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.

Autophagy-related protein Atg11 preserves asymmetric inheritance of spindle microtubule-organizing centers

Asymmetric spindle pole body (SPB) inheritance requires a cascade of events that involve kinases, phosphatases and structural scaffold proteins including molecular motors and microtubule-associated proteins present in the nucleus and/or the cytoplasm. Higher levels of an SPB component Spc72 and the spindle positioning factor Kar9 at the old SPB, which migrates to the daughter cell, ensure asymmetric SPB inheritance. Timely SPB duplication followed by its asymmetric inheritance is a key to correct spindle alignment leading to high-fidelity chromosome segregation. By combining in silico analysis of known protein-protein interactions of autophagy (Atg)-related proteins with those that constitute the chromosome segregation machinery, and growth dynamics of 35 atg mutants in the presence of a microtubule poison, we identified Atg11 as a potential regulator of chromosome transmission. Cells lacking Atg11 did not show any kinetochore defects but displayed a high rate of chromosome loss and delayed anaphase onset. Atg11 positively interacted with Kar9 and Kip2 and negatively with Dyn1 and Kar3 in mediating proper chromosome segregation suggesting a role of Atg11 in spindle positioning. Indeed, atg11∆ cells displayed an inverted SPB inheritance. We further show that Atg11 promotes asymmetric localization of Spc72 and Kar9 on the old SPB. Atg11 physically interacted with Spc72 and transiently localized close to the old SPB during metaphase-to-anaphase progression. Taken together, our study uncovers an autophagy-independent role of Atg11 in spindle alignment and emphasizes the importance of unbiased screens to identify factors mediating the complex and intricate crosstalk between processes fundamental to genomic integrity.

Unraveling the kinetochore nanostructure in Schizosaccharomyces pombe using multi-color single-molecule localization microscopy

The key to ensuring proper chromosome segregation during mitosis is the kinetochore complex. This large and tightly regulated multi-protein complex links the centromeric chromatin to the microtubules attached to the spindle pole body and as such leads the segregation process. Understanding the architecture, function and regulation of this vital complex is therefore essential. However, due to its complexity and dynamics, only its individual subcomplexes could be studied in high-resolution structural detail so far.In this study we construct a nanometer-precise in situ map of the human-like regional kinetochore of Schizosaccharomyces pombe (S. pombe) using multi-color single-molecule localization microscopy (SMLM). We measure each kinetochore protein of interest (POI) in conjunction with two reference proteins, cnp1CENP-A at the centromere and sad1 at the spindle pole. This arrangement allows us to determine the cell cycle and in particularly the mitotic plane, and to visualize individual centromere regions separately. From these data, we determine protein distances within the complex using Bayesian inference, establish the stoichiometry of each POI for individual chromosomes and, consequently, build an in situ kinetochore model for S.pombe with so-far unprecedented precision. Being able to quantify the kinetochore proteins within the full in situ kinetochore structure, we provide valuable new insights in the S.pombe kinetochore architecture.

Three-dimensional structure of the kinetochore-fibers in human mitotic spindles

During cell division, kinetochore microtubules (KMTs) provide a physical linkage between the spindle poles and the chromosomes. KMTs in mammalian cells are organized into bundles, so-called kinetochore-fibers (k-fibers), but the ultrastructure of these fibers is currently not well characterized. Here we show by large-scale electron tomography that each k-fiber in HeLa cells in metaphase is composed of approximately nine KMTs, only half of which reach the spindle pole. Our comprehensive reconstructions allowed us to analyze the three-dimensional (3D) morphology of k-fibers in detail, and we find that they exhibit remarkable variation. K-fibers display differences in circumference and KMT density along their length, with the pole-facing side showing a splayed-out appearance. We further observed that the association of KMTs with non-KMTs predominantly occurs in the spindle pole regions. Our 3D reconstructions have implications for models of KMT behavior and k-fiber self-organization as covered in a parallel publication applying complementary live-cell imaging in combination with biophysical modeling (Conway et al., 2021). The presented data will also serve as a resource for further studies on mitosis in human cells.

Pcp1/pericentrin controls the SPB number in fission yeast meiosis and ploidy homeostasis

During sexual reproduction, the zygote must inherit exactly one centrosome (spindle pole body [SPB] in yeasts) from the gametes, which then duplicates and assembles a bipolar spindle that supports the subsequent cell division. Here, we show that in the fission yeast Schizosaccharomyces pombe, the fusion of SPBs from the gametes is blocked in polyploid zygotes. As a result, the polyploid zygotes cannot proliferate mitotically and frequently form supernumerary SPBs during subsequent meiosis, which leads to multipolar nuclear divisions and the generation of extra spores. The blockage of SPB fusion is caused by persistent SPB localization of Pcp1, which, in normal diploid zygotic meiosis, exhibits a dynamic association with the SPB. Artificially induced constitutive localization of Pcp1 on the SPB is sufficient to cause blockage of SPB fusion and formation of extra spores in diploids. Thus, Pcp1-dependent SPB quantity control is crucial for sexual reproduction and ploidy homeostasis in fission yeast.

SFI1 and centrin form a distal end complex critical for proper centriole architecture and ciliogenesis

Over the course of evolution, the function of the centrosome has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, as illustrated by the presence of centrioles in humans and a spindle pole body in yeast (SPB). Consistently, the composition of these two core elements has diverged greatly, with the exception of centrin, a protein known to form a complex with Sfi1 in yeast to structurally initiate SPB duplication. Even though SFI1 has been localized to human centrosomes, whether this complex exists at centrioles and whether its function has been conserved is still unclear. Here, using conventional fluorescence and super-resolution microscopies, we demonstrate that human SFI1 is a bona fide centriolar protein localizing to the very distal end of the centriole, where it associates with a pool of distal centrin. We also found that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the specific loss of the distal pool of centrin, without altering centriole duplication in human cells, in contrast to its function for SPB. Instead, we found that SFI1/centrin complexes are essential for correct centriolar architecture as well as for ciliogenesis. We propose that SFI1/centrin complexes may guide centriole growth to ensure centriole integrity and function as a basal body.

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.

Interferon Regulatory Factor 2 (IRF2) Inhibits the Invasion and Migration of Renal Clear Cell Carcinoma Cells by Downregulation of Spindle Pole Body Component 24 (SPC24)

It has been reported that the increased expression of SPC24 (spindle pole body component 24) was involved in the initiation and development of various cancers. However, the role of SPC24 in ccRCC (clear cell renal cell carcinoma) remains largely unknown. In the present study, the changes and correlation of SPC24 and IRF2 (interferon regulatory factor 2) with ccRCC were evaluated by using GEPIA, TCGA and GTEx database. Then the involvement of SPC24 and IRF2 in invasion and migration was investigated in CaKi-1 cells, a human renal adenocarcinoma cell line. The bioinformatics assay revealed that the expression of SPC24 and IRF2 in kidney tissue of patients with renal clear cell cancer was significantly increased, and the expression of SPC24 and IRF2 in kidney tissue was positively and negatively related to cancer phase and survival rate in patients with ccRCC respectively. Notably, in vitro experimental study demonstrated that SPC25 promoted the invasion and migration of CaKi-1 cells, a human renal adenocarcinoma cell line. Furthermore, IRF2 shows potential binding site with SPC24 promoter, IRF2 overexpression significantly decreased SPC24 mRNA level, whereas inhibition of IRF2 with specific small hairpin RNA (shRNA) significantly increased SPC24 mRNA level. Functionally, inhibition of SPC24 with specific shRNA reversed the stimulatory effect of IRF2 shRNA on the invasion and migration of cells, whereas SPC24 over-expression reversed the inhibitory effect of IRF2 overexpression on the invasion and migration of cells. Finally, ChIP (chromatin immunoprecipitation) assay shows that IRF2 could directly bind with SPC24 promoter. In conclusion, these results demonstrated that IRF2/SPC24 signaling pathway contributes to the increased invasion and migration in ccRCC.