Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

Download Full-text

Bimodal Interaction of Mammalian Polo-Like Kinase 1 and a Centrosomal Scaffold, Cep192, in the Regulation of Bipolar Spindle Formation

Molecular and Cellular Biology ◽

10.1128/mcb.00068-15 ◽

2015 ◽

Vol 35 (15) ◽

pp. 2626-2640 ◽

Cited By ~ 23

Author(s):

Lingjun Meng ◽

Jung-Eun Park ◽

Tae-Sung Kim ◽

Eun Hye Lee ◽

Suk-Youl Park ◽

...

Keyword(s):

Xenopus Laevis ◽

Spindle Assembly ◽

Human Cells ◽

Mitotic Progression ◽

Spindle Formation ◽

Content Type ◽

Bipolar Spindle ◽

Egg Extracts ◽

Cultured Human Cells ◽

Bipolar Spindles

Serving as microtubule-organizing centers, centrosomes play a key role in forming bipolar spindles. The mechanism of how centrosomes promote bipolar spindle assembly in various organisms remains largely unknown. A recent study withXenopus laevisegg extracts suggested that the Plk1 ortholog Plx1 interacts with the phospho-T46 (p-T46) motif ofXenopusCep192 (xCep192) to form an xCep192-mediated xAurA-Plx1 cascade that is critical for bipolar spindle formation. Here, we demonstrated that in cultured human cells, Cep192 recruits AurA and Plk1 in a cooperative manner, and this event is important for the reciprocal activation of AurA and Plk1. Strikingly, Plk1 interacted with Cep192 through either the p-T44 (analogous toXenopusp-T46) or the newly identified p-S995 motif via its C-terminal noncatalytic polo-box domain. The interaction between Plk1 and the p-T44 motif was prevalent in the presence of Cep192-bound AurA, whereas the interaction of Plk1 with the p-T995 motif was preferred in the absence of AurA binding. Notably, the loss of p-T44- and p-S995-dependent Cep192-Plk1 interactions induced an additive defect in recruiting Plk1 and γ-tubulin to centrosomes, which ultimately led to a failure in proper bipolar spindle formation and mitotic progression. Thus, we propose that Plk1 promotes centrosome-based bipolar spindle formation by forming two functionally nonredundant complexes with Cep192.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0497 ◽

2017 ◽

Vol 28 (25) ◽

pp. 3647-3659 ◽

Cited By ~ 23

Author(s):

Masashi Yukawa ◽

Tomoki Kawakami ◽

Masaki Okazaki ◽

Kazunori Kume ◽

Ngang Heok Tang ◽

...

Keyword(s):

Fission Yeast ◽

Chromosome Segregation ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

Temperature Sensitive ◽

Bipolar Spindle ◽

Pole Body ◽

Cross Linker ◽

Spindle Elongation

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.

Download Full-text

Kinetochore-mediated outward force promotes spindle pole separation in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e19-07-0366 ◽

2019 ◽

Vol 30 (22) ◽

pp. 2802-2813 ◽

Cited By ~ 4

Author(s):

Yutaka Shirasugi ◽

Masamitsu Sato

Keyword(s):

Fission Yeast ◽

Motor Proteins ◽

Spindle Assembly ◽

Spindle Pole ◽

Double Knockout ◽

Bipolar Spindle ◽

Spindle Poles ◽

Bipolar Spindles ◽

Meiosis I

Bipolar spindles are organized by motor proteins that generate microtubule-dependent forces to separate the two spindle poles. The fission yeast Cut7 (kinesin-5) is a plus-end-directed motor that generates the outward force to separate the two spindle poles, whereas the minus-end-directed motor Pkl1 (kinesin-14) generates the inward force. Balanced forces by these antagonizing kinesins are essential for bipolar spindle organization in mitosis. Here, we demonstrate that chromosomes generate another outward force that contributes to the bipolar spindle assembly. First, it was noted that the cut7 pkl1 double knockout failed to separate spindle poles in meiosis I, although the mutant is known to succeed it in mitosis. It was assumed that this might be because meiotic kinetochores of bivalent chromosomes joined by cross-overs generate weaker tensions in meiosis I than the strong tensions in mitosis generated by tightly tethered sister kinetochores. In line with this idea, when meiotic mono-oriented kinetochores were artificially converted to a mitotic bioriented layout, the cut7 pkl1 mutant successfully separated spindle poles in meiosis I. Therefore, we propose that spindle pole separation is promoted by outward forces transmitted from kinetochores to spindle poles through microtubules.

Download Full-text

Theory of cytoskeletal reorganization during crosslinker-mediated mitotic spindle assembly

10.1101/419135 ◽

2018 ◽

Cited By ~ 1

Author(s):

A. R. Lamson ◽

C. J. Edelmaier ◽

M. A. Glaser ◽

M. D. Betterton

Keyword(s):

Mitotic Spindle ◽

Large Scale ◽

Dynamic Instability ◽

Kinetic Monte Carlo ◽

Spindle Assembly ◽

Spindle Pole ◽

Balance Model ◽

Cytoskeletal Reorganization ◽

Bipolar Spindle ◽

Mitotic Spindle Assembly

AbstractCells grow, move, and respond to outside stimuli by large-scale cytoskeletal reorganization. A prototypical example of cytoskeletal remodeling is mitotic spindle assembly, during which micro-tubules nucleate, undergo dynamic instability, bundle, and organize into a bipolar spindle. Key mechanisms of this process include regulated filament polymerization, crosslinking, and motor-protein activity. Remarkably, using passive crosslinkers, fission yeast can assemble a bipolar spindle in the absence of motor proteins. We develop a torque-balance model that describes this reorganization due to dynamic microtubule bundles, spindle-pole bodies, the nuclear envelope, and passive crosslinkers to predict spindle-assembly dynamics. We compare these results to those obtained with kinetic Monte Carlo-Brownian dynamics simulations, which include crosslinker-binding kinetics and other stochastic effects. Our results show that rapid crosslinker reorganization to microtubule overlaps facilitates crosslinker-driven spindle assembly, a testable prediction for future experiments. Combining these two modeling techniques, we illustrate a general method for studying cytoskeletal network reorganization.

Download Full-text

Novelsfi1Alleles Uncover Additional Functions for Sfi1p in Bipolar Spindle Assembly and Function

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0918 ◽

2007 ◽

Vol 18 (6) ◽

pp. 2047-2056 ◽

Cited By ~ 24

Author(s):

Victoria E. Anderson ◽

John Prudden ◽

Simon Prochnik ◽

Thomas H. Giddings ◽

Kevin G. Hardwick

Keyword(s):

Essential Gene ◽

Light And Electron Microscopy ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

The Novel ◽

Synthetic Lethal ◽

Bipolar Spindle ◽

Novel Alleles ◽

And Function

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 μm apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle assembly.

Download Full-text

Synergistic role of fission yeast Alp16GCP6 and Mzt1MOZART1 in γ-tubulin complex recruitment to mitotic spindle pole bodies and spindle assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0577 ◽

2016 ◽

Vol 27 (11) ◽

pp. 1753-1763 ◽

Cited By ~ 19

Author(s):

Hirohisa Masuda ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Synthetic Lethality ◽

Spindle Assembly ◽

Spindle Pole ◽

Microtubule Assembly ◽

Spindle Microtubule ◽

Spindle Pole Bodies ◽

Mitotic Spindle Pole

In fission yeast, γ-tubulin ring complex (γTuRC)–specific components Gfh1GCP4, Mod21GCP5, and Alp16GCP6 are nonessential for cell growth. Of these deletion mutants, only alp16Δ shows synthetic lethality with temperature-sensitive mutants of Mzt1MOZART1, a component of the γTuRC required for recruitment of the complex to microtubule-organizing centers. γ-Tubulin small complex levels at mitotic spindle pole bodies (SPBs, the centrosome equivalent in fungi) and microtubule levels for preanaphase spindles are significantly reduced in alp16Δ cells but not in gfh1Δ or mod21Δ cells. Furthermore, alp16Δ cells often form monopolar spindles and frequently lose a minichromosome when the spindle assembly checkpoint is inactivated. Alp16GCP6 promotes Mzt1-dependent γTuRC recruitment to mitotic SPBs and enhances spindle microtubule assembly in a manner dependent on its expression levels. Gfh1GCP4 and Mod21GCP5 are not required for Alp16GCP6-dependent γTuRC recruitment. Mzt1 has an additional role in the activation of the γTuRC for spindle microtubule assembly. The ratio of Mzt1 to γTuRC levels for preanaphase spindles is higher than at other stages of the cell cycle. Mzt1 overproduction enhances spindle microtubule assembly without affecting γTuRC levels at mitotic SPBs. We propose that Alp16GCP6 and Mzt1 act synergistically for efficient bipolar spindle assembly to ensure faithful chromosome segregation.

Download Full-text

Kinetochore-microtubule stability governs the metaphase requirement for Eg5

Molecular Biology of the Cell ◽

10.1091/mbc.e14-03-0785 ◽

2014 ◽

Vol 25 (13) ◽

pp. 2051-2060 ◽

Cited By ~ 26

Author(s):

A. Sophia Gayek ◽

Ryoma Ohi

Keyword(s):

Physical Properties ◽

Human Cell ◽

Mitotic Spindle ◽

Mammalian Cells ◽

Human Cell Lines ◽

Bipolar Spindle ◽

Daughter Cells ◽

Microtubule Stability ◽

The Stability ◽

Bipolar Spindles

The mitotic spindle is a bipolar, microtubule (MT)-based cellular machine that segregates the duplicated genome into two daughter cells. The kinesin-5 Eg5 establishes the bipolar geometry of the mitotic spindle, but previous work in mammalian cells suggested that this motor is unimportant for the maintenance of spindle bipolarity. Although it is known that Kif15, a second mitotic kinesin, enforces spindle bipolarity in the absence of Eg5, how Kif15 functions in this capacity and/or whether other biochemical or physical properties of the spindle promote its bipolarity have been poorly studied. Here we report that not all human cell lines can efficiently maintain bipolarity without Eg5, despite their expressing Kif15. We show that the stability of chromosome-attached kinetochore-MTs (K-MTs) is important for bipolar spindle maintenance without Eg5. Cells that efficiently maintain bipolar spindles without Eg5 have more stable K-MTs than those that collapse without Eg5. Consistent with this observation, artificial destabilization of K-MTs promotes spindle collapse without Eg5, whereas stabilizing K-MTs improves bipolar spindle maintenance without Eg5. Our findings suggest that either rapid K-MT turnover pulls poles inward or slow K-MT turnover allows for greater resistance to inward-directed forces.

Download Full-text

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

FEBS Letters ◽

10.1016/j.febslet.2014.06.027 ◽

2014 ◽

Vol 588 (17) ◽

pp. 2814-2821 ◽

Cited By ~ 12

Author(s):

Ngang Heok Tang ◽

Naoyuki Okada ◽

Chii Shyang Fong ◽

Kunio Arai ◽

Masamitsu Sato ◽

...

Keyword(s):

Fission Yeast ◽

Mitotic Spindle ◽

Spindle Pole Body ◽

Spindle Assembly ◽

Spindle Pole ◽

Pole Body ◽

Mitotic Spindle Assembly

Download Full-text

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The Journal of Cell Biology ◽

10.1083/jcb.202006085 ◽

2021 ◽

Vol 220 (2) ◽

Author(s):

Takumi Chinen ◽

Kaho Yamazaki ◽

Kaho Hashimoto ◽

Ken Fujii ◽

Koki Watanabe ◽

...

Keyword(s):

A549 Cells ◽

Spindle Assembly ◽

Spindle Pole ◽

Scaffold Proteins ◽

Spindle Formation ◽

Bipolar Spindle ◽

Spindle Poles ◽

Pericentriolar Material ◽

Mitotic Cells

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.

Download Full-text

Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A

mSphere ◽

10.1128/msphere.00056-17 ◽

2017 ◽

Vol 2 (2) ◽

Cited By ~ 12

Author(s):

Jinglin L. Xie ◽

Teresa R. O’Meara ◽

Elizabeth J. Polvi ◽

Nicole Robbins ◽

Leah E. Cowen

Keyword(s):

Candida Albicans ◽

Small Molecules ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Bloodstream Infections ◽

Filamentous Growth ◽

Genetic Pathways ◽

Content Type ◽

Virulence Trait ◽

The Impact

ABSTRACT The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream infections. Treatment of these infections is extremely difficult, as fungi are closely related to humans, and there are limited drugs that kill the fungus without host toxicity. The capacity of C. albicans to transition between yeast and filamentous forms is a key virulence trait. Thus, understanding the genetic pathways that regulate morphogenesis could provide novel therapeutic targets to treat C. albicans infections. Here, we establish the small molecule staurosporine as an inducer of filamentous growth. We unveil distinct regulatory circuitry required for staurosporine-induced filamentation that appears to be unique to this filament-inducing cue. Thus, this work highlights the fact that small molecules, such as staurosporine, can improve our understanding of the pathways required for key virulence programs, which may lead to the development of novel therapeutics. Protein kinases are key regulators of signal transduction pathways that participate in diverse cellular processes. In fungal pathogens, kinases regulate signaling pathways that govern drug resistance, stress adaptation, and pathogenesis. The impact of kinases on the fungal regulatory circuitry has recently garnered considerable attention in the opportunistic fungal pathogen Candida albicans, which is a leading cause of human morbidity and mortality. Complex regulatory circuitry governs the C. albicans morphogenetic transition between yeast and filamentous growth, which is a key virulence trait. Here, we report that staurosporine, a promiscuous kinase inhibitor that abrogates fungal drug resistance, also influences C. albicans morphogenesis by inducing filamentation in the absence of any other inducing cue. We further establish that staurosporine exerts its effect via the adenylyl cyclase Cyr1 and the cyclic AMP (cAMP)-dependent protein kinase A (PKA). Strikingly, filamentation induced by staurosporine does not require the known upstream regulators of Cyr1, Ras1 or Pkc1, or effectors downstream of PKA, including Efg1. We further demonstrate that Cyr1 is capable of activating PKA to enable filamentation in response to staurosporine through a mechanism that does not require degradation of the transcriptional repressor Nrg1. We establish that staurosporine-induced filamentation is accompanied by a defect in septin ring formation, implicating cell cycle kinases as potential staurosporine targets underpinning this cellular response. Thus, we establish staurosporine as a chemical probe to elucidate the architecture of cellular signaling governing fungal morphogenesis and highlight the existence of novel circuitry through which the Cyr1 and PKA govern a key virulence trait. IMPORTANCE The impact of fungal pathogens on human health is devastating. One of the most pervasive fungal pathogens is Candida albicans, which kills ~40% of people suffering from bloodstream infections. Treatment of these infections is extremely difficult, as fungi are closely related to humans, and there are limited drugs that kill the fungus without host toxicity. The capacity of C. albicans to transition between yeast and filamentous forms is a key virulence trait. Thus, understanding the genetic pathways that regulate morphogenesis could provide novel therapeutic targets to treat C. albicans infections. Here, we establish the small molecule staurosporine as an inducer of filamentous growth. We unveil distinct regulatory circuitry required for staurosporine-induced filamentation that appears to be unique to this filament-inducing cue. Thus, this work highlights the fact that small molecules, such as staurosporine, can improve our understanding of the pathways required for key virulence programs, which may lead to the development of novel therapeutics.

Download Full-text