A Novel Hyperactive Nud1 Mitotic Exit Network Scaffold Causes Spindle Position Checkpoint Bypass in Budding Yeast

Mitotic exit is a critical cell cycle transition that requires the careful coordination of nuclear positioning and cyclin B destruction in budding yeast for the maintenance of genome integrity. The mitotic exit network (MEN) is a Ras-like signal transduction pathway that promotes this process during anaphase. A crucial step in MEN activation occurs when the Dbf2-Mob1 protein kinase complex associates with the Nud1 scaffold protein at the yeast spindle pole bodies (SPBs; centrosome equivalents) and thereby becomes activated. This requires prior priming phosphorylation of Nud1 by Cdc15 at SPBs. Cdc15 activation, in turn, requires both the Tem1 GTPase and the Polo kinase Cdc5, but how Cdc15 associates with SPBs is not well understood. We have identified a hyperactive allele of NUD1, nud1-A308T, that recruits Cdc15 to SPBs in all stages of the cell cycle in a CDC5-independent manner. This allele leads to early recruitment of Dbf2-Mob1 during metaphase and requires known Cdc15 phospho-sites on Nud1. The presence of nud1-A308T leads to loss of coupling between nuclear position and mitotic exit in cells with mispositioned spindles. Our findings highlight the importance of scaffold regulation in signaling pathways to prevent improper activation.

Centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-ACnp1 incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-ACnp1 overexpression bypasses heterochromatin dependency, suggesting heterochromatin ensures exposure to conditions or locations permissive for CENP-ACnp1 assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-ACnp1 incorporation. We demonstrate that heterochromatin-independent de novo CENP-ACnp1 chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-ACnp1 assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-ACnp1 incorporation because target sequences are exposed to high levels of CENP-ACnp1 and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity.

Downregulation of the Tem1 GTPase by Amn1 after cytokinesis involves both nuclear import and SCF-mediated degradation

ABSTRACT At mitotic exit the cell cycle engine is reset to allow crucial processes, such as cytokinesis and replication origin licensing, to take place before a new cell cycle begins. In budding yeast, the cell cycle clock is reset by a Hippo-like kinase cascade called the mitotic exit network (MEN), whose activation is triggered at spindle pole bodies (SPBs) by the Tem1 GTPase. Yet, MEN activity must be extinguished once MEN-dependent processes have been accomplished. One factor contributing to switching off the MEN is the Amn1 protein, which binds Tem1 and inhibits it through an unknown mechanism. Here, we show that Amn1 downregulates Tem1 through a dual mode of action. On one side, it evicts Tem1 from SPBs and escorts it into the nucleus. On the other, it promotes Tem1 degradation as part of a Skp, Cullin and F-box-containing (SCF) ubiquitin ligase. Tem1 inhibition by Amn1 takes place after cytokinesis in the bud-derived daughter cell, consistent with its asymmetric appearance in the daughter cell versus the mother cell. This dual mechanism of Tem1 inhibition by Amn1 may contribute to the rapid extinguishing of MEN activity once it has fulfilled its functions.

The role of Aspergillus nidulans polo-like kinase PlkA in microtubule-organizing center control

Centrosomes are important microtubule-organizing centers (MTOC) in animal cells. In addition, non-centrosomal MTOCs (ncMTOCs) were described in many cell types. Functional analogs of centrosomes in fungi are the spindle pole bodies (SPBs). In Aspergillus nidulans additional MTOCs were discovered at septa (sMTOC). Although the core components are conserved in both MTOCs, their composition and organization are different and dynamic. Here, we show that the polo-like kinase PlkA binds the γ-tubulin ring complex (γ-TuRC) receptor protein ApsB and contributes to targeting ApsB to both MTOCs. PlkA coordinates SPB outer plaque with sMTOC activities. PlkA kinase activity was required for astral MT formation involving ApsB recruitment. PlkA also interacted with the γ-TuRC inner plaque receptor protein PcpA. Mitosis was delayed without PlkA, and the PlkA protein was required for proper mitotic spindle morphology, although this function was independent of its catalytic activity. Our results suggest polo-like kinase as a regulator of MTOC activities and as a scaffolding unit through interaction with γ-tubulin ring complex receptors.

Cross-compartment signal propagation in the mitotic exit network

In budding yeast, the mitotic exit network (MEN), a GTPase signaling cascade, integrates spatial and temporal cues to promote exit from mitosis. This signal integration requires transmission of a signal generated on the cytoplasmic face of spindle pole bodies (SPBs; yeast equivalent of centrosomes) to the nucleolus, where the MEN effector protein Cdc14 resides. Here, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. The kinase-primed transmission of the MEN signal from the cytoplasm to the nucleolus exemplifies how signaling cascades can bridge distant inputs and responses.

The collapse of the spindle following ablation in S. pombe is mediated by microtubules and the motor protein dynein

A microtubule-based machine called the mitotic spindle segregates chromosomes when eukaryotic cells divide. In the fission yeast S. pombe, which undergoes closed mitosis, the spindle forms a single bundle of microtubules inside the nucleus. During elongation, the spindle extends via antiparallel microtubule sliding by molecular motors. These extensile forces from the spindle are thought to resist compressive forces from the nucleus. We probe the mechanism and maintenance of this force balance via laser ablation of spindles at various stages of mitosis. We find that spindle pole bodies collapse toward each other following ablation, but spindle geometry is often rescued, allowing spindles to resume elongation and segregate chromosomes. While this basic behavior has been previously observed, many questions remain about this phenomenon’s dynamics, mechanics, and molecular requirements. In this work, we find that previously hypothesized viscoelastic relaxation of the nucleus cannot fully explain spindle shortening in response to laser ablation. Instead, spindle collapse requires microtubule dynamics and is powered at least partly by the minus-end directed motor protein dynein. These results suggest a role for dynein in redundantly supporting force balance and bipolarity in the S. pombe spindle.

Cross-compartment signal propagation in the Mitotic Exit Network

ABSTRACTIn budding yeast, the Mitotic Exit Network (MEN), a GTPase signaling cascade integrates spatial and temporal cues to promote exit from mitosis. This signal integration requires transmission of a signal generated on the cytoplasmic face of spindle pole bodies (SPBs; yeast equivalent of centrosomes) to the nucleolus, where the MEN effector protein Cdc14 resides. Here, we show that the MEN activating signal at SPBs is relayed to Cdc14 in the nucleolus through the dynamic localization of its terminal kinase complex Dbf2-Mob1. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. The kinase-primed transmission of the MEN signal from the cytoplasm to the nucleolus exemplifies how signaling cascades can bridge distant inputs and responses.

Loss of Kinesin-8 improves the robustness of the acentrosomal spindle

Chromosome segregation in female meiosis is inherently error-prone, among other reasons, because the acentrosomal spindle assembles and segregates chromosomes without the major microtubule-organizing centres in eukaryotes, the centrosomes, which causes high rate of aneuploidy. The molecular basis underlying formation of acentrosomal spindles is not as well-understood as that of centrosomal spindles and, consequently, strategies to improve spindle robustness are difficult to address. Recently, we noticed during fission yeast meiosis the formation of unexpected microtubules arrays, independent of the spindle pole bodies (yeast centrosome equivalent), with ability to segregate chromosomes. Here, we establish such microtubules formation as bonafide self-assembled spindles that depend on the canonical microtubule crosslinker Ase1/PRC1, share similar structural polarity and harbour the microtubule polymerase Alp14/XMAP215, while being independent of conventional γ-tubulin-mediated nucleation mechanisms. Remarkably, acentrosomal spindle robustness was reinforced by deletion of the Klp6/Kinesin-8, which, consequently, led to a reduced meiotic aneuploidy rate. Our results enlighten the molecular basis of acentrosomal meiosis, a crucial event in understanding gametogenesis.

Dynein collective behavior in mitotic nuclear positioning of Saccharomyces cerevisiae

1AbstractPositioning the nucleus at the bud-neck prior during Saccharomyces cerevisiae mitosis during anaphase involves pulling forces of cytoplasmic dynein localized in the daughter cell. While genetic analysis has revealed a complex network positioning the nucleus, quantification of the forces acting on the nucleus and dyneins numbers driving the process has remained difficult. In order to better understand the role of motor-microtubule mechanics during nuclear positioning and the role of dynein, we have used a computational model of nuclear mobility in S. cerevisiae and reconciled it to the mobility of labelled spindle pole bodies (SPBs) measured by quantifying fluorescence microscopy time-series. We model the apparent random-walk mobility of SPBs by combining diffusion of the nucleus and active pushing of MTs at the cell membrane. By minimizing the deviation between tracks of fluorescently tagged SPBs and simulations, we estimate the effective cytoplasmic viscosity to be 0.5 Pa s. The directed transport of nuclei during the budding process is similarly quantified by tracking the daughter SPB (SPB-D) in experiment. Using force-balance, we find 2 to 8 motors are required to pull the nucleus to the bud-neck. Simulations of the cytoplasmic MT (cMT) ‘search and capture’ by dynein suggest single motor binding is followed by a rapid saturation of number of bound motors. The short time and length of MT interactions with the cortex and minimal collective dynein force required, predict a functional role for dynein clustering in nuclear positioning.