The joy of the 11th International Fission Yeast Meeting in Hiroshima (POMBE2023 Hiroshima) after a long wait due to the COVID-19 pandemic.

The 11th International Fission Yeast Meeting took place at Astel Plaza in Hiroshima, Japan, from May 28th to June 2nd, 2023. This highly anticipated gathering, originally scheduled for May 2021, had been postponed for 2 years due to the COVID-19 pandemic. Researchers from 21 countries, including 211 overseas and 157 domestic participants (overall gender ratio is roughly 60% male vs. 40% female), eagerly awaited the opportunity to meet in person, as virtual interactions had been the only means of communication during this challenging period. The meeting featured four kick-off special lectures, 101 regular talks, and 152 poster presentations. Additionally, a discussion session on upfront frontier research in fission yeast provided an interactive platform for both speakers and attendees. Throughout the event, participants shared cutting-edge knowledge, celebrated significant research findings, and relished the invaluable experience of an in-person meeting. The vibrant and friendly atmosphere, characteristic of this esteemed international conference, fostered collaboration and reinforced the significance of studying this exceptional model organism. Undoubtedly, the outcomes of this meeting will greatly contribute to our understanding of complex biological systems, not only in fission yeast but also in general eukaryotes.

Seismic seiche-related oscillations in Lake Biwa, Japan, after the 2011 Tohoku earthquake.

Seismic seiche-related oscillations caused by Rayleigh waves from large earthquakes are yet to be explored and elucidated comprehensively, then need to accumulate continuously. Herein, we investigated water level fluctuations in Lake Biwa of Japan from surface seiches following the 2011 Tohoku earthquake. Lake Biwa is the largest freshwater resource in Japan, and a small change in its water level can affect local ecosystems. Field observations were conducted during 2010-2012 using a water level gauge with a 1 mm resolution and 2 min data sampling interval. Fast Fourier transform and maximum entropy methods were used for data spectral analysis to distinguish the effects of inherent oscillations on water levels generated by the earthquake. We considered that water level changes were influenced by long-period Rayleigh waves. We observed a wave with a 3.08-3.10 h duration, which was close to the duration determined for the Rayleigh waves (3.08 h). The 3.08-3.10 h wave was caused by forced oscillation of Rayleigh waves characterised by a frequency close to the natural frequency and excited by the earthquake. Overall, our findings suggest that water level fluctuations can be good indicators of high-magnitude earthquakes.

Complementation of fission yeast kinesin-5/Cut7 with human Eg5 provides a versatile platform for screening of anticancer compounds.

Kinesin-5 family proteins are essential for bipolar spindle assembly to ensure mitotic fidelity. Here, we demonstrate evolutionary functional conservation of kinesin-5 between human and fission yeast. Human Eg5 expressed in the nucleus replaces fission yeast counterpart Cut7. Intriguingly, Eg5 overproduction results in cytotoxicity. This phenotype provides a useful platform for the development of novel kinesin-5 inhibitors as anticancer drugs.

The Putative RNA-Binding Protein Dri1 Promotes the Loading of Kinesin-14/Klp2 to the Mitotic Spindle and Is Sequestered into Heat-Induced Protein Aggregates in Fission Yeast.

Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

KIFC1 regulates ZWINT to promote tumor progression and spheroid formation in colorectal cancer.

Colorectal cancer (CRC) is the second leading cause of cancer-related mortality worldwide. Kinesin Family Member C1 (KIFC1) has been proposed as a promising therapeutic target due to its pivotal role in centrosome clustering to mediate cancer cell progression. This study aimed to analyze the expression and biological function of KIFC1 in CRC. Immunohistochemically, 67 (52%) of 129 CRC cases were positive for KIFC1 and statistically associated with poorer overall survival. KIFC1 small interfering RNA (siRNA)-transfected cells demonstrated lower cell proliferation as compared to the negative control cells. A specific KIFC1 inhibitor, kolavenic acid analog (KAA) drastically inhibited CRC cell proliferation. Microarray analysis revealed that KAA-treated CRC cells presented reduced ZW10 interacting kinetochore protein (ZWINT) expression as compared to control cells. Immunohistochemical analysis demonstrated that 61 (47%) of 129 CRC cases were positive for ZWINT and ZWINT expression was significantly correlated with KIFC1 expression. ZWINT-positive cases exhibited significantly worse overall survival. KIFC1 siRNA-transfected cells showed reduced ZWINT expression while ZWINT siRNA-transfected cells decreased cell proliferation. Both KIFC1 and ZWINT knockdown cells attenuated spheroid formation ability. This study provides new insights into KIFC1 regulating ZWINT in CRC progression and its potential as a therapeutic target.

Escape from mitotic catastrophe by actin-dependent nuclear displacement in fission yeast.

Eukaryotic cells position the nucleus within the proper intracellular space, thereby safeguarding a variety of cellular processes. In fission yeast, the interphase nucleus is placed in the cell middle in a microtubule-dependent manner. By contrast, how the mitotic nucleus is positioned remains elusive. Here we show that several cell-cycle mutants that arrest in mitosis all displace the nucleus toward one end of the cell. Intriguingly, the actin cytoskeleton is responsible for nuclear movement. Time-lapse live imaging indicates that mitosis-specific F-actin cables possibly push the nucleus through direct interaction with the nuclear envelope, and subsequently actomyosin ring constriction further shifts the nucleus away from the center. This nuclear movement is beneficial, because if the nuclei were retained in the center, unseparated chromosomes would be intersected by the contractile actin ring and the septum, imposing the lethal cut phenotype. Thus, fission yeast escapes from mitotic catastrophe by means of actin-dependent nuclear movement.

How Essential Kinesin-5 Becomes Non-Essential in Fission Yeast: Force Balance and Microtubule Dynamics Matter.

The bipolar mitotic spindle drives accurate chromosome segregation by capturing the kinetochore and pulling each set of sister chromatids to the opposite poles. In this review, we describe recent findings on the multiple pathways leading to bipolar spindle formation in fission yeast and discuss these results from a broader perspective. The roles of three mitotic kinesins (Kinesin-5, Kinesin-6 and Kinesin-14) in spindle assembly are depicted, and how a group of microtubule-associated proteins, sister chromatid cohesion and the kinetochore collaborate with these motors is shown. We have paid special attention to the molecular pathways that render otherwise essential Kinesin-5 to become non-essential: how cells build bipolar mitotic spindles without the need for Kinesin-5 and where the alternate forces come from are considered. We highlight the force balance for bipolar spindle assembly and explain how outward and inward forces are generated by various ways, in which the proper fine-tuning of microtubule dynamics plays a crucial role. Overall, these new pathways have illuminated the remarkable plasticity and adaptability of spindle mechanics. Kinesin molecules are regarded as prospective targets for cancer chemotherapy and many specific inhibitors have been developed. However, several hurdles have arisen against their clinical implementation. This review provides insight into possible strategies to overcome these challenges.

Kolavenic acid analog restores growth in HSET-overproducing fission yeast cells and multipolar mitosis in MDA-MB-231 human cells.

Although cancer cells often harbor supernumerary centrosomes, they form pseudo-bipolar spindles via centrosome clustering, instead of lethal multipolar spindles, and thus avoid cell death. Kinesin-14 HSET/KIFC1 is a crucial protein involved in centrosome clustering. Accordingly, a compound that targets HSET could potentially inhibit cancer cell proliferation in a targeted manner. Here, we report three natural compounds derived from Solidago altissima that restored the growth of fission yeast cells exhibiting lethal HSET overproduction (positive screening), namely solidagonic acid (SA) (1), kolavenic acid analog (KAA: a stereo isomer at C-9 and C-10 of 6ß-tigloyloxykolavenic acid) (2), and kolavenic acid (KA) (3). All three compounds suppressed fission yeast cell death and enabled reversion of the mitotic spindles from a monopolar to bipolar morphology. Compound 2, which exerted the strongest activity against HSET-overproducing yeast cells, also inhibited centrosome clustering in MDA-MB-231 human breast adenocarcinoma cells, which contained large numbers of supernumerary centrosomes. These natural compounds may be useful as bioprobes in studies of HSET function. Moreover, compound 2 is a prime contender in the development of novel agents for cancer treatment.

Two XMAP215/TOG Microtubule Polymerases, Alp14 and Dis1, Play Non-Exchangeable, Distinct Roles in Microtubule Organisation in Fission Yeast.

Proper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal "cut" phenotype. By implementing an artificial targeting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Interestingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.

Kinesin-8 and Dis1/TOG collaborate to limit spindle elongation from prophase to anaphase A for proper chromosome segregation in fission yeast.

High-fidelity chromosome segregation relies on proper microtubule regulation. Kinesin-8 has been shown to destabilise microtubules to reduce metaphase spindle length and chromosome movements in multiple species. XMAP215/chTOG polymerases catalyse microtubule growth for spindle assembly, elongation and kinetochore-microtubule attachment. Understanding of their biochemical activity has advanced, but little work directly addresses the functionality and interplay of these conserved factors. We utilised the synthetic lethality of fission yeast kinesin-8 (Klp5-Klp6) and XMAP215/chTOG (Dis1) to study their individual and overlapping roles. We found that the non-motor kinesin-8 tailbox is essential for mitotic function; mutation compromises plus-end-directed processivity. Klp5-Klp6 induces catastrophes to control microtubule length and, surprisingly, Dis1 collaborates with kinesin-8 to slow spindle elongation. Together, they enforce a maximum spindle length for a viable metaphase-anaphase transition and limit elongation during anaphase A to prevent lagging chromatids. Our work provides mechanistic insight into how kinesin-8 negatively regulates microtubules and how this functionally overlaps with Dis1 and highlights the importance of spindle length control in mitosis.

Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast.

Bipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation in a process called anaphase B. Two kinesin motors, Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules. However, how these three proteins orchestrate this process remains unknown. Here we explore the functional interplay among fission yeast Kinesin-5/Cut7, Kinesin-6/Klp9 and Ase1. Using total internal reflection fluorescence microscopy, we show that Klp9 forms homotetramers and that Klp9 is a processive plus end-directed motor. klp9Δase1Δ is synthetically lethal. Surprisingly, this lethality is not ascribable to the defective motor activity of Klp9; instead, it is dependent upon a nuclear localisation signal and coiled coil domains within the non-motor region. We isolated a cut7 mutant (cut7-122) that displays temperature sensitivity only in the absence of Klp9. Interestingly, cut7-122 alone is impaired in spindle elongation during anaphase B, and furthermore, cut7-122klp9Δ double mutants exhibit additive defects. We propose that Klp9 plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut7 in force generation, while the other is motor-independent that ensures structural integrity of spindle microtubules together with Ase1.

Generation of temperature sensitive mutations with error-prone PCR in a gene encoding a component of the spindle pole body in fission yeast.

Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions of essential genes. We report here asimple procedure to generate ts mutations using error-prone PCR within pcp1 that encodes aspindle pole body (SPB) component in Schizosaccharomyces pombe. This manipulation is not restricted to pcp1, and can be suited to any essential genes involved in other processes.

Prevalence of Autoimmune Gastritis in Individuals Undergoing Medical Checkups in Japan.

Objective Based on both endoscopic findings and serum auto-antibody levels, we determined the prevalence of autoimmune gastritis (AIG), which has not been previously reported, in individuals who underwent health checkup examinations in Japan. Methods At total of 6,739 subjects (4,288 males, 2,451 females; mean age 52.1 years) underwent an upper gastrointestinal endoscopic examination as part of an annual medical checkup. Those suspected to have AIG based on endoscopic evidence of proximal-predominant gastric mucosal atrophy were further examined for the presence of anti-parietal cells and anti-intrinsic factor antibodies, with a final diagnosis of AIG made in cases found to be positive for either or both of those factors. Results Of the 6,739 examined subjects, 46 were suspected to have AIG based on the endoscopic findings, of whom 33 were finally diagnosed with AIG, for an overall prevalence 0.49% (females 0.65%, males 0.40%). Seven with AIG also had thyroid disease, including Hashimoto's and Basedow disease, while none with AIG showed anemia in blood test findings. The prevalence of AIG was not different regardless of the H. pylori infection status (negative, positive, post-eradicated). Conclusion In individuals who underwent an upper gastrointestinal endoscopic examination as part of an annual checkup in Japan, the prevalence of AIG was 0.49%. We concluded that it is not uncommon for asymptomatic and healthy individuals to have AIG, and propose that additional studies are needed to clarify its prevalence as well as to establish the criteria used for diagnosis.

Suppressor Analysis Uncovers That MAPs and Microtubule Dynamics Balance with the Cut7/Kinesin-5 Motor for Mitotic Spindle Assembly in Schizosaccharomyces pombe.

The Kinesin-5 motor Cut7 in Schizosaccharomyces pombe plays essential roles in spindle pole separation, leading to the assembly of bipolar spindle. In many organisms, simultaneous inactivation of Kinesin-14s neutralizes Kinesin-5 deficiency. To uncover the molecular network that counteracts Kinesin-5, we have conducted a genetic screening for suppressors that rescue the cut7-22 temperature sensitive mutation, and identified 10 loci. Next generation sequencing analysis reveals that causative mutations are mapped in genes encoding α-, ß-tubulins and the microtubule plus-end tracking protein Mal3/EB1, in addition to the components of the Pkl1/Kinesin-14 complex. Moreover, the deletion of various genes required for microtubule nucleation/polymerization also suppresses the cut7 mutant. Intriguingly, Klp2/Kinesin-14 levels on the spindles are significantly increased in cut7 mutants, whereas these increases are negated by suppressors, which may explain the suppression by these mutations/deletions. Consistent with this notion, mild overproduction of Klp2 in these double mutant cells confers temperature sensitivity. Surprisingly, treatment with a microtubule-destabilizing drug not only suppresses cut7 temperature sensitivity but also rescues the lethality resulting from the deletion of cut7, though a single klp2 deletion per se cannot compensate for the loss of Cut7. We propose that microtubule assembly and/or dynamics antagonize Cut7 functions, and that the orchestration between these two factors is crucial for bipolar spindle assembly.

Effects of Helicobacter pylori eradication on serum lipid levels.

The purpose was to clarify the effects of Helicobacter pylori (H. pylori) eradication on the changes in serum lipid levels by comparing subjects with and without continuous H. pylori infection. The study subjects were 774 individuals (males 536, females 238, mean age 52.6 years) who visited between April 2013 and March 2016 for annual medical checkups. Serum total cholesterol, high-density lipoprotein cholesterol (HDLC), low-density lipoprotein cholesterol (LDLC), and triglyceride levels, and LDLC/HDLC ratio were compared between the subjects with and without H. pylori infection, as well as those with H. pylori eradication subjects. The HDLC level in the H. pylori-positive group was significantly lower as compared to the H. pylori-negative group. The serum level of HDLC in subjects with successful eradication of H. pylori tended to be higher, while the serum levels of total cholesterol, LDLC, and triglycerides tended to be lower in comparison to subjects with continuous H. pylori infection. In addition, the LDLC/HDLC ratio in the H. pylori-positive group was significantly higher than that in the H. pylori-negative group, and successful H. pylori eradication tended to reduce that ratio. In conclusion, successful eradication of H. pylori may have favorable effects on lipid metabolism.

Fission yeast cells overproducing HSET/KIFC1 provides a useful tool for identification and evaluation of human kinesin-14 inhibitors.

Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.

Two spatially distinct kinesin-14 proteins, Pkl1 and Klp2, generate collaborative inward forces against kinesin-5 Cut7 in S. pombe.

Kinesin motors play central roles in bipolar spindle assembly. In many eukaryotes, spindle pole separation is driven by kinesin-5, which generates outward force. This outward force is balanced by antagonistic inward force elicited by kinesin-14 and/or dynein. In fission yeast, two kinesin-14 proteins, Pkl1 and Klp2, play an opposing role against the kinesin-5 motor protein Cut7. However, how the two kinesin-14 proteins coordinate individual activities remains elusive. Here, we show that although deletion of either pkl1 or klp2 rescues temperature-sensitive cut7 mutants, deletion of only pkl1 can bypass the lethality caused by cut7 deletion. Pkl1 is tethered to the spindle pole body, whereas Klp2 is localized along the spindle microtubule. Forced targeting of Klp2 to the spindle pole body, however, compensates for Pkl1 functions, indicating that cellular localizations, rather than individual motor specificities, differentiate between the two kinesin-14 proteins. Interestingly, human kinesin-14 (KIFC1 or HSET) can replace either Pkl1 or Klp2. Moreover, overproduction of HSET induces monopolar spindles, reminiscent of the phenotype of Cut7 inactivation. Taken together, this study has uncovered the biological mechanism whereby two different Kinesin-14 motor proteins exert their antagonistic roles against kinesin-5 in a spatially distinct manner.

A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast.

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end-directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end-directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

Temporal Patterns of Individual Neuronal Firing in Rat Dorsal Column Nuclei Provide Information Required for Somatosensory Discrimination.

A wealth of mechanical information from the body generates various forms of sensory experience during touch or kinesthesia. Dorsal column nuclei (DCN) in the medulla are the first relay station for somatosensory inputs from peripheral receptors. These nuclei integrate somatosensory information and send the output to higher-order centers; therefore, investigating the firing patterns of DCN neurons should elucidate coding principles within the somatosensory system. In this study, we quantified the firing patterns of DCN neurons and examined whether the firing patterns of particular neurons are altered when moving tactile stimuli are applied in different directions. The activities of 17 neurons in the DCN of anesthetized rats were selected and their firing patterns were analyzed using LvR, which refers to the local variation of intervals of action potentials (i.e., the cross-correlation between consecutive intervals of action potentials) compensated by the refractoriness constant, R. The LvR of the 17 neurons ranged widely from 0.35 to 2.28. Of the 17 neurons, 12 responded to hair deflection (hair neurons), whereas five responded specifically to movement of forelimb joints. In 11 of 12 hair neurons, moving stimuli were applied in two to four different directions, which yielded 25 pairs of comparisons. Of these, 14 pairs (56%) showed significant differences in LvR. Among these 14 pairs, the range of LvR fluctuation was 0.13 ± 0.06 (mean ± standard deviation) and its effect size (Cohen's d) was 0.6 ± 0.2. These results suggest that the firing pattern of individual DCN neurons may contribute to somatosensory discrimination.

Identification of three signaling molecules required for calcineurin-dependent monopolar growth induced by the DNA replication checkpoint in fission yeast.

Cell polarity is coordinately regulated with the cell cycle. Growth polarity of the fission yeast Schizosaccharomyces pombe transits from monopolar to bipolar during G2 phase, termed NETO (new end take off). Upon perturbation of DNA replication, the checkpoint kinase Cds1/CHK2 induces NETO delay through activation of Ca2+/calmodulin-dependent protein phosphatase calcineurin (CN). CN in turn regulates its downstream targets including the microtubule (MT) plus-end tracking CLIP170 homologue Tip1 and the Casein kinase 1γ Cki3. However, whether and which Ca2+ signaling molecules are involved in the NETO delay remains elusive. Here we show that 3 genes (trp1322, vcx1 and SPAC6c3.06c encoding TRP channel, antiporter and P-type ATPase, respectively) play vital roles in the NETO delay. Upon perturbation of DNA replication, these 3 genes are required for not only the NETO delay but also for the maintenance of cell viability. Trp1322 and Vcx1 act downstream of Cds1 and upstream of CN for the NETO delay, whereas SPAC6c3.06c acts downstream of CN. Consistently, Trp1322 and Vcx1, but not SPAC6c3.06c, are essential for activation of CN. Interestingly, we have found that elevated extracellular Ca2+ per se induces a NETO delay, which depends on CN and its downstream target genes. These findings imply that Ca2+-CN signaling plays a central role in cell polarity control by checkpoint activation.