Pineapples, or self-organized, Taxol-stabilized microtubule assemblies, reveal the richness of self-organizing mechanisms that operate on assembled microtubules during cell division and provide a biochemically tractable system for investigating these mechanisms during meiosis and cytokinesis.
Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B–containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.
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Poly(ADP-ribose) [pADPr] is a large, structurally complex polymer of repeating ADP-ribose units. It is biosynthesized from NAD+ by poly(ADP-ribose) polymerases [PARPs] and degraded to ADP-ribose by poly(ADP-ribose) glycohydrolase. PADPr is involved in many cellular processes and exerts biological function through covalent modification and non-covalent binding to specific proteins. Very little is known about molecular recognition and structure activity relationships for non-covalent interaction between pADPr and its binding proteins, in part because of lack of access to the polymer on large scale and to units of defined lengths. We prepared polydisperse pADPr from PARP1 and tankyrase 1 at hundreds of milligram scale by optimizing enzymatic synthesis and scaling up chromatographic purification methods. We developed and calibrated an anion exchange chromatography method to assign pADPr size and scaled it up to purify defined length polymers on the milligram scale. Furthermore, we present a pADPr profiling method to characterize the polydispersity of pADPr produced by PARPs under different reaction conditions and find that substrate proteins affect the pADPr size distribution. These methods will facilitate structural and biochemical studies of pADPr and its binding proteins.
PARP; PARG; pADPr; PAR; ADPr; poly(adp-ribose); poly(ADP-ribose) polymerase; poly(ADP-ribose) glycohydrolase; polymer; NAD+; ADP ribose
The microtubules that comprise mitotic spindles in animal cells are nucleated at centrosomes and by spindle assembly factors that are activated in the vicinity of chromatin. Indirect evidence also has suggested that microtubules might be nucleated from pre-existing microtubules throughout the spindle, but this process has not been observed directly. Here, we demonstrate microtubule nucleation from the sides of existing microtubules in meiotic Xenopus egg extracts. Daughter microtubules grow at a low branch angle and with the same polarity as mother filaments. Branching microtubule nucleation requires gamma-tubulin and augmin and is stimulated by GTP-bound Ran and its effector TPX2, factors previously implicated in chromatin-stimulated nucleation. Because of the rapid amplification of microtubule numbers and the preservation of microtubule polarity, microtubule-dependent microtubule nucleation is well suited for spindle assembly and maintenance.
Cytotoxic cancer chemotherapy drugs are believed to gain selectivity by targeting cells that proliferate rapidly. However, the proliferation rate is low in many chemosensitive human cancers, and it is not clear how a drug that only kills dividing cells could promote tumor regression. Four potential solutions to this “proliferation rate paradox” are discussed for the microtubule-stabilizing drug paclitaxel: drug retention in tumors, killing of quiescent cells, targeting of noncancer cells in the tumor, and bystander effects. Testing these potential mechanisms of drug action will facilitate rational improvement of antimitotic chemotherapy and perhaps cytotoxic chemotherapy more generally.
Kinetochores mediate chromosome segregation at mitosis. They are thought to contain both active, force-producing and passive, frictional interfaces with microtubules whose relative locations have been unclear. We inferred mechanical deformation within single kinetochores during metaphase oscillations by measuring average separations between fluorescently labeled kinetochore subunits in living cells undergoing mitosis. Inter-subunit distances were shorter in kinetochores moving toward poles than those moving away. Inter-subunit separation decreased abruptly when kinetochores switched to poleward movement, and decreased further when pulling force increased, suggesting that active force generation during poleward movement compresses kinetochores. The data revealed an active force -generating interface within kinetochores, and a separate passive frictional interface located at least 20 nm away poleward. Together, these interfaces allow persistent attachment with intermittent active force generation.
Actin filaments and microtubules polymerize and depolymerize by adding and removing subunits at polymer ends, and these dynamics drive cytoplasmic organization, cell division and cell motility. Since Wegner proposed the treadmilling theory for actin in 1976, it has largely been assumed that the chemical state of the bound nucleotide determines the rates of subunit addition and removal. This chemical kinetics view is difficult to reconcile with observations revealing multiple structural states of the polymer that influence polymerization dynamics, but are not strictly coupled to the bound nucleotide state. We refer to these phenomena as “structural plasticity”, and discuss emerging evidence that they play a central role in polymer dynamics and function.
Small molecule inhibitors of Kinesin-5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti-cancer drug candidates. Clinical trials of K5Is revealed dose-limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture. We found K5I treatment caused unusually rapid death of HL60 cells exclusively during mitotic arrest. This mitotic death occurred via the intrinsic apoptosis pathway with molecular events that include cytochrome c leakage into the cytoplasm, caspase activation, and Parp1 cleavage. Bcl-2 overexpression protected from death. We probed mitochondrial physiology to find candidate triggers of cytochrome c release, and observed a decrease of membrane potential (ΔΨm) before mitochondrial outer membrane permeabilization (MOMP). Interestingly, this loss of ΔΨm was not blocked by overexpressing Bcl-2, suggesting it might be a cause of Bax/Bak activation, not a consequence. Taken together, these results show that K5I induces intrinsic apoptosis during mitotic arrest in HL60 with loss of ΔΨm as an upstream event of MOMP.
Kinesin-5; mitosis; apoptosis; HL60
Plk1, but not Cdk1, phosphorylates PRC1 on Thr-602 to prevent premature midzone assembly in metaphase. Microtubules enhance this phosphorylation. Plk1, PRC1, and microtubules together regulate homeostatic MT bundling throughout cell division.
To achieve mitosis and cytokinesis, microtubules must assemble into distinct structures at different stages of cell division—mitotic spindles to segregate the chromosomes before anaphase and midzones to keep sister genomes apart and guide the cleavage furrow after anaphase. This temporal regulation is believed to involve Cdk1 kinase, which is inactivated in a switch-like way after anaphase. We found that inhibiting Plk1 caused premature assembly of midzones in cells still in metaphase, breaking the temporal regulation of microtubules. The antiparallel microtubule-bundling protein PRC1 plays a key role in organizing the midzone complex. We found that Plk1 negatively regulates PRC1 through phosphorylation of a single site, Thr-602, near the C-terminus of PRC1. We also found that microtubules stimulated Thr-602 phosphorylation by Plk1. This creates a potential negative feedback loop controlling PRC1 activity. It also made the extent of Thr-602 phosphorylation during mitotic arrest dependent on the mechanism of the arresting drug. Unexpectedly, we could not detect a preanaphase regulatory role for Cdk1 sites on PRC1. We suggest that PRC1 is regulated by Plk1, rather than Cdk1 as previously proposed, because its activity must be spatiotemporally regulated both preanaphase and postanaphase, and Cdk1 activity is too binary for this purpose.
Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 µM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.
Combining microtubule-targeting anti-mitotic drugs with targeted apoptosis potentiators is a promising new chemotherapeutic strategy to treat cancer. In this study we investigate the cellular mechanism by which Navitoclax (previously called ABT-263), a Bcl-2 family inhibitor, potentiates apoptosis triggered by paclitaxel and an inhibitor of Kinesin-5 (KSP), across a panel of epithelial cancer lines. Using time-lapse microscopy, we show that Navitoclax has little effect on cell death during interphase, but strongly accelerates apoptosis during mitotic arrest, and greatly increases the fraction of apoptosis-resistant cells that die. By systematically knocking down individual Bcl-2 proteins we determined that Mcl-1 and Bcl-xL are the primary negative regulators of apoptosis during prolonged mitotic arrest. Mcl-1 levels decrease during mitotic arrest due to an imbalance between synthesis and turnover, and turnover depends in part on the MULE/HUWE1 E3 ligase. The combination of Mcl-1 loss with inhibition of Bcl-xL by Navitoclax causes rapid apoptosis in all lines tested. Variation in expression levels of Mcl-1 and Bcl-xL largely determine variation in response to anti-mitotics alone, and anti-mitotics combined with Navitoclax, across our panel. We conclude that Bcl-xL is a critical target of Bcl-2 family inhibitors for enhancing the lethality of anti-mitotic drugs in epithelial cancers, and combination treatment with Navitoclax and a spindle specific anti-mitotic, such as a Kinesin-5 inhibitor, might be more effective than paclitaxel alone.
Navitoclax; paclitaxel; apoptosis response
The structural composition of the midbody changes during progression throughout cytokinesis. A detailed description is given of how known midbody proteins localize during late steps in cytokinesis. How these assembly events are regulated is investigated, together with their functional implications.
The midbody is a transient structure that connects two daughter cells at the end of cytokinesis, with the principal function being to localize the site of abscission, which physically separates two daughter cells. Despite its importance, understanding of midbody assembly and its regulation is still limited. Here we describe how the structural composition of the midbody changes during progression throughout cytokinesis and explore the functional implications of these changes. Deriving from midzones, midbodies are organized by a set of microtubule interacting proteins that colocalize to a zone of microtubule overlap in the center. We found that these proteins split into three subgroups that relocalize to different parts of the midbody: the bulge, the dark zone, and the flanking zone. We characterized these relocalizations and defined domain requirements for three key proteins: MKLP1, KIF4, and PRC1. Two cortical proteins—anillin and RhoA—localized to presumptive abscission sites in mature midbodies, where they may regulate the endosomal sorting complex required for transport machinery. Finally, we characterized the role of Plk1, a key regulator of cytokinesis, in midbody assembly. Our findings represent the most detailed description of midbody assembly and maturation to date and may help elucidate how abscission sites are positioned and regulated.
Midzones, also called central spindles, are an array of antiparallel microtubules that form during animal cell cytokinesis between the separated chromosomes . Midzones can be considered platforms that recruit specific proteins and orchestrate cytokinetic events, such as keeping sister nuclei apart, furrow ingression, and abscission [2–3]. Despite this important role, many aspects of midzone biology remain unknown, including the dynamic organization of midzone microtubules. Investigating midzone microtubule dynamics has been difficult in part because their plus-ends are interdigitated and buried in a dense matrix, making them difficult to observe. We employed monopolar cytokinesis to reveal that midzone plus-ends appear non-dynamic. We identified the chromokinesin KIF4 as a negative regulator of midzone plus-end dynamics, whose activity controls midzone length, but not stability. KIF4 is required to terminate midzone elongation in late anaphase. In the absence of KIF4, midzones elongate abnormally, and their overlap regions are unfocused. Electron dense material and midbodies are both absent from the elongated midzones, and actin filaments from the furrow cortex are not disassembled after ingression. KIF4 mediated midzone length regulation appears to occur by terminating midzone elongation at a specific time during cytokinesis, making midzones and mitotic spindles differ in their dynamics and length regulating mechanisms.
ETOC: Despite the use of antimitotic drugs, our understanding of the stress response, especially during mitotic arrest, is lacking. We report a molecular mechanism resulting in DNA damage during mitotic arrest that occurs via the apoptotic machinery but in the absence of cell death; this mechanism triggers p53 induction after cells slip from mitotic arrest.
Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.
Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes using dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex, or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length–dependent pulling forces, which move and orient centrosomes.
We identified a clarified extract containing the soluble factors for microtubule assembly. We found that microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.
The assembly of microtubules during mitosis requires many identified components, such as γ-tubulin ring complex (γ-TuRC), components of the Ran pathway (e.g., TPX2, HuRP, and Rae1), and XMAP215/chTOG. However, it is far from clear how these factors function together or whether more factors exist. In this study, we used biochemistry to attempt to identify active microtubule nucleation protein complexes from Xenopus meiotic egg extracts. Unexpectedly, we found both microtubule assembly and bipolar spindle assembly required glycogen, which acted both as a crowding agent and as metabolic source glucose. By also reconstituting microtubule assembly in clarified extracts, we showed microtubule assembly does not require ribosomes, mitochondria, or membranes. Our clarified extracts will provide a powerful tool for activity-based biochemical fractionations for microtubule assembly.
Variability in cell-to-cell behavior within clonal populations can be attributed to the inherent stochasticity of biochemical reactions. Most single-cell studies have examined variation in behavior due to randomness in gene transcription. Here we investigate the mechanism of cell fate choice and the origin of cell-to-cell variation during mitotic arrest, when transcription is silenced. Prolonged mitotic arrest is commonly observed in cells treated with anti-mitotic drugs. Cell fate during mitotic arrest is determined by two alternative pathways, one promoting cell death, the other promoting cyclin B1 degradation, which leads to mitotic slippage and survival. It has been unclear whether these pathways are mechanistically coupled or independent. In this study we experimentally uncoupled these two pathways using zVAD-fmk to block cell death or Cdc20 knockdown to block slippage. We then used time-lapse imaging to score the kinetics of single cells adopting the remaining fate. We also used kinetic simulation to test whether the behaviors of death versus slippage in cell populations where both pathways are active can be quantitatively recapitulated by a model that assumes stochastic competition between the pathways. Our data are well fit by a model where the two pathways are mechanistically independent, and cell fate is determined by a stochastic kinetic competition between them that results in cell-to-cell variation.
Current anti-mitotics work by perturbing spindle assembly, which activates the spindle assembly checkpoint, causes mitotic arrest, and triggers apoptosis. Cancer cells can resist such killing by premature exit, before cells initiate apoptosis, due to a weak checkpoint or rapid slippage. We reasoned blocking mitotic exit downstream of the checkpoint might circumvent this resistance. Using single-cell approaches, we showed that blocking mitotic exit by Cdc20 knockdown slowed cyclin B1 proteolysis, thus allowed more time for death initiation. Killing by Cdc20 knockdown did not require checkpoint activity, and can occur by intrinsic apoptosis, or an alternative death pathway when Bcl2 was over-expressed. We conclude targeting Cdc20, or otherwise blocking mitotic exit, may be a better cancer therapeutic strategy than perturbing spindle assembly.
The mitotic spindle assembles to a steady-state length at metaphase through the integrated action of molecular mechanisms that generate and respond to mechanical forces. While molecular mechanisms that produce force have been described, our understanding of how they integrate with each other, and with the assembly-disassembly mechanisms that regulate length, is poor. We review current understanding of the basic architecture and dynamics of the metaphase spindle, and some of the elementary force producing mechanisms. We then discuss models for force integration, and spindle length determination. We also emphasize key missing data that notably includes absolute values of forces, and how they vary as a function of position, within the spindle.
The spindle matrix does not make a significant mechanical contribution to metaphase spindle length.
Several recent models for spindle length regulation propose an elastic pole to pole spindle matrix that is sufficiently strong to bear or antagonize forces generated by microtubules and microtubule motors. We tested this hypothesis using microneedles to skewer metaphase spindles in Xenopus laevis egg extracts. Microneedle tips inserted into a spindle just outside the metaphase plate resulted in spindle movement along the interpolar axis at a velocity slightly slower than microtubule poleward flux, bringing the nearest pole toward the needle. Spindle velocity decreased near the pole, which often split apart slowly, eventually letting the spindle move completely off the needle. When two needles were inserted on either side of the metaphase plate and rapidly moved apart, there was minimal spindle deformation until they reached the poles. In contrast, needle separation in the equatorial direction rapidly increased spindle width as constant length spindle fibers pulled the poles together. These observations indicate that an isotropic spindle matrix does not make a significant mechanical contribution to metaphase spindle length determination.
During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin [1, 2]. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors [3–5]. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin.
Although the molecules involved in mitosis are becoming better characterized, we still lack an understanding of the emergent mechanical properties of the mitotic spindle. For example, we cannot explain how spindle length is determined. To gain insight into how forces are generated and responded to in the spindle, we developed a method to apply controlled mechanical compression to metaphase mitotic spindles in living mammalian cells, while monitoring microtubules and kinetochores by fluorescence microscopy.
Compression caused reversible spindle widening and lengthening to a new steady-state. Widening was a passive mechanical response, and lengthening an active mechanochemical process requiring microtubule polymerization but not kinesin-5 activity. Spindle morphology during lengthening and drug perturbations suggested that kinetochore fibers are pushed outwards by pole-directed forces generated within the spindle. Lengthening of kinetochore fibers occurred by inhibition of microtubule depolymerization at poles, with no change in sliding velocity, inter-kinetochore stretching, or kinetochore dynamics.
We propose that spindle length is controlled by a mechanochemical switch at the poles that regulates the depolymerization rate of kinetochore-fibers in response to compression, and discuss models for how this switch is controlled. Poleward force appears to be exerted along kinetochore fibers by some mechanism other than kinesin-5 activity, and we speculate that it may arise from polymerization pressure from growing plus-ends of interpolar microtubules whose minus-ends are anchored in the fiber. These insights provide a framework for conceptualizing mechanical integration within the spindle.
Bioassay-guided fractionation of Physocarpus capitatus yielded two new cucurbitacins (3 and 4) along with the known cucurbitacin F (1) and dihydrocucurbitacin F (2). Preliminary mechanism of action studies indicate that the cucurbitacins cause actin aggregates and inhibit cell division.
Microtubules play a central role in centering the nucleus or mitotic in eukaryotic cells. However, despite common use of microtubules for centring, physical mechanisms can vary greatly, and depend on cell size and cell type. In the small fission yeast cells, the nucleus can be centered by pushing forces that are generated when growing microtubules hit the cell boundary. This mechanism may not be possible in larger cells, because the compressive force that microtubules can sustain are limited by buckling, so maximal force decreases with microtubule length. In a well-studied intermediate sized cell, the C. elegans fertilized egg, centrosomes are centered by cortex-attached motors that pull on microtubules. This mechanism is widely assumed to be general for larger cells. However, re-evaluation of classic experiments in a very large cell, the fertilized amphibian egg, argues against such generality. In these large eggs, movement of asters away from a part of the cell boundary that they are touching cannot be mediated by cortical pulling, because the astral microtubules are too short to reach the opposite cell boundary. A century ago, Herlant and Brachet discovered that multiple asters within a single egg center relative to the cell boundary, but also relative to each other. Here, we summarize current understanding of microtubule organization during the first cell cycle in a fertilized Xenopus egg, discuss how microtubule asters move towards the center of this very large cell, and how multiple asters shape and position themselves relative to each other.