Human SFI1 and Centrin form a complex critical for centriole architecture and ciliogenesis.

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.

Kinesin-14 family proteins and microtubule dynamics define S. pombe mitotic and meiotic spindle assembly, and elongation.

To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases, such as cancer or developmental disorders. Here, we compared mitotic and meiotic spindles in fission yeast. We show that, even though mitotic and meiotic spindles underwent the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of the kinesin-14 family protein Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of the kinesin-5 family protein Cut7 remains constant. We identified the second kinesin-14 family protein Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines the differences between mitotic and meiotic spindles in fission yeast Schizosaccharomyces pombe, and provides prospect for future comparative studies.This article has an associated First Person interview with the first author of the paper.

Increasing ergosterol levels delays formin-dependent assembly of F-actin cables and disrupts division plane positioning in fission yeast.

In most eukaryotes, cytokinesis is mediated by the constriction of a contractile acto-myosin ring (CR), which promotes the ingression of the cleavage furrow. Many components of the CR interact with plasma membrane lipids suggesting that lipids may regulate CR assembly and function. Although there is clear evidence that phosphoinositides play an important role in cytokinesis, much less is known about the role of sterols in this process. Here, we studied how sterols influence division plane positioning and CR assembly in fission yeast. We show that increasing ergosterol levels in the plasma membrane blocks the assembly of F-actin cables from cytokinetic precursor nodes, preventing their compaction into a ring. Abnormal F-actin cables form after a delay, leading to randomly placed septa. Since the formin Cdc12 was detected on cytokinetic precursors and the phenotype can be partially rescued by inhibiting the Arp2/3 complex, which competes with formins for F-actin nucleation, we propose that ergosterol may inhibit formin dependent assembly of F-actin cables from cytokinetic precursors.

Molecular control of fission yeast cytokinesis.

Cytokinesis gives rise to two independent daughter cells at the end of the cell division cycle. The fission yeast Schizosaccharomyces pombe has emerged as one of the most powerful systems to understand how cytokinesis is controlled molecularly. Like in most eukaryotes, fission yeast cytokinesis depends on an acto-myosin based contractile ring that assembles at the division site under the control of spatial cues that integrate information on cell geometry and the position of the mitotic apparatus. Cytokinetic events are also tightly coordinated with nuclear division by the cell cycle machinery. These spatial and temporal regulations ensure an equal cleavage of the cytoplasm and an accurate segregation of the genetic material in daughter cells. Although this model system has specificities, the basic mechanisms of contractile ring assembly and function deciphered in fission yeast are highly valuable to understand how cytokinesis is controlled in other organisms that rely on a contractile ring for cell division.

Molecular control of the Wee1 regulatory pathway by the SAD kinase Cdr2.

Cell growth and division are tightly coordinated to maintain cell size constant during successive cell cycles. In Schizosaccharomyces pombe, the SAD kinase Cdr2 regulates the cell size at division and the positioning of the division plane. Cdr2 forms nodes on the medial cortex containing factors that constitute an inhibitory pathway for Wee1. This pathway is regulated by polar gradients of the DYRK kinase Pom1, and involves a direct inhibitor of Wee1, the SAD kinase Cdr1. Cdr2 also interacts with the anillin Mid1, which defines the division plane, and with additional components of the medial cortical nodes, including Blt1, which participate in the mitotic-promoting and cytokinetic functions of nodes. Here, we show that the interaction of Cdr2 with Wee1 and Mid1 requires the UBA domain of Cdr2, which is necessary for its kinase activity. In contrast, Cdr1 associates with the C-terminus of Cdr2, which is composed of basic and KA-1 lipid-binding domains. Mid1 also interacts with the C-terminus of Cdr2 and might bridge the N- and C-terminal domains, whereas Blt1 associates with the central spacer region. We propose that the association of Cdr2 effectors with different domains might constrain Cdr1 and Wee1 spatially to promote Wee1 inhibition upon Cdr2 kinase activation.

Cell cycle control of spindle pole body duplication and splitting by Sfi1 and Cdc31 in fission yeast.

Spindle pole biogenesis and segregation are tightly coordinated to produce a bipolar mitotic spindle. In yeasts, the spindle pole body (SPB) half-bridge composed of Sfi1 and Cdc31 duplicates to promote the biogenesis of a second SPB. Sfi1 accumulates at the half-bridge in two phases in Schizosaccharomyces pombe, from anaphase to early septation and throughout G2 phase. We found that the function of Sfi1-Cdc31 in SPB duplication is accomplished before septation ends and G2 accumulation starts. Thus, Sfi1 early accumulation at mitotic exit might correspond to half-bridge duplication. We further show that Cdc31 phosphorylation on serine 15 in a Cdk1 (encoded by cdc2) consensus site is required for the dissociation of a significant pool of Sfi1 from the bridge and timely segregation of SPBs at mitotic onset. This suggests that the Cdc31 N-terminus modulates the stability of Sfi1-Cdc31 arrays in fission yeast, and impacts on the timing of establishment of spindle bipolarity.

Oscillatory AAA+ ATPase Knk1 constitutes a novel morphogenetic pathway in fission yeast.

Cellular morphogenesis relies partly on cell polarization by the cytoskeleton. In the fission yeast Schizosaccharomyces pombe, it is well established that microtubules (MTs) deliver the spatial cue Tea1, a kelch repeat protein, to the tip regions to direct the growth machinery at the cell tips driving the linear extension of the rod-shaped organism to maintain a straight long axis. Here, we report the characterization of Knk1 (kink), a previously unidentified member of the superfamily of ATPases associated with various cellular activities (AAA(+)), whose deletion causes a unique morphological defect characterized by the formation of kinks close to cell tips. Through genetic analysis, we place Knk1 into a novel pathway controlling cell shape independently of MTs and Tea1. Knk1 localizes at cell tips. Its localization is mediated by the Knk1 N terminus and is enhanced upon ATP binding to the C-terminal ATPase domain. Furthermore, Knk1 tip recruitment is regulated by SRC-like adaptor 2 (Sla2) and cell division cycle 42 (Cdc42) independently of Sla2's role in endocytosis. Finally, we discovered that Knk1 shows an anticorrelated oscillatory behavior between the two cell tips at a periodicity that is different from the reported oscillatory Cdc42 dynamics.

A spatial gradient coordinates cell size and mitotic entry in fission yeast.

Many eukaryotic cell types undergo size-dependent cell cycle transitions controlled by the ubiquitous cyclin-dependent kinase Cdk1 (refs 1-4). The proteins that control Cdk1 activity are well described but their links with mechanisms monitoring cell size remain elusive. In the fission yeast Schizosaccharomyces pombe, cells enter mitosis and divide at a defined and reproducible size owing to the regulated activity of Cdk1 (refs 2, 3). Here we show that the cell polarity protein kinase Pom1, which localizes to cell ends, regulates a signalling network that contributes to the control of mitotic entry. This network is located at cortical nodes in the middle of interphase cells, and these nodes contain the Cdk1 inhibitor Wee1, the Wee1-inhibitory kinases Cdr1 (also known as Nim1) and Cdr2, and the anillin-like protein Mid1. Cdr2 establishes the hierarchical localization of other proteins in the nodes, and receives negative regulatory signals from Pom1. Pom1 forms a polar gradient extending from the cell ends towards the cell middle and acts as a dose-dependent inhibitor of mitotic entry, working through the Cdr2 pathway. As cells elongate, Pom1 levels decrease at the cell middle, leading to mitotic entry. We propose that the Pom1 polar gradient and the medial cortical nodes generate information about cell size and coordinate this with mitotic entry by regulating Cdk1 through Pom1, Cdr2, Cdr1 and Wee1.

Mechanisms controlling division-plane positioning.

A critical and irreversible step in the cell division cycle is cytokinesis which physically separates the two daughter cells. This event is consequently subject to tight spatial and temporal regulation. This review focuses on the spatial regulatory mechanisms controlling the position of the division plane. Studies performed in prokaryotic and eukaryotic systems have revealed that various signal-emitting spatial cues - mitotic spindle, nucleus, nucleoid or cell tips - can favour or inhibit the assembly of the cytokinetic apparatus in their vicinity. Most often, several mechanisms operate in parallel to integrate spatial information and promote faithful genome segregation as well as proper cytoplasmic division. We primarily describe the spatial regulatory mechanisms operating in the fission yeast model system, where a detailed molecular understanding of cytokinesis has been achieved. In this system, spatial regulations target a major factor controlling the position of the division plane, the anillin-like protein Mid1. These mechanisms are then compared to spatial regulatory mechanisms prevailing in animal cells and rod-shaped bacteria.

Mid1p-dependent regulation of the M-G1 transcription wave in fission yeast.

The control of gene expression at certain times during the mitotic cell division cycle is a common feature in eukaryotes. In fission yeast, at least five waves of gene expression have been described, with one transcribed at the M-G1 interval under the control of the PBF transcription factor complex. PBF consists of at least three transcription factors, two forkhead-like proteins Sep1p and Fkh2p, and a MADS box-like protein Mbx1p, and binds to PCB motifs found in the gene promoters. Mbx1p is under the direct control of the polo-like kinase Plo1p and the Cdc14p-like phosphatase Clp1p (Flp1p). Here, we show that M-G1 gene expression in fission yeast is also regulated by the anillin-like protein, Mid1p (Dmf1p). Mid1p binds in vivo to both Fkh2p and Sep1p, and to the promoter regions of M-G1 transcribed genes. Mid1p promoter binding is dependent on Fkh2p, Plo1p and Clp1p. The absence of mid1(+) in cells results in partial loss of M-G1 specific gene expression, suggesting that it has a negative role in controlling gene expression. This phenotype is exacerbated by also removing clp1(+), suggesting that Mid1p and Clp1p have overlapping functions in controlling transcription. As mid1(+) is itself expressed at M-G1, these observations offer a new mechanism whereby Mid1p contributes to controlling cell cycle-specific gene expression as part of a feedback loop.

Septin filament compaction into rings requires the anillin Mid2 and contractile ring constriction.

Septin filaments assemble into high-order molecular structures that associate with membranes, acting as diffusion barriers and scaffold proteins crucial for many cellular processes. How septin filaments organize in such structures is still not understood. Here, we used fission yeast to explore septin filament organization during cell division and its cell cycle regulation. Live-imaging and polarization microscopy analysis uncovered that septin filaments are initially recruited as a diffuse meshwork surrounding the acto-myosin contractile ring (CR) in anaphase, which undergoes compaction into two rings when CR constriction is initiated. We found that the anillin-like protein Mid2 is necessary to promote this compaction step, possibly acting as a bundler for septin filaments. Moreover, Mid2-driven septin compaction requires inputs from the septation initiation network as well as CR constriction and the ß(1,3)-glucan synthase Bgs1. This work highlights that anillin-mediated septin ring assembly is under strict cell cycle control.

Physical mechanisms redirecting cell polarity and cell shape in fission yeast.

The cylindrical rod shape of the fission yeast Schizosaccharomyces pombe is organized and maintained by interactions between the microtubule, cell membrane, and actin cytoskeleton [1]. Mutations affecting any components in this pathway lead to bent, branched, or round cells [2]. In this context, the cytoskeleton controls cell polarity and thus dictates cell shape. Here, we use soft-lithography techniques to construct microfluidic channels to control cell shape. We show that when wild-type rod-shaped cells are physically forced to grow in a bent fashion, they will reorganize their cytoskeleton and redirect cell polarity to make new ectopic cell tips. Moreover, when bent or round mutant cells are physically forced to conform to the wild-type rod-shape, they will reverse their mutational phenotypes by reorganizing their cytoskeleton to maintain proper wild-type-like localization of microtubules, cell-membrane proteins, and actin. Our study provides direct evidence that the cytoskeleton controls cell polarity and cell shape and demonstrates that cell shape also controls the organization of the cytoskeleton in a feedback loop. We present a model of the feedback loop to explain how fission yeast maintain a rod shape and how perturbation of specific parameters of the loop can lead to different cell shapes.

Efficient formation of bipolar microtubule bundles requires microtubule-bound gamma-tubulin complexes.

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal gamma-tubulin complexes (gamma-TuCs). In interphase mto2Delta cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from gamma-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.

Kar9 asymmetrical loading on spindle poles mediates proper spindle alignment in budding yeast.

In the February 21 issue of Cell, demonstrate that asymmetrical loading of Kar9 onto astral microtubules (MTs) emanating from the bud-ward-directed spindle pole ensures delivery of this spindle pole to the bud. Kar9 mediates alignment of the spindle with the cell polarity axis through a Myo2-dependent mechanism that reorients astral MTs toward the bud.

Mid2p stabilizes septin rings during cytokinesis in fission yeast.

Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2delta mutants had delays in cell-cell separation. mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.

Kinesin-6 regulates cell-size-dependent spindle elongation velocity to keep mitosis duration constant in fission yeast.

The length of the mitotic spindle scales with cell size in a wide range of organisms during embryonic development. Interestingly, in C. elegans embryos, this goes along with temporal regulation: larger cells speed up spindle assembly and elongation. We demonstrate that, similarly in fission yeast, spindle length and spindle dynamics adjust to cell size, which allows to keep mitosis duration constant. Since prolongation of mitosis was shown to affect cell viability, this may resemble a mechanism to regulate mitosis duration. We further reveal how the velocity of spindle elongation is regulated: coupled to cell size, the amount of kinesin-6 Klp9 molecules increases, resulting in an acceleration of spindle elongation in anaphase B. In addition, the number of Klp9 binding sites to microtubules increases overproportionally to Klp9 molecules, suggesting that molecular crowding inversely correlates to cell size and might have an impact on spindle elongation velocity control.

Choosing appropriate size of I-Gel® for initial success insertion: a prospective comparative study.

PURPOSE: The optimal size of the I-Gel® remains unclear since the manufacturer's weight-based formula (size 3 for weight < 50 kg, size 4 for weight 50-90 kg, and size 5 for weight > 90 kg) for the laryngeal mask airway I-Gel® is not evidence-based. We hypothesised that sex may also guide the choice of I-Gel® size. METHODS: Insertion success rates of the I-Gel® chosen according to the weight-based formula were prospectively recorded and compared with those of a patients' cohort ventilated with an I-Gel® chosen according to the sex-based formula recorded. Two periods of 18 months were randomised in three independent hospitals in France to study each choice strategy. Patients requiring I-Gel® size change were compared with those who where successfully ventilated with the initially chosen device. Complications linked to the I-Gel® and factors for changing the size of the I-Gel® were also recorded and analysed. RESULTS: Data from 900 patients were prospectively collected in the three participating centres. The overall initial ventilation was inadequate in 80 cases, including 7% (n = 31) in the weight-based group and 3% (n = 13) in the sex-based group (P = 0.01). In the weight-based group, changing size of I-Gel® was successful in 28 (90%) cases. In the sex-based group, changing size of I-Gel® was useful in 1 case only. Endotracheal tube insertion was necessary in 15 cases despite changing I-Gel® size, including 3 cases in the weight-based group and 12 cases in the sex-based group. Ease of insertion and postoperative pharyngo-laryngeal problems were similar between groups with or without changing size of I-Gel®. CONCLUSION: Adequate ventilation is achieved most of the time using size selection for the I-Gel® laryngeal mask airway according to the manufacturer's weight-based formula. However, our results suggest that the sex-based formula in healthy, anaesthetised, adult patients may also be appropriate for I-Gel® size choice.

Ase1p organizes antiparallel microtubule arrays during interphase and mitosis in fission yeast.

Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.

C-terminal anchoring of mid1p to membranes stabilizes cytokinetic ring position in early mitosis in fission yeast.

mid1p is a key factor for the central positioning of the cytokinetic ring in Schizosaccharomyces pombe. In interphase and early mitosis, mid1p forms a medial cortical band overlying the nucleus, which may represent a landmark for cytokinetic ring assembly. It compacts before anaphase into a tight ring with other cytokinetic ring components. We show here that mid1p binds to the medial cortex by at least two independent means. First, mid1p C-terminus association with the cortex requires a putative amphipathic helix adjacent to mid1p nuclear localization sequence (NLS), which is predicted to insert directly into the lipid bilayer. This association is stabilized by the polybasic NLS. mid1p mutated within the helix and the NLS forms abnormal filaments in early mitosis that are not properly anchored to the medial cortex. Misplaced rings assemble in late mitosis, indicating that mid1p C-terminus binding to membranes stabilizes cytokinetic ring position. Second, the N terminus of mid1p has the ability to associate faintly with the medial cortex and is sufficient to form tight rings. In addition, we show that mid1p oligomerizes. We propose that membrane-bound oligomers of mid1p assemble recruitment "platforms" for cytokinetic ring components at the medial cortex and stabilize the ring position during its compaction.