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.

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.

Fission yeast spindle dynamics and chromosome segregation fidelity show distinct thermosensitivity.

Cellular processes rely on proteins with temperature-dependent stability and activity. While thermosensitivity in biological networks is well-explored, the effect of temperature on complex mechanochemical assemblies, like the spindle, is rarely studied. We examined fission yeast spindle dynamics and chromosome segregation from 15°C to 40°C. Our findings reveal that these parameters follow U-shaped temperature-dependent curves but reach their minima at different temperatures. Specifically, spindle dynamics peak around 35°C, whereas chromosome segregation defects are minimized at 25°C. This suggests a scenario in which mitotic errors are tolerated to expedite rapid cell cycle progression.

Prolongation of mitosis is associated with enhanced endogenous DNA damage in fission yeast.

Mitosis is usually shorter than other phases of the cell cycle and maintains a consistent duration despite variations in cell size and spindle size. This suggests the existence of a compensatory mechanism that ensures a short duration, possibly as a protective measure against irreversible damage, such as DNA damage. To explore the link between prolonged mitosis and DNA damage, we develop a microscopy-based assay utilizing Rad52-GFP as a marker for mitotic DNA damage. Through this assay, we provide evidence that mutants with prolonged mitosis exhibit increased Rad52 puncta, indicating an elevation in endogenous DNA damage.

Reliable and robust control of nucleus centering is contingent on nonequilibrium force patterns.

Cell centers their division apparatus to ensure symmetric cell division, a challenging task when the governing dynamics is stochastic. Using fission yeast, we show that the patterning of nonequilibrium polymerization forces of microtubule (MT) bundles controls the precise localization of spindle pole body (SPB), and hence the division septum, at the onset of mitosis. We define two cellular objectives, reliability, the mean SPB position relative to the geometric center, and robustness, the variance of the SPB position, which are sensitive to genetic perturbations that change cell length, MT bundle number/orientation, and MT dynamics. We show that simultaneous control of reliability and robustness is required to minimize septum positioning error achieved by the wild type (WT). A stochastic model for the MT-based nucleus centering, with parameters measured directly or estimated using Bayesian inference, recapitulates the maximum fidelity of WT. Using this, we perform a sensitivity analysis of the parameters that control nuclear centering.

Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B.

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

Kinesin-14 HSET may not oppose kinesin-5 Eg5 activity in RPE-1 cells.

Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines.

Kinesin-5 and kinesin-14 are partially antagonistic in spindle assembly in the holocentric silkworm B. mori cells.

We previously showed that the silkworm holocentric spindles are square-shaped, compared to the canonical oval shape of human monocentric spindles (Vanpoperinghe et al. 2021). Further, while kinesin-5 depletion resulted in monopolar spindles in both cells, kinesin-14 depletion affected only the silkworm cells, resulting in mal-shaped spindles (Vanpoperinghe et al. 2021). We now extend our study to quantify the effect of kinesin-5 and kinesin-14 on spindle assembly dynamics and chromosome segregation in holocentric silkworm BmN4 cells. We find that mal-shaped spindle and prolonged mitosis duration are highly correlated with chromosome segregation error, leading to aneuploidy and cell death in BmN4 cells. Further, double RNAi-mediated depletion of kinesin-5 and kinesin-14 partially rescue the monopolar spindle and mal-shaped spindle phenotypes in kinesin-5 and kinesin 14-depleted cells, respectively.

Chromosome segregation: organizing overlap at the midzone.

Sets of overlapping microtubules support the segregation of chromosomes by linking the poles of mitotic spindles. Recent work examines the effect of putting these linkages under pressure by the activation of dicentric chromosomes and sheds new light on the structural role of several well-known spindle midzone proteins.

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.

Live-cell imaging reveals square shape spindles and long mitosis duration in the silkworm holocentric cells.

Proper chromosome segregation during mitosis requires both the assembly of a microtubule (MT)-based spindle and the assembly of DNA-centromere-based kinetochore structure. Kinetochore-to-MT attachment enables chromosome separation. Monocentric cells, such as found in human, have one unique kinetochore per chromosome. Holocentric cells, such as found in the silkworm, in contrast, have multiple kinetochore structures per chromosome. Interestingly, some human cancer chromosomes contain more than one kinetochore, a condition called di- and tricentric. Thus, comparing how wild-type mono- and holocentric cells perform mitosis may provide novel insights into cancer di- and tricentric cell mitosis. We present here live-cell imaging of human RPE1 and silkworm BmN4 cells, revealing striking differences in spindle architecture and dynamics, and highlighting differential kinesin function between mono- and holocentric cells.

Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth.

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in Schizosaccharomyces pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

Spindle scaling mechanisms.

The mitotic spindle robustly scales with cell size in a plethora of different organisms. During development and throughout evolution, the spindle adjusts to cell size in metazoans and yeast in order to ensure faithful chromosome separation. Spindle adjustment to cell size occurs by the scaling of spindle length, spindle shape and the velocity of spindle assembly and elongation. Different mechanisms, depending on spindle structure and organism, account for these scaling relationships. The limited availability of critical spindle components, protein gradients, sequestration of spindle components, or post-translational modification and differential expression levels have been implicated in the regulation of spindle length and the spindle assembly/elongation velocity in a cell size-dependent manner. In this review, we will discuss the phenomenon and mechanisms of spindle length, spindle shape and spindle elongation velocity scaling with cell size.

Quantifying Tubulin Concentration and Microtubule Number Throughout the Fission Yeast Cell Cycle.

The fission yeast Schizosaccharomycespombe serves as a good genetic model organism for the molecular dissection of the microtubule (MT) cytoskeleton. However, analysis of the number and distribution of individual MTs throughout the cell cycle, particularly during mitosis, in living cells is still lacking, making quantitative modelling imprecise. We use quantitative fluorescent imaging and analysis to measure the changes in tubulin concentration and MT number and distribution throughout the cell cycle at a single MT resolution in living cells. In the wild-type cell, both mother and daughter spindle pole body (SPB) nucleate a maximum of 23 ± 6 MTs at the onset of mitosis, which decreases to a minimum of 4 ± 1 MTs at spindle break down. Interphase MT bundles, astral MT bundles, and the post anaphase array (PAA) microtubules are composed primarily of 1 ± 1 individual MT along their lengths. We measure the cellular concentration of αß-tubulin subunits to be ~5 µM throughout the cell cycle, of which one-third is in polymer form during interphase and one-quarter is in polymer form during mitosis. This analysis provides a definitive characterization of αß-tubulin concentration and MT number and distribution in fission yeast and establishes a foundation for future quantitative comparison of mutants defective in MTs.

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.

Fission yeast neddylation ligase Dcn1 facilitates cohesin cleavage and chromosome segregation at anaphase.

Post-translational protein modification such as phosphorylation and ubiquitination are critical during mitosis to ensure proper timing and progression of chromosome segregation. It has been recently recognized that another type of protein modification - neddylation - may also regulate mitosis and chromosome segregation. The conserved protein DCN1 (defective cullin neddylation 1) has been shown, when knocked-down by RNAi, to result in multinucleated cells and/or blockage of cell proliferation. However, how DCN1 functions in mitosis and chromosome segregation is not known. We report here the fission yeast dcn1+ and its role in mitosis and chromosome segregation. Dcn1-GFP localizes to the nucleus throughout the cell cycle. dcn1-deletion (dcn1Δ) leads to chromosome and kinetochore lagging at anaphase, resulting from delayed and attenuated cohesin cleavage and sister chromatids separation. These results put Dcn1 upstream of the anaphase promoting complex/cyclosome (APC/C) pathway. We propose a mechanism for Dcn1 function at mitosis.

Multiple Motifs Compete for EB-Dependent Microtubule Plus End Binding.

Microtubule (MT) dynamics are regulated by a plethora of microtubule-associated proteins (MAPs). An important MT regulator is the end binding protein EB, which serves as a scaffold to recruit other MAPs to MT plus ends. In this issue of Structure, Kumar et al. (2017) describe LxxPTPh, a new linear sequence motif that can bind EBs. The finding opens up the possibility of discovering new MT regulators.

SIN-Dependent Dissociation of the SAD Kinase Cdr2 from the Cell Cortex Resets the Division Plane.

Proper division plane positioning is crucial for faithful chromosome segregation but also influences cell size, position, or fate [1]. In fission yeast, medial division is controlled through negative signaling by the cell tips during interphase and positive signaling by the centrally placed nucleus at mitotic entry [2-4]: the cell geometry network (CGN), controlled by the inhibitory cortical gradient of the DYRK kinase Pom1 emanating from the cell tips, first promotes the medial localization of cytokinetic ring precursors organized by the SAD kinase Cdr2 to pre-define the division plane [5-8]; then, massive nuclear export of the anillin-like protein Mid1 at mitosis entry confirms or readjusts the division plane according to nuclear position and triggers the assembly of a medial contractile ring [5, 9-11]. Strikingly, the Hippo-like septation initiation network (SIN) induces Cdr2 dissociation from cytokinetic precursors at this stage [12-14]. We show here that SIN-dependent phosphorylation of Cdr2 promotes its interaction with the 14-3-3 protein Rad24 that sequesters it in the cytoplasm during cell division. If this interaction is compromised, cytokinetic precursors are asymmetrically distributed in the cortex of newborn cells, leading to asymmetrical division if nuclear signaling is abolished. We conclude that, through this new function, the SIN resets the division plane in newborn cells to ensure medial division.