Prasad V. Jallepalli

Polo Kinase and Separase Regulate the Mitotic Licensing of Centriole Duplication in Human Cells

It has been proposed that separase-dependent centriole disengagement at anaphase licenses centrosomes for duplication in the next cell cycle. Here we test whether such a mechanism exists in intact human cells. Loss of separase blocked centriole disengagement during mitotic exit and delayed assembly of new centrioles during the following S phase; however, most engagements were eventually dissolved. We identified Polo-like kinase 1 (Plk1) as a parallel activator of centriole disengagement. Timed inhibition of Plk1 mapped its critical period of action to late G2 or early M phase, i.e., prior to securin destruction and separase activation at anaphase onset. Crucially, when cells exited mitosis after downregulation of both separase and Plk1, centriole disengagement failed completely, and subsequent centriole duplication in interphase was also blocked. Our results indicate that Plk1 and separase act at different times during M phase to license centrosome duplication, reminiscent of their roles in removing cohesin from chromosomes.

Chemical genetics reveals the requirement for Polo-like kinase 1 activity in positioning RhoA and triggering cytokinesis in human cells.

Polo-like kinases (Plks) play crucial roles in mitosis and cell division. Whereas lower eukaryotes typically contain a single Plk, mammalian cells express several closely related but functionally distinct Plks. We describe here a chemical genetic system in which a single Plk family member, Plk1, can be inactivated with high selectivity and temporal resolution by using an allele-specific, small-molecule inhibitor, as well as the application of this system to dissect Plk1s role in cytokinesis. To do this, we disrupted both copies of the PLK1 locus in human cells through homologous recombination and then reconstituted Plk1 activity by using either the wild-type kinase (Plk1wt) or a mutant version whose catalytic pocket has been enlarged to accommodate bulky purine analogs (Plk1as). When cultured in the presence of these analogs, Plk1as cells accumulate in prometaphase with defects that parallel those found in PLK1Δ/Δ cells. In addition, acute treatment of Plk1as cells during anaphase prevents recruitment of both Plk1 itself and the Rho guanine nucleotide exchange factor (RhoGEF) Ect2 to the central spindle, abolishes RhoA GTPase localization to the equatorial cortex, and suppresses cleavage furrow formation and cell division. Our studies define and illuminate a late mitotic function of Plk1 that, although difficult or impossible to detect in Plk1-depleted cells, is readily revealed with chemical genetics.

Mps1 directs the assembly of Cdc20 inhibitory complexes during interphase and mitosis to control M phase timing and spindle checkpoint signaling

The spindle assembly checkpoint (SAC) in mammals uses cytosolic and kinetochore-based signaling pathways to inhibit anaphase. In this study, we use chemical genetics to show that the protein kinase Mps1 regulates both aspects of the SAC. Human MPS1-null cells were generated via gene targeting and reconstituted with either the wild-type kinase (Mps1(wt)) or a mutant version (Mps1(as)) sensitized to bulky purine analogues. Mps1 inhibition sharply accelerated anaphase onset, such that cells completed mitosis in 12 min, and prevented Cdc20s association with either Mad2 or BubR1 during interphase, i.e., before the appearance of functional kinetochores. Furthermore, intramitotic Mps1 inhibition evicted Bub1 and all other known SAC transducers from the outer kinetochore, but contrary to a recent study, did not perturb aurora B-dependent phosphorylation. We conclude that Mps1 has two complementary roles in SAC regulation: (1) initial cytoplasmic activation of Cdc20 inhibitors and (2) recruitment of factors that promote sustained anaphase inhibition and chromosome biorientation to unattached kinetochores.

Cohesin acetylation speeds the replication fork

Cohesin not only links sister chromatids but also inhibits the transcriptional machinery’s interaction with and movement along chromatin. In contrast, replication forks must traverse such cohesin-associated obstructions to duplicate the entire genome in S phase. How this occurs is unknown. Through single-molecule analysis, we demonstrate that the replication factor C (RFC)–CTF18 clamp loader (RFCCTF18) controls the velocity, spacing and restart activity of replication forks in human cells and is required for robust acetylation of cohesin’s SMC3 subunit and sister chromatid cohesion. Unexpectedly, we discovered that cohesin acetylation itself is a central determinant of fork processivity, as slow-moving replication forks were found in cells lacking the Eco1-related acetyltransferases ESCO1 or ESCO2 (refs 8–10) (including those derived from Roberts’ syndrome patients, in whom ESCO2 is biallelically mutated) and in cells expressing a form of SMC3 that cannot be acetylated. This defect was a consequence of cohesin’s hyperstable interaction with two regulatory cofactors, WAPL and PDS5A (refs 12, 13); removal of either cofactor allowed forks to progress rapidly without ESCO1, ESCO2, or RFCCTF18. Our results show a novel mechanism for clamp-loader-dependent fork progression, mediated by the post-translational modification and structural remodelling of the cohesin ring. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage and may contribute to the abnormalities of the Roberts’ syndrome cohesinopathy.

The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.

Plk1 self-organization and priming phosphorylation of HsCYK-4 at the spindle midzone regulate the onset of division in human cells

Self-regulated movement of Polo-like kinase 1 to the midzone of the mitotic spindle initiates a local signaling cascade that activates the cell division machinery at the cells equator.

Chromothripsis: Chromosomes in Crisis

During oncogenesis, cells acquire multiple genetic alterations that confer essential tumor-specific traits, including immortalization, escape from antimitogenic signaling, neovascularization, invasiveness, and metastatic potential. In most instances, these alterations are thought to arise incrementally over years, if not decades. However, recent progress in sequencing cancer genomes has begun to challenge this paradigm, because a radically different phenomenon, termed chromothripsis, has been suggested to cause complex intra- and interchromosomal rearrangements on short timescales. In this Review, we review established pathways crucial for genome integrity and discuss how their dysfunction could precipitate widespread chromosome breakage and rearrangement in the course of malignancy.

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.

Combination of Chemical Genetics and Phosphoproteomics for Kinase Signaling Analysis Enables Confident Identification of Cellular Downstream Targets

Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates.

Sister acts: coordinating DNA replication and cohesion establishment

The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesins ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated.