Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC–spindle interaction to ensure proper microtubule dynamics

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase–anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC–microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.

Download Full-text

Kinesins relocalize the chromosomal passenger complex to the midzone for spindle disassembly

The Journal of Cell Biology ◽

10.1083/jcb.201708114 ◽

2018 ◽

Vol 217 (5) ◽

pp. 1687-1700 ◽

Cited By ~ 7

Author(s):

Itziar Ibarlucea-Benitez ◽

Luke S. Ferro ◽

David G. Drubin ◽

Georjana Barnes

Keyword(s):

Single Molecule ◽

Molecular Mechanisms ◽

Chromosomal Passenger Complex ◽

Late Anaphase ◽

Chromosomal Passenger ◽

Spindle Disassembly ◽

Spindle Midzone ◽

Chromosome Separation

Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly.

Download Full-text

The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

The Journal of Cell Biology ◽

10.1083/jcb.201312039 ◽

2014 ◽

Vol 204 (6) ◽

pp. 965-975 ◽

Cited By ~ 27

Author(s):

Rania S. Rizk ◽

Katherine A. DiScipio ◽

Kathleen G. Proudfoot ◽

Mohan L. Gupta

Keyword(s):

Mitotic Spindle ◽

Molecular Mechanisms ◽

Spindle Microtubule ◽

Microtubule Polymerization ◽

Sister Chromatids ◽

Final Length ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Spindle Function ◽

Yeast Mutants

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

Download Full-text

Analysis of the distribution of spindle microtubules in the diatom Fragilaria.

The Journal of Cell Biology ◽

10.1083/jcb.79.3.737 ◽

1978 ◽

Vol 79 (3) ◽

pp. 737-763 ◽

Cited By ~ 39

Author(s):

D H Tippit ◽

D Schulz ◽

J D Pickett-Heaps

Keyword(s):

Spatial Arrangement ◽

Spindle Pole ◽

Serial Sections ◽

Late Prophase ◽

Central Spindle ◽

Sources Of Error ◽

Late Anaphase ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Intercellular Variability

The spindle of the colonial diatom Fragilaria contains two distinct sets of spindle microtubules (MTs): (a) MTs comprising the central spindle, which is composed of two half-spindles interdigitated to form a region of "overlap"; (b) MTs which radiate laterally from the poles. The central spindles from 28 cells are reconstructed by tracking each MT of the central spindle through consecutive serial sections. Because the colonies of Fragilaria are flat ribbons of contiguous cells (clones), it is possible, by using single ribbons of cells, to compare reconstructed spindles at different mitotic stages with minimal intercellular variability. From these reconstructions we have determined: (a) the changes in distribution of MTs along the spindle during mitosis; (b) the change in the total number of MTs during mitosis; (c) the length of each MT (measured by the number of sections each traverses) at different mitotic stages; (d) the frequency of different classes of MTs (i.e., free, continuous, etc.); (e) the spatial arrangement of MTs from opposite poles in the overlap; (f) the approximate number of MTs, separate from the central spindle, which radiate from each spindle pole. From longitudinal sections of the central spindle, the lengths of the whole spindle, half-spindle, and overlap were measured from 80 cells at different mitotic stages. Numerous sources of error may create inaccuracies in these measurements; these problems are discussed. The central spindle at prophase consists predominantly of continuous MTs (pole to pole). Between late prophase and prometaphase, spindle length increases, and the spindle is transformed into two half-spindles (mainly polar MTs) interdigitated to form the overlap. At late anaphase-telophase, the overlap decreases concurrent with spindle elongation. Our interpretation is that the MTs of the central spindle slide past one another at both late prophase and late anaphase. These changes in MT distribution have the effect of elongating the spindle and are not involved in the poleward movement of the chromosomes. Some aspects of tracking spindle MTs, the interaction of MTs in the overlap, formation of the prophase spindle, and our interpretation of rearrangements of MTs, are discussed.

Download Full-text

TOG–tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation

The Journal of Cell Biology ◽

10.1083/jcb.201610090 ◽

2017 ◽

Vol 216 (6) ◽

pp. 1641-1657 ◽

Cited By ~ 28

Author(s):

Amy E. Byrnes ◽

Kevin C. Slep

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly Checkpoint ◽

Polymerase Activity ◽

Microtubule Dynamics ◽

Spindle Formation ◽

Microtubule Binding ◽

Microtubule Polymerization ◽

Tubulin Binding ◽

Specific Order ◽

Spindle Function

XMAP215, CLASP, and Crescerin use arrayed tubulin-binding tumor overexpressed gene (TOG) domains to modulate microtubule dynamics. We hypothesized that TOGs have distinct architectures and tubulin-binding properties that underlie each family’s ability to promote microtubule polymerization or pause. As a model, we investigated the pentameric TOG array of a Drosophila melanogaster XMAP215 member, Msps. We found that Msps TOGs have distinct architectures that bind either free or polymerized tubulin, and that a polarized array drives microtubule polymerization. An engineered TOG1-2-5 array fully supported Msps-dependent microtubule polymerase activity. Requisite for this activity was a TOG5-specific N-terminal HEAT repeat that engaged microtubule lattice-incorporated tubulin. TOG5–microtubule binding maintained mitotic spindle formation as deleting or mutating TOG5 compromised spindle architecture and increased the mitotic index. Mad2 knockdown released the spindle assembly checkpoint triggered when TOG5–microtubule binding was compromised, indicating that TOG5 is essential for spindle function. Our results reveal a TOG5-specific role in mitotic fidelity and support our hypothesis that architecturally distinct TOGs arranged in a sequence-specific order underlie TOG array microtubule regulator activity.

Download Full-text

Mitosis in the pinnate diatom surirella ovalis

The Journal of Cell Biology ◽

10.1083/jcb.73.3.705 ◽

1977 ◽

Vol 73 (3) ◽

pp. 705-727 ◽

Cited By ~ 66

Author(s):

DH Tippit ◽

JD Pickett-Heaps

Keyword(s):

Spindle Pole ◽

Late Prophase ◽

Unknown Mechanism ◽

Electron Dense Material ◽

Central Spindle ◽

Late Anaphase ◽

Sliding Mechanism ◽

Chromosome Attachment ◽

Microtubule Center ◽

Spindle Function

Mitosis in Surirella is described; this organism displays a number of unusual features including an unorthodox method of chromosome attachment to the spindle, and the differentiation of an extranuclear central spindle from a large spherical organelle named the microtubule center (MC). The MC, present during interphase, breaks down at late prophase as the central spindle is formed. Later, the spindle enters the nucleus; the chromatin, in association with microtubules (MTs) from the poles, increasingly aggregates around the middle "overlap" region of the central spindle, and by metaphase completely encircles it. Throughout, MTs usually associate laterally with the chromatin. We were not able to identify kinetochore MTs with confidence at either metaphase or anaphase. Instead, at anaphase the leading point of the chromosomes is embedded in a ring of electron-dense material, named the "collar," which encircles each half spindle and extends from the chromatin to the pole. Anaphase separation of the chromosomes is achieved by at least three separate mechanisms: (a) between metaphase and late anaphase the central spindle increases in length by the addition of MT subunits; (b) at late anaphase the central spindle elongates concurrent with a reduction in the overlap; this apparently results from an MT/MT sliding mechanism; (c) each set of chromosomes moves to the poles by a thus far unknown mechanism; however, we anticipate some interaction of the collar and central spindle. At telophase, the polar complexes, (i.e., structures at the spindle pole) separate from the spindle, and later a new MC is formed near each polar complex, after which the polar complexes break down. Aspects of the complex differentiation of the MC, spindle formation, and some unusual characteristics of the diatom spindle as they relate to anaphase motion and spindle function are discussed.

Download Full-text

The Drosophila kinesin-like protein KLP3A is a midbody component required for central spindle assembly and initiation of cytokinesis.

The Journal of Cell Biology ◽

10.1083/jcb.129.3.709 ◽

1995 ◽

Vol 129 (3) ◽

pp. 709-723 ◽

Cited By ~ 99

Author(s):

B C Williams ◽

M F Riedy ◽

E V Williams ◽

M Gatti ◽

M L Goldberg

Keyword(s):

Motor Protein ◽

Spindle Assembly ◽

Male Meiosis ◽

Cleavage Furrow ◽

Critical Component ◽

Central Spindle ◽

Late Anaphase ◽

Spindle Elongation ◽

Anaphase B ◽

Kinesin Superfamily

We describe here a new member of the kinesin superfamily in Drosophila, KLP3A (Kinesin-Like-Protein-at-3A). The KLP3A protein localizes to the equator of the central spindle during late anaphase and telophase of male meiosis. Mutations in the KLP3A gene disrupt the interdigitation of microtubules in spermatocyte central spindles. Despite this defect, anaphase B spindle elongation is not obviously aberrant. However, cytokinesis frequently fails after both meiotic divisions in mutant testes. Together, these findings strongly suggest that the KLP3A presumptive motor protein is a critical component in the establishment or stabilization of the central spindle. Furthermore, these results imply that the central spindle is the source of signals that initiate the cleavage furrow in higher cells.

Download Full-text

Spindle scaling mechanisms

Essays in Biochemistry ◽

10.1042/ebc20190064 ◽

2020 ◽

Vol 64 (2) ◽

pp. 383-396

Author(s):

Lara K. Krüger ◽

Phong T. Tran

Keyword(s):

Cell Size ◽

Spindle Assembly ◽

Dependent Manner ◽

Post Translational Modification ◽

Size Dependent ◽

A Cell ◽

Chromosome Separation ◽

Spindle Elongation ◽

Protein Gradients ◽

Spindle Structure

Abstract 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.

Download Full-text

Regulation of Microtubule Dynamics by Bim1 and Bik1, the Budding Yeast Members of the EB1 and CLIP-170 Families of Plus-End Tracking Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.e10-02-0083 ◽

2010 ◽

Vol 21 (12) ◽

pp. 2013-2023 ◽

Cited By ~ 33

Author(s):

Kristina A. Blake-Hodek ◽

Lynne Cassimeris ◽

Tim C. Huffaker

Keyword(s):

Budding Yeast ◽

Family Members ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

Microtubule Polymerization

Microtubule dynamics are regulated by plus-end tracking proteins (+TIPs), which bind microtubule ends and influence their polymerization properties. In addition to binding microtubules, most +TIPs physically associate with other +TIPs, creating a complex web of interactions. To fully understand how +TIPs regulate microtubule dynamics, it is essential to know the intrinsic biochemical activities of each +TIP and how +TIP interactions affect these activities. Here, we describe the activities of Bim1 and Bik1, two +TIP proteins from budding yeast and members of the EB1 and CLIP-170 families, respectively. We find that purified Bim1 and Bik1 form homodimers that interact with each other to form a tetramer. Bim1 binds along the microtubule lattice but with highest affinity for the microtubule end; however, Bik1 requires Bim1 for localization to the microtubule lattice and end. In vitro microtubule polymerization assays show that Bim1 promotes microtubule assembly, primarily by decreasing the frequency of catastrophes. In contrast, Bik1 inhibits microtubule assembly by slowing growth and, consequently, promoting catastrophes. Interestingly, the Bim1-Bik1 complex affects microtubule dynamics in much the same way as Bim1 alone. These studies reveal new activities for EB1 and CLIP-170 family members and demonstrate how interactions between two +TIP proteins influence their activities.

Download Full-text

Mitosis in the cellular slime mold Polysphondylium violaceum.

The Journal of Cell Biology ◽

10.1083/jcb.64.2.480 ◽

1975 ◽

Vol 64 (2) ◽

pp. 480-491 ◽

Cited By ~ 41

Author(s):

U P Roos

Keyword(s):

Spindle Pole ◽

Equatorial Region ◽

Cellular Slime Mold ◽

Central Spindle ◽

Spindle Pole Bodies ◽

Cellular Slime ◽

Late Telophase ◽

Spindle Elongation ◽

Well Differentiated ◽

Opaque Body

Myxamebas of Polysphondylium violaceum were grown in liquid medium and processed for electron microscopy. Mitosis is characterized by a persistent nuclear envelope, ring-shaped extranuclear spindle pole bodies (SPBs), a central spindle spatially separated from the chromosomal microtubules, well-differentiated kinetochores, and dispersion of the nucleoli. SPBs originate from the division, during prophase, of an electron-opaque body associated with the interphase nucleus. The nuclear nevelope becomes fenestrated in their vicinity, allowing the build-up of the intranuclear, central spindle and chromosomal microtubules as the SPBs migrate to opposite poles. At metaphase the chromosomes are in amphitelic orientation, each sister chromatid being directly connected to the corresponding SPB by a single microtubule. During ana- and telophase the central spindle elongates, the daughter chromosomes approach the SPBs, and the nucleus constricts in the equatorial region. The cytoplasm cleaves by furrowing in late telophase, which is in other respects characterized by a re-establishment of the interphase condition. Spindle elongation and poleward movement of chromosomes are discussed in relation to hypotheses of the mechanism of mitosis.

Download Full-text

Spatiotemporal control of functional specification and distribution of spindle microtubules with 13, 14 and 15 protofilaments during mitosis in the ciliate Nyctotherus

Journal of Cell Science ◽

10.1242/jcs.76.1.337 ◽

1985 ◽

Vol 76 (1) ◽

pp. 337-355

Author(s):

U. Eichenlaub-Ritter

Keyword(s):

Differential Sensitivity ◽

Microtubule Depolymerization ◽

Functional Specification ◽

Microtubule Stability ◽

Early Anaphase ◽

Late Telophase ◽

Spindle Microtubules ◽

Elongation Phase ◽

Spatiotemporal Control ◽

Macronuclear Division

The formation of microtubules with more than 13 protofilaments in the ciliate Nyctotherus ovalis Leidy seems to be a highly ordered process. Such microtubules are restricted to the nucleoplasm and, moreover, to certain stages of nuclear division. They assemble during anaphase of micronuclear mitosis and during the elongation phase of macronuclear division. The number of microtubules with more than 13 protofilaments in the micronuclear nucleoplasm increases as anaphase progresses. Furthermore, assembly of microtubules with 14 and 15 protofilaments seems to proceed concomitantly with net disassembly of 13-protofilament microtubules, because the total amount of polymerized tubulin in the interpolar spindle region remains approximately constant between mid anaphase and late telophase. In addition, evidence for spatial control of the distribution of microtubules with different protofilament numbers in the micronuclear stembody has been found. The percentage of microtubules with 13 protofilaments per stembody cross-section is highest at the ends of the stembody, while the percentage of microtubules with either 14 or 15 protofilaments increases as the middle of the stembody is approached. Temporal control of polymerization of microtubules with high protofilament numbers seems to be exerted independently in the two types of nuclei. For example, when the macronucleus starts to elongate it contains microtubules with more than 13 protofilaments but the metaphase micronucleus still possesses only microtubules with 13 protofilaments at this stage. Control of fidelity of protofilament numbers is not lost in the early stages of micronuclear or macronuclear division when cells are exposed to 2H2O or media containing taxol. Even microtubules that reassemble during recovery of metaphase micronuclei from nocodazole-induced microtubule depolymerization, in either the absence or presence of 2H2O and taxol, possess 13 protofilaments. Similarly, if the introduction of microtubules with 14 and 15 protofilaments is inhibited during early micronuclear anaphase and delayed for 60 min by exposure to nocodazole, such microtubules still assemble during telophase when recovery is permitted. Microtubules that have been assembled under normal conditions show differential sensitivity to nocodazole. During metaphase, nocodazole induces disassembly of most microtubules. There is an increase in microtubule stability that coincides with the appearance of microtubules with high protofilament numbers during early anaphase. However, considerable numbers of 13-protofilament microtubules, as well as microtubules with 14 and 15 protofilaments, exhibit such stability during anaphase.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text