutive heterochromatin follows a stereotyped developmental program in which different repetitive sequences use distinct interactions and independent pathways to arrive at a heterochromatic state. This differential emergence of heterochromatin on various repetitive sequences changes their replication order and remodels the DNA replication schedule during embryonic development.

At the Mid-Blastula Transition (MBT), externally developing embryos refocus from increasing cell number to elaboration of the body plan. Studies in Drosophila reveal a sequence of changes in regulators of Cyclin:Cdk1 that increasingly restricts the activity of this cell cycle kinase to slow cell cycles during early embryogenesis. By reviewing these events, we provide an outline of the mechanisms slowing the cell cycle at and around the time of MBT. The perspectives developed should provide a guiding paradigm for the study of other MBT changes as the embryo transits from maternal control to a regulatory program centered on the expression of zygotic genes.

Summary The timing mechanism for mitotic progression is still poorly understood. The spindle assembly checkpoint (SAC), whose reversal upon chromosome alignment is thought to time anaphase [1–3], is functional during the rapid mitotic cycles of the Drosophila embryo; but its genetic inactivation had no consequence on the timing of the early mitoses. Mitotic cyclins—Cyclin A, Cyclin B, and Cyclin B3—influence mitotic progression and are degraded in a stereotyped sequence [4–11]. RNAi knockdown of Cyclins A and B resulted in a Cyclin B3-only mitosis in which anaphase initiated prior to chromosome alignment. Furthermore, in such a Cyclin B3-only mitosis, colchicine-induced SAC activation failed to block Cyclin B3 destruction, chromosome decondensation, or nuclear membrane re-assembly. Injection of Cyclin B proteins restored the ability of SAC to prevent Cyclin B3 destruction. Thus, SAC function depends on particular cyclin types. Changing Cyclin B3 levels showed that it accelerated progress to anaphase, even in the absence of SAC function. The impact of Cyclin B3 on anaphase initiation appeared to decline with developmental progress. Our results show that different cyclin types affect anaphase timing differently in the early embryonic divisions. The early-destroyed cyclins—Cyclins A and B—restrain anaphase-promoting complex/cyclosome (APC/C) function, whereas the late-destroyed cyclin, Cyclin B3, stimulates function. We propose that the destruction schedule of cyclin types guides mitotic exit by affecting both Cdk1 and APC/C, whose activities change as each cyclin type is lost.

Chromosome segregation in mitosis is orchestrated by dynamic interactions between spindle microtubules and centromeres, which in turn are governed by protein kinase-and phosphatase-signaling cascades. Previous studies showed that overexpression of human phosphatase HsCdc14A, an antagonist of cyclin-dependent kinase 1, affects several aspects of cell division. However, the molecular mechanism underlying HsCdc14A regulation in mitosis has remained elusive. Here we show that HsCdc14A activity is regulated by an auto-inhibitory mechanism via its intra-molecular association. Our biochemical study demonstrated that Polo-like kinase 1 (PLK1) interacts with and phosphorylates HsCdc14A. This phosphorylation partially releases the auto-inhibition of HsCdc14A judged by its phosphatase activity in vitro. To examine the functional relevance of such phospho-regulation of HsCdc14A in vivo, a phospho-mimicking mutant of HsCdc14A was expressed in HeLa cells. Importantly, overexpression of the phospho-mimicking mutants caused aberrant chromosome alignment with a prometaphase delay, suggesting the temporal regulation of HsCdc14A activity is critical for orchestrating mitotic events. Given the fact that HsCdc14A forms an intra-molecular association and PLK1-mediated phospho-regulation promotes HsCdc14A phosphatase activity, we propose that PLK1-HsCdc14A interaction provides a temporal regulation of HsCdc14A in chromosome segregation during mitosis.Mitosis is orchestrated by signaling cascades that coordinate mitotic processes and ensure accurate chromosome segregation. The key switch for the onset of mitosis is the archetypal cyclin-dependent kinase, Cdc2. Besides the master mitotic kinase Cdc2, there are three protein serine/threonine kinase families, the Polo kinases, Aurora kinases, and the NEK2 kinases. The process of mitosis is complex and involves multiple independent regulatory steps, most of which are controlled by reversible protein phosphorylation. In higher eukaryotes, mitosis involves condensation of chromosomes, disassembly of the nuclear lamina, breakdown of the nuclear envelope, and disassembly of many forms of nuclear bodies, including nucleoli. Completion of mitosis requires alignment and proper segregation of chromosomes into daughter cells followed by reassembly of nuclei and cytokinesis. These and many other events, such as centrosome separation and spindle assembly, are tightly regulated, and several critical checkpoints occur during mitosis to ensure fidelity.In the budding yeast Saccharomyces cerevisiae, Cdc14p plays a key role in the exit from mitosis by dephosphorylating cyclindependent kinase targets (e.g. Refs.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator for an accurate kinetochore-microtubule attachment. However, the regulatory mechanism underlying precise MCAK depolymerase activity control during mitosis remains elusive. Here, we describe a novel pathway involving an Aurora B-PLK1 axis for regulation of MCAK activity in mitosis. Aurora B phosphorylates PLK1 on Thr210 to activate its kinase activity at the kinetochores during mitosis. Aurora B-orchestrated PLK1 kinase activity was examined in real-time mitosis using a fluorescence resonance energy transfer-based reporter and quantitative analysis of native PLK1 substrate phosphorylation. Active PLK1, in turn, phosphorylates MCAK at Ser715 which promotes its microtubule depolymerase activity essential for faithful chromosome segregation. Importantly, inhibition of PLK1 kinase activity or expression of a non-phosphorylatable MCAK mutant prevents correct kinetochore-microtubule attachment, resulting in abnormal anaphase with chromosome bridges. We reason that the Aurora B-PLK1 signaling at the kinetochore orchestrates MCAK activity, which is essential for timely correction of aberrant kinetochore attachment to ensure accurate chromosome segregation during mitosis.

Chromosome segregation in mitosis is orchestrated by the interaction of the kinetochore with spindle microtubules. Our recent study shows that NEK2A interacts with MAD1 at the kinetochore and possibly functions as a novel integrator of spindle checkpoint signaling. However, it is unclear how NEK2A regulates kinetochore-microtubule attachment in mitosis. Here we show that NEK2A phosphorylates human Sgo1 and such phosphorylation is essential for faithful chromosome congression in mitosis. NEK2A binds directly to HsSgo1 in vitro and co-distributes with HsSgo1 to the kinetochore of mitotic cells. Our in vitro phosphorylation experiment demonstrated that HsSgo1 is a substrate of NEK2A and the phosphorylation sites were mapped to Ser 14 and Ser 507 as judged by the incorporation of 32 P. Although such phosphorylation is not required for assembly of HsSgo1 to the kinetochore, expression of non-phosphorylatable mutant HsSgo1 perturbed chromosome congression and resulted in a dramatic increase in microtubule attachment errors, including syntelic and monotelic attachments. These findings reveal a key role for the NEK2A-mediated phosphorylation of HsSgo1 in orchestrating dynamic kinetochore-microtubule interaction. We propose that NEK2A-mediated phosphorylation of human Sgo1 provides a link between centromeric cohesion and spindle microtubule attachment at the kinetochores.

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