Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division.

Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche.

Cyclin F-dependent degradation of E2F7 is critical for DNA repair and G2-phase progression.

E2F7 and E2F8 act as tumor suppressors via transcriptional repression of genes involved in S-phase entry and progression. Previously, we demonstrated that these atypical E2Fs are degraded by APC/CCdh1 during G1 phase of the cell cycle. However, the mechanism driving the downregulation of atypical E2Fs during G2 phase is unknown. Here, we show that E2F7 is targeted for degradation by the E3 ubiquitin ligase SCFcyclin F during G2. Cyclin F binds via its cyclin domain to a conserved C-terminal CY motif on E2F7. An E2F7 mutant unable to interact with SCFcyclin F remains stable during G2. Furthermore, SCFcyclin F can also interact and induce degradation of E2F8. However, this does not require the cyclin domain of SCFcyclin F nor the CY motifs in the C-terminus of E2F8, implying a different regulatory mechanism than for E2F7. Importantly, depletion of cyclin F causes an atypical-E2F-dependent delay of the G2/M transition, accompanied by reduced expression of E2F target genes involved in DNA repair. Live cell imaging of DNA damage revealed that cyclin F-dependent regulation of atypical E2Fs is critical for efficient DNA repair and cell cycle progression.

Opinion: On the Way towards the New Paradigm of Atherosclerosis.

Atherosclerosis is a multicausal disease characterized by the formation of cholesterol-containing plaque in the pronounced intima nearest to the heart's elastic-type arteries that have high levels of blood circulation. Plaques are formed due to arterial pressure-induced damage to the endothelium in areas of turbulent blood flow. It is found in the majority of the Western population, including young people. This denies the monogenic mechanism of atherogenesis. In 1988, Orekhov et al. and Kawai et al. discovered that the presence of atherogenic (modified, including oxidized ones) LDLs is necessary for atherogenesis. On the basis of our discovery, suggesting that the overloading of enterocytes with lipids could lead to the formation of modified LDLs, we proposed a new hypothesis explaining the main factors of atherogenesis. Indeed, when endothelial cells are damaged and then pass through the G2 phase of their cell cycle they secrete proteins into their basement membrane. This leads to thickening of the basement membrane and increases its affinity to LDL especially for modified ones. When the enterocyte transcytosis pathway is overloaded with fat, very large chylomicrons are formed, which have few sialic acids, circulate in the blood for a long time, undergo oxidation, and can induce the production of autoantibodies. It is the sialic acids that shield the short forks of the polysaccharide chains to which autoantibodies are produced. Here, these data are evaluated from the point of view of our new model.

The telomere deprotection response is functionally distinct from the genomic DNA damage response.

Loss of chromosome end protection through telomere erosion is a hallmark of aging and senescence. Here we developed an experimental system that mimics physiological telomere deprotection in human cells and discovered that the telomere deprotection response is functionally distinct from the genomic DNA damage response. We found that, unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion-resistant intermediate state activate differential ataxia telangiectasia mutated (ATM) signaling where CHK2 is not phosphorylated. Also unlike genomic breaks, we found that deprotected telomeres do not contribute to the G2/M checkpoint and are instead passed through cell division to induce p53-dependent G1 arrest in the daughter cells. Telomere deprotection is therefore an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest occurs in stable diploid G1 phase cells before genome instability can occur.

Holliday junction recognition protein interacts with and specifies the centromeric assembly of CENP-T.

The centromere is an evolutionarily conserved eukaryotic protein machinery essential for precision segregation of the parental genome into two daughter cells during mitosis. Centromere protein A (CENP-A) organizes the functional centromere via a constitutive centromere-associated network composing the CENP-T complex. However, how CENP-T assembles onto the centromere remains elusive. Here we show that CENP-T binds directly to Holliday junction recognition protein (HJURP), an evolutionarily conserved chaperone involved in loading CENP-A. The binding interface of HJURP was mapped to the C terminus of CENP-T. Depletion of HJURP by CRISPR-elicited knockout minimized recruitment of CENP-T to the centromere, indicating the importance of HJURP in CEPN-T loading. Our immunofluorescence analyses indicate that HJURP recruits CENP-T to the centromere in S/G2 phase during the cell division cycle. Significantly, the HJURP binding-deficient mutant CENP-T6L failed to locate to the centromere. Importantly, CENP-T insufficiency resulted in chromosome misalignment, in particular chromosomes 15 and 18. Taken together, these data define a novel molecular mechanism underlying the assembly of CENP-T onto the centromere by a temporally regulated HJURP-CENP-T interaction.

Cell Cycle Arrest in G2/M Phase Enhances Replication of Interferon-Sensitive Cytoplasmic RNA Viruses via Inhibition of Antiviral Gene Expression.

Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of these viruses using an array of cell lines with different levels of impairment of antiviral signaling and a panel of chemical compounds arresting the cell cycle at different phases. We observed that all compounds inducing cell cycle arrest in G2/M phase strongly enhanced the replication of VSV-ΔM51 in cells with functional antiviral signaling. G2/M arrest strongly inhibited type I and type III interferon (IFN) production as well as expression of IFN-stimulated genes in response to exogenously added IFN. Moreover, G2/M arrest enhanced the replication of Sendai virus (a paramyxovirus), which is also highly sensitive to the type I IFN response but did not stimulate the replication of a wild-type VSV that is more effective at evading antiviral responses. In contrast, the positive effect of G2/M arrest on virus replication was not observed in cells defective in IFN signaling. Altogether, our data show that replication of IFN-sensitive cytoplasmic viruses can be strongly stimulated during G2/M phase as a result of inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. The G2/M phase thus could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest.IMPORTANCE Vesicular stomatitis virus (VSV) (a rhabdovirus) and its variant VSV-ΔM51 are widely used model systems to study mechanisms of virus-host interactions. Here, we investigated how the cell cycle affects replication of VSV and VSV-ΔM51. We show that G2/M cell cycle arrest strongly enhances the replication of VSV-ΔM51 (but not of wild-type VSV) and Sendai virus (a paramyxovirus) via inhibition of antiviral gene expression, likely due to mitotic inhibition of transcription, a global repression of cellular transcription during G2/M phase. Our data suggest that the G2/M phase could represent an "Achilles' heel" of the infected cell, a phase when the cell is inadequately protected. This model could explain at least one of the reasons why many viruses have been shown to induce G2/M arrest, and it has important implications for oncolytic virotherapy, suggesting that frequent cell cycle progression in cancer cells could make them more permissive to viruses.

Pinostrobin suppresses the Ca2+-signal-dependent growth arrest in yeast by inhibiting the Swe1-mediated G2 cell-cycle regulation.

Pinostrobin, a flavonoid compound known for its diverse pharmacological actions, including anti-leukemic and anti-inflammatory activities, has been repeatedly isolated by various screenings, but its action mechanism is still obscure. Previously, pinostrobin was rediscovered in our laboratory using a yeast-based assay procedure devised specifically for the inhibitory effect on the activated Ca2+ signaling that leads the cells to severe growth retardation in the G2 phase. Here, we attempted to identify target of pinostrobin employing the genetic techniques available in the yeast. Using various genetically engineered yeast strains in which the Ca2+-signaling cascade can be activated by the controlled expression of the various signaling molecules of the cascade, its target was narrowed down to Swe1, the cell-cycle regulatory protein kinase. The Swe1 kinase is situated at the downstream of the Ca2+-signaling cascade and downregulates the Cdc28/Clb complex by phosphorylating the Cdc28 moiety of the complex in the G2 phase. We further demonstrated that pinostrobin inhibits the protein kinase activity of Swe1 in vivo as estimated by the decreased level of Cdc28 phosphorylation at Tyr-19. Since the yeast SWE1 gene is an ortholog for the human WEE1 gene, our finding implied a potentiality of pinostrobin as the G2 checkpoint abrogator in cancer chemotherapy.

Sequential posttranslational modifications program FEN1 degradation during cell-cycle progression.

We propose that cell-cycle-dependent timing of FEN1 nuclease activity is essential for cell-cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of posttranslational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which in turn stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination-defective, nondegradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases, and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell-cycle progression and to prevent transformation.

Doxorubicin-induced DNA Damage Causes Extensive Ubiquitination of Ribosomal Proteins Associated with a Decrease in Protein Translation.

Protein posttranslational modifications (PTMs) play a central role in the DNA damage response. In particular, protein phosphorylation and ubiquitination have been shown to be essential in the signaling cascade that coordinates break repair with cell cycle progression. Here, we performed whole-cell quantitative proteomics to identify global changes in protein ubiquitination that are induced by DNA double-strand breaks. In total, we quantified more than 9,400 ubiquitin sites and found that the relative abundance of ∼10% of these sites was altered in response to DNA double-strand breaks. Interestingly, a large proportion of ribosomal proteins, including those from the 40S as well as the 60S subunit, were ubiquitinated in response to DNA damage. In parallel, we discovered that DNA damage leads to the inhibition of ribosome function. Taken together, these data uncover the ribosome as a major target of the DNA damage response.

Transient transcriptional silencing alters the cell cycle to promote germline stem cell differentiation in Drosophila.

Transcriptional silencing is a conserved process used by embryonic germ cells to repress somatic fate and maintain totipotency and immortality. In Drosophila, this transcriptional silencing is mediated by polar granule component (pgc). Here, we show that in the adult ovary, pgc is required for timely germline stem cell (GSC) differentiation. Pgc is expressed transiently in the immediate GSC daughter (pre-cystoblast), where it mediates a pulse of transcriptional silencing. This transcriptional silencing mediated by pgc indirectly promotes the accumulation of Cyclin B (CycB) and cell cycle progression into late-G2 phase, when the differentiation factor bag of marbles (bam) is expressed. Pgc mediated accumulation of CycB is also required for heterochromatin deposition, which protects the germ line genome against selfish DNA elements. Our results suggest that transient transcriptional silencing in the pre-cystoblast "re-programs" it away from self-renewal and toward the gamete differentiation program.

Phosphorylation of CDC25C by AMP-activated protein kinase mediates a metabolic checkpoint during cell-cycle G2/M-phase transition.

From unicellular to multicellular organisms, cell-cycle progression is tightly coupled to biosynthetic and bioenergetic demands. Accumulating evidence has demonstrated the G1/S-phase transition as a key checkpoint where cells respond to their metabolic status and commit to replicating the genome. However, the mechanism underlying the coordination of metabolism and the G2/M-phase transition in mammalian cells remains unclear. Here, we show that the activation of AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, significantly delays mitosis entry. The cell-cycle G2/M-phase transition is controlled by mitotic cyclin-dependent kinase complex (CDC2-cyclin B), which is inactivated by WEE1 family protein kinases and activated by the opposing phosphatase CDC25C. AMPK directly phosphorylates CDC25C on serine 216, a well-conserved inhibitory phosphorylation event, which has been shown to mediate DNA damage-induced G2-phase arrest. The acute induction of CDC25C or suppression of WEE1 partially restores mitosis entry in the context of AMPK activation. These findings suggest that AMPK-dependent phosphorylation of CDC25C orchestrates a metabolic checkpoint for the cell-cycle G2/M-phase transition.

Fluorescent indicators for simultaneous reporting of all four cell cycle phases.

A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.

Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming.

Primordial germ cells (PGCs) are reprogrammed into pluripotent embryonic germ cells (EGCs) under specific culture conditions, but the detailed mechanisms of PGC reprogramming have not yet been fully clarified. Previous studies have demonstrated that AKT, an important intracellular signaling molecule, promotes reprogramming of PGCs into EGCs. Because AKT likely inhibits p53 functions to enhance PGC reprogramming, and p53 negatively regulates cell cycle progression, we analyzed cell cycle changes in PGCs following AKT activation and found that the ratio of PGCs in the G1/G0 phase was decreased while that of PGCs in the G2/M phase was increased after AKT activation. We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1­S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation. The results suggested that the characteristic cell cycle changes of PGCs by AKT activation are, at least in part, due to decreased expression of p27kip1 . We also investigated changes in histone H3K27 tri-methylation (H3K27me3) by AKT activation in PGCs, because we previously found that decreased H3K27me3 was involved in PGC reprogramming via upregulation of cyclin D1. We observed that AKT activation in PGCs resulted in H3K27 hypomethylation. In addition, DZNeP, an inhibitor of the H3K27 trimethyl transferase Ezh2, stimulated EGC formation. These results together suggested that AKT activation promotes G1-S transition and downregulates H3K27me3 to enhance PGC reprogramming.

DAZ-interacting Protein 1 (Dzip1) Phosphorylation by Polo-like Kinase 1 (Plk1) Regulates the Centriolar Satellite Localization of the BBSome Protein during the Cell Cycle.

The function of the primary cilia, which is assembled in most vertebrate cells, is achieved by transport in and out of kinds of signaling receptors. The BBSome protein complex could recognize and target membrane proteins to the cilia, but how the BBSome itself is transported into the cilia is poorly understood. Here we demonstrate that the centrosome protein Dzip1 mediates the assembly of the BBSome-Dzip1-PCM1 complex in the centriolar satellites (CS) at the G0 phase for ciliary translocation of the BBSome. Phosphorylation of Dzip1 at Ser-210 by Plk1 (polo-like kinase 1) during the G2 phase promotes disassembly of this complex, resulting in removal of Dzip1 and the BBSome from the CS. Inhibiting the kinase activity of Plk1 maintains the CS localization of the BBSome and Dzip1 at the G2 phase. Collectively, our findings reveal the cell cycle-dependent regulation of BBSome transport to the CS and highlight a potential mechanism that the BBSome-mediated signaling pathways are accordingly regulated during the cell cycle.

Endoplasmic reticulum stress induces G2 cell-cycle arrest via mRNA translation of the p53 isoform p53/47.

p53 downstream pathways control G1 and G2 cell-cycle arrest, DNA repair, or apoptosis. However, it is still not clear how cells differentiate the cell-biological outcome of p53 activation in response to different types of stresses. The p53/47 isoform lacks the first 39 amino acids of full-length p53 including the Mdm2 binding site and the first trans-activation domain, and tetramers including p53/47 exhibit altered activity and biochemical properties. Here we show that endoplasmic reticulum stress promotes PERK-dependent induction of p53/47 mRNA translation and p53/47 homo-oligomerization. p53/47 induces 14-3-3sigma and G2 arrest but does not affect G1 progression. This is contrary to p53FL, which promotes G1 arrest but has no effect on the G2. These results show a unique role for p53/47 in the p53 pathway and illustrate how a cellular stress leads to a defined cell-biological outcome through expression of a p53 isoform.

Relative importance of phosphatidylinositol-3 kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK3/1) signaling during maturational steroid-induced meiotic G2-M1 transition in zebrafish oocytes.

Participation and relative importance of phosphatidylinositol-3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling, either alone or in combination, have been investigated during 17α,20ß-dihydroxy-4-pregnen-3-one (DHP)-induced meiotic G2-M1 transition in denuded zebrafish oocyte. Results demonstrate that concomitant with rapid phosphorylation (activation) of Akt (Ser473) and MAPK (ERK1/2) at as early as 15 min of incubation, DHP stimulation promotes enhanced an GVBD response and histone H1 kinase activation between 1 and 5 h in full-grown oocytes in vitro. While p-Akt reaches its peak at 60 to 90 min and undergoes downregulation to the basal level by 240 min, ERK1/2 phosphorylation (activation) increases gradually until 120 min and remains high thereafter. Although, priming with MEK1/2 inhibitor U0126 is without effect, PI3K inhibitors, wortmannin or LY294002, delay the GVBD response significantly (P < 0.001) until 3 h but not at 5 h of incubation. Interestingly, blocking PI3K and MEK function together could abrogate steroid-induced oocyte maturation at all time points tested. While DHP stimulation promotes phospho-PKA catalytic (p-PKAc) dephosphorylation (inactivation) between 30-120 min of incubation, simultaneous inhibition of PI3K and MEK1/2 kinases abrogates DHP action. Conversely, elevated intra-oocyte cAMP, through priming with either adenylyl cyclase (AC) activator forskolin (FK) or dibutyryl cAMP (db-cAMP), abrogates steroid-induced Akt and ERK1/2 phosphorylation. Taken together, these results suggest that DHP-induced Akt and ERK activation precedes the onset of meiosis (GVBD response) in a cAMP-sensitive manner and PI3K/Akt and MEK/MAPK pathways together have a pivotal influence in the downregulation of PKA and resumption of meiotic maturation in zebrafish oocytes in vitro.

Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) are co-receptors for Wnt ligands. Upon ligand binding, LRP5/6 undergo glycogen synthase kinase 3 (GSK3)/casein kinase I (CKI)-mediated phosphorylation at multiple PPP(S/T)P motifs in the intracellular domain, which is essential for canonical Wnt signal transduction. On the other hand, in the Wnt-off state, the mitosis-specific CDK14-Cyclin Y kinase complex phosphorylates Ser-1490 of LRP5/6 at G2/M, thereby priming the receptor for Wnt-induced phosphorylation. However, it remains unclear how CDK14/Cyclin Y is recruited to LRP5/6 and whether there are other cofactors involved in this process. Previously, we identified Caprin-2 as a positive regulator of canonical Wnt signaling by promoting GSK3-depedent LRP5/6 phosphorylation upon Wnt stimulation. Here we uncovered that Caprin-2 positively regulates constitutive LRP5/6 Ser-1490 phosphorylation by complexing with CDK14/Cyclin Y. Caprin-2-mediated LRP5/6 phosphorylation is cell cycle-dependent in a pattern similar to that of CDK14/Cyclin Y-dependent LRP5/6 phosphorylation. Moreover, knockdown of Caprin-2 disrupts not only the interaction between CDK14 and Cyclin Y but also the interaction between CDK14/Cyclin Y and LRP6. Overall, our findings revealed an unrecognized role of Caprin-2 in facilitating LRP5/6 constitutive phosphorylation at G2/M through forming a quaternary complex with CDK14, Cyclin Y, and LRP5/6.

MEK1 inactivates Myt1 to regulate Golgi membrane fragmentation and mitotic entry in mammalian cells.

The pericentriolar stacks of Golgi cisternae are separated from each other in G2 and fragmented extensively during mitosis. MEK1 is required for Golgi fragmentation in G2 and for the entry of cells into mitosis. We now report that Myt1 mediates MEK1's effects on the Golgi complex. Knockdown of Myt1 by siRNA increased the efficiency of Golgi complex fragmentation by mitotic cytosol in permeabilized and intact HeLa cells. Myt1 knockdown eliminated the requirement of MEK1 in Golgi fragmentation and alleviated the delay in mitotic entry due to MEK1 inhibition. The phosphorylation of Myt1 by MEK1 requires another kinase but is independent of RSK, Plk, and CDK1. Altogether our findings reveal that Myt1 is inactivated by MEK1 mediated phosphorylation to fragment the Golgi complex in G2 and for the entry of cells into mitosis. It is known that Myt1 inactivation is required for CDK1 activation. Myt1 therefore is an important link by which MEK1 dependent fragmentation of the Golgi complex in G2 is connected to the CDK1 mediated breakdown of Golgi into tubules and vesicles in mitosis.

JNK2 controls fragmentation of the Golgi complex and the G2/M transition through phosphorylation of GRASP65.

In mammalian cells, the Golgi complex is composed of stacks that are connected by membranous tubules. During G2, the Golgi complex is disassembled into isolated stacks. This process is required for entry into mitosis, indicating that the correct inheritance of the organelle is monitored by a 'Golgi mitotic checkpoint'. However, the regulation and the molecular mechanisms underlying this Golgi disassembly are still poorly understood. Here, we show that JNK2 has a crucial role in the G2-specific separation of the Golgi stacks through phosphorylation of Ser277 of the Golgi-stacking protein GRASP65 (also known as GORASP1). Inhibition of JNK2 by RNA interference or by treatment with three unrelated JNK inhibitors causes a potent and persistent cell cycle block in G2. JNK activity becomes dispensable for mitotic entry if the Golgi complex is disassembled by brefeldin A treatment or by GRASP65 depletion. Finally, measurement of the Golgi fluorescence recovery after photobleaching demonstrates that JNK is required for the cleavage of the tubules connecting Golgi stacks. Our findings reveal that a JNK2-GRASP65 signalling axis has a crucial role in coupling Golgi inheritance and G2/M transition.

How do fission yeast cells grow and connect growth to the mitotic cycle?

To maintain size homeostasis in a unicellular culture, cells should coordinate growth to the division cycle. This is achieved via size control mechanisms (also known as size checkpoints), i.e. some events during the mitotic cycle supervene only if the cell has reached a critical size. Rod-shaped cells like those of fission yeast are ideal model organisms to study these checkpoints via time-lapse microphotography. By applying this method, once we can analyse the growth process between two consecutive divisions at a single (or even at an 'average') cellular level, moreover, we can also position the size checkpoint(s) at the population level. Finally, any of these controls can be abolished in appropriate cell cycle mutants, either in steady-state or in induction synchronised cultures. In the latter case, we produce abnormally oversized cells, and microscopic experiments with them clearly show the existence of a critical size above which the size checkpoint ceases (becomes cryptic). In this review, we delineate the development of our knowledge both on the growth mode of fission yeast and on the operating size control(s) during its mitotic cycle. We finish these historical stories with our recent findings, arguing that three different size checkpoints exist in the fission yeast cell cycle, namely in late G1, in mid G2 and in late G2, which has been concluded by analysing these controls in several cell cycle mutants.