Vincent Vanoosthuyse

A Novel Protein Phosphatase 1-Dependent Spindle Checkpoint Silencing Mechanism

Summary The spindle checkpoint is a surveillance system acting in mitosis to delay anaphase onset until all chromosomes are properly attached to the mitotic spindle [1, 2]. When the checkpoint is activated, the Mad2 and Mad3 proteins directly bind and inhibit Cdc20, which is an essential activator of an E3 ubiquitin ligase known as the anaphase-promoting complex (APC) [3]. When the checkpoint is satisfied, Cdc20-APC is activated and polyubiquitinates securin and cyclin, leading to the dissolution of sister chromatid cohesion and mitotic progression. Several protein kinases play critical roles in spindle checkpoint signaling, but the mechanism (or mechanisms) by which they inhibit mitotic progression remains unclear [4]. Furthermore, it is not known whether their activity needs to be reversed by protein phosphatases before anaphase onset can occur. Here we employ fission yeast to show that Aurora (Ark1) kinase activity is directly required to maintain spindle checkpoint arrest, even in the presence of many unattached kinetochores. Upon Ark1 inhibition, checkpoint complexes are disassembled and cyclin B is rapidly degraded. Importantly, checkpoint silencing and cyclin B degradation require the kinetochore-localized isoform of protein phosphatase 1 (PP1Dis2). We propose that PP1Dis2-mediated dephosphorylation of checkpoint components forms a novel spindle checkpoint silencing mechanism.

Spindle checkpoint silencing requires association of PP1 to both Spc7 and kinesin-8 motors.

The spindle checkpoint is the prime cell-cycle control mechanism that ensures sister chromatids are bioriented before anaphase takes place. Aurora B kinase, the catalytic subunit of the chromosome passenger complex, both destabilizes kinetochore attachments that do not generate tension and simultaneously maintains the spindle checkpoint signal. However, it is unclear how the checkpoint is silenced following chromosome biorientation. We demonstrate that association of type 1 phosphatase (PP1(Dis2)) with both the N terminus of Spc7 and the nonmotor domains of the Klp5-Klp6 (kinesin-8) complex is necessary to counteract Aurora B kinase to efficiently silence the spindle checkpoint. The role of Klp5 and Klp6 in checkpoint silencing is specific to this class of kinesin and independent of their motor activities. These data demonstrate that at least two distinct pools of PP1, one kinetochore associated and the other motor associated, are needed to silence the spindle checkpoint.

Interaction of Calmodulin, a Sorting Nexin and Kinase-Associated Protein Phosphatase with the Brassica oleracea S Locus Receptor Kinase

Recognition of self-pollen during the self-incompatibility response in Brassica oleracea is mediated by the binding of a secreted peptide (the S locus cysteine-rich protein) to the S locus receptor kinase (SRK), a member of the plant receptor kinase (PRK) superfamily. Here, we describe the characterization of three proteins that interact with the cytosolic kinase domain of SRK. A B. oleracea homolog of Arabidopsis kinase-associated protein phosphatase was shown to interact with and dephosphorylate SRK and was itself phosphorylated by SRK. Yeast (Saccharomyces cerevisiae) two-hybrid screens identified two additional interactors, calmodulin and a sorting nexin, both of which have been implicated in receptor kinase down-regulation in animals. A calmodulin-binding site was identified in sub-domain VIa of the SRK kinase domain. The binding site is conserved and functional in several other members of the PRK family. The sorting nexin also interacted with diverse members of the PRK family, suggesting that all three of the interacting proteins described here may play a general role in signal transduction by this family of proteins.

Making inroads into plant receptor kinase signalling pathways

Cell-membrane-located receptor kinases play important roles in many plant signal-transduction pathways. Exciting progress has been made in recent years with the characterization of four ligand-receptor systems involved in physiological processes as diverse as self-pollen rejection, stem-cell maintenance and differentiation at the shoot meristem, the response to the brassinosteroid hormones and the innate response to bacterial pathogens. These new findings emphasize the remarkably high diversity of these signalling pathways, although some downstream components are shared. This observation supports the idea that the wide diversification of plant receptors is associated with a high degree of specialization, one receptor potentially regulating a single developmental process. However, the possibility that one receptor might have a dual recognition function cannot be ruled out.

Kinetochore Targeting of Fission Yeast Mad and Bub Proteins Is Essential for Spindle Checkpoint Function but Not for All Chromosome Segregation Roles of Bub1p

ABSTRACT Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a “wait anaphase” signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.

An acetylated form of histone H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe

Histone variant H2A.Z has a conserved role in genome stability, although it remains unclear how this is mediated. Here we demonstrate that the fission yeast Swr1 ATPase inserts H2A.Z (Pht1) into chromatin and Kat5 acetyltransferase (Mst1) acetylates it. Deletion or an unacetylatable mutation of Pht1 leads to genome instability, primarily caused by chromosome entanglement and breakage at anaphase. This leads to the loss of telomere-proximal markers, though telomere protection and repeat length are unaffected by the absence of Pht1. Strikingly, the chromosome entanglement in pht1Δ anaphase cells can be rescued by forcing chromosome condensation before anaphase onset. We show that the condensin complex, required for the maintenance of anaphase chromosome condensation, prematurely dissociates from chromatin in the absence of Pht1. This and other findings suggest an important role for H2A.Z in the architecture of anaphase chromosomes.

Control of Shugoshin Function during Fission-Yeast Meiosis

Meiosis consists of a single round of DNA replication followed by two consecutive nuclear divisions. During the first division (MI), sister kinetochores must orient toward the same pole to favor reductional segregation. Correct chromosome segregation during the second division (MII) requires the retention of centromeric cohesion until anaphase II. The spindle checkpoint protein Bub1 is essential for both processes in fission yeast . When bub1 is deleted, the Shugoshin protein Sgo1 is not recruited to centromeres, cohesin Rec8 does not persist at centromeres, and sister-chromatid cohesion is lost by the end of MI. Deletion of bub1 also affects kinetochore orientation because sister centromeres can move to opposite spindle poles in approximately 30% of MI divisions. We show here that these two functions are separable within the Bub1 protein. The N terminus of Bub1 is necessary and sufficient for Sgo1 targeting to centromeres and the protection of cohesion, whereas the C-terminal kinase domain acts together with Sgo2, the second fission-yeast Shugoshin protein, to promote sister-kinetochore co-orientation during MI. Additional analyses suggest that the protection of centromeric cohesion does not operate when sister kinetochores attach to opposite spindle poles during MI. Sgo1-mediated protection of centromere cohesion might therefore be regulated by the mode of kinetochore attachment.

Bub3p Facilitates Spindle Checkpoint Silencing in Fission Yeast

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3 Delta cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.

Overcoming inhibition in the spindle checkpoint

Spindle checkpoint silencing is a critical step during mitosis that initiates chromosome segregation, yet surprisingly little is known about its mechanism. Protein phosphatase I (PP1) was shown recently to be a key player in this process, and in this issue of Genes & Deverlopment, Akiyoshi and colleagues (pp. 2887-2899) identify budding yeast Fin1p as a kinetochore-localized regulator of PP1 activity toward checkpoint targets. Here we review recent mechanistic insights and propose a working model for spindle checkpoint silencing.

CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation.

Functional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3′ end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1Dis2 with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72.