Publications by authors named "Jeffrey K Moore"

35 Publications

Ase1 domains dynamically slow anaphase spindle elongation and recruit Bim1 to the midzone.

Mol Biol Cell 2020 11 30;31(24):2733-2747. Epub 2020 Sep 30.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045.

How cells regulate microtubule cross-linking activity to control the rate and duration of spindle elongation during anaphase is poorly understood. In this study, we test the hypothesis that PRC1/Ase1 proteins use distinct microtubule-binding domains to control the spindle elongation rate. Using the budding yeast Ase1, we identify unique contributions for the spectrin and carboxy-terminal domains during different phases of spindle elongation. We show that the spectrin domain uses conserved basic residues to promote the recruitment of Ase1 to the midzone before anaphase onset and slow spindle elongation during early anaphase. In contrast, a partial Ase1 carboxy-terminal truncation fails to form a stable midzone in late anaphase, produces higher elongation rates after early anaphase, and exhibits frequent spindle collapses. We find that the carboxy-terminal domain interacts with the plus-end tracking protein EB1/Bim1 and recruits Bim1 to the midzone to maintain midzone length. Overall, our results suggest that the Ase1 domains provide cells with a modular system to tune midzone activity and control elongation rates.
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http://dx.doi.org/10.1091/mbc.E20-07-0493-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927185PMC
November 2020

Mechanisms of microtubule dynamics and force generation examined with computational modeling and electron cryotomography.

Nat Commun 2020 07 28;11(1):3765. Epub 2020 Jul 28.

Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA.

Microtubules are dynamic tubulin polymers responsible for many cellular processes, including the capture and segregation of chromosomes during mitosis. In contrast to textbook models of tubulin self-assembly, we have recently demonstrated that microtubules elongate by addition of bent guanosine triphosphate tubulin to the tips of curving protofilaments. Here we explore this mechanism of microtubule growth using Brownian dynamics modeling and electron cryotomography. The previously described flaring shapes of growing microtubule tips are remarkably consistent under various assembly conditions, including different tubulin concentrations, the presence or absence of a polymerization catalyst or tubulin-binding drugs. Simulations indicate that development of substantial forces during microtubule growth and shortening requires a high activation energy barrier in lateral tubulin-tubulin interactions. Modeling offers a mechanism to explain kinetochore coupling to growing microtubule tips under assisting force, and it predicts a load-dependent acceleration of microtubule assembly, providing a role for the flared morphology of growing microtubule ends.
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http://dx.doi.org/10.1038/s41467-020-17553-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387542PMC
July 2020

Microtubule dynamics at low temperature: evidence that tubulin recycling limits assembly.

Mol Biol Cell 2020 05 26;31(11):1154-1166. Epub 2020 Mar 26.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045.

How temperature specifically affects microtubule dynamics and how these lead to changes in microtubule networks in cells have not been established. We investigated these questions in budding yeast, an organism found in diverse environments and therefore predicted to exhibit dynamic microtubules across a broad temperature range. We measured the dynamics of GFP-labeled microtubules in living cells and found that lowering temperature from 37°C to 10°C decreased the rates of both polymerization and depolymerization, decreased the amount of polymer assembled before catastrophes, and decreased the frequency of microtubule emergence from nucleation sites. Lowering to 4°C caused rapid loss of almost all microtubule polymer. We provide evidence that these effects on microtubule dynamics may be explained in part by changes in the cofactor-dependent conformational dynamics of tubulin proteins. Ablation of tubulin-binding cofactors (TBCs) further sensitizes cells and their microtubules to low temperatures, and we highlight a specific role for TBCB/Alf1 in microtubule maintenance at low temperatures. Finally, we show that inhibiting the maturation cycle of tubulin by using a point mutant in β-tubulin confers hyperstable microtubules at low temperatures and rescues the requirement for TBCB/Alf1 in maintaining microtubule polymer at low temperatures. Together, these results reveal an unappreciated step in the tubulin cycle.
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http://dx.doi.org/10.1091/mbc.E19-11-0634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353160PMC
May 2020

Reduced TUBA1A Tubulin Causes Defects in Trafficking and Impaired Adult Motor Behavior.

eNeuro 2020 Mar/Apr;7(2). Epub 2020 Apr 27.

Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora CO 80045

Newly born neurons express high levels of TUBA1A α-tubulin to assemble microtubules for neurite extension and to provide tracks for intracellular transport. In the adult brain, expression decreases dramatically. A mouse that harbors a loss-of-function mutation in the gene encoding TUBA1A ( ) allows us to ask whether TUBA1A is important for the function of mature neurons. α-Tubulin levels are about half of wild type in juvenile brains, but are close to normal in older animals. In postnatal day (P)0 cultured neurons, reduced TUBA1A allows for assembly of less microtubules in axons resulting in more pausing during organelle trafficking. While mouse behavior is indistinguishable from wild-type siblings at weaning, mice develop adult-onset ataxia. Neurons important for motor function in remain indistinguishable from wild-type with respect to morphology and number and display no evidence of axon degeneration. neuromuscular junction (NMJ) synapses are the same size as wild-type before the onset of ataxia, but are reduced in size in older animals. Together, these data indicate that the TUBA1A-rich microtubule tracks that are assembled during development are essential for mature neuron function and maintenance of synapses over time.
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http://dx.doi.org/10.1523/ENEURO.0045-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7218002PMC
April 2020

Tubulin mutations in brain development disorders: Why haploinsufficiency does not explain TUBA1A tubulinopathies.

Cytoskeleton (Hoboken) 2020 03 31;77(3-4):40-54. Epub 2019 Oct 31.

Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

The neuronal cytoskeleton performs incredible feats during nervous system development. Extension of neuronal processes, migration, and synapse formation rely on the proper regulation of microtubules. Mutations that disrupt the primary α-tubulin expressed during brain development, TUBA1A, are associated with a spectrum of human brain malformations. One model posits that TUBA1A mutations lead to a reduction in tubulin subunits available for microtubule polymerization, which represents a haploinsufficiency mechanism. We propose an alternative model for the majority of tubulinopathy mutations, in which the mutant tubulin polymerizes into the microtubule lattice to dominantly "poison" microtubule function. Nine distinct α-tubulin and ten β-tubulin genes have been identified in the human genome. These genes encode similar tubulin proteins, called isotypes. Multiple tubulin isotypes may partially compensate for heterozygous deletion of a tubulin gene, but may not overcome the disruption caused by missense mutations that dominantly alter microtubule function. Here, we describe disorders attributed to haploinsufficiency versus dominant negative mechanisms to demonstrate the hallmark features of each disorder. We summarize literature on mouse models that represent both knockout and point mutants in tubulin genes, with an emphasis on how these mutations might provide insight into the nature of tubulinopathy patient mutations. Finally, we present data from a panel of TUBA1A tubulinopathy mutations generated in yeast α-tubulin that demonstrate that α-tubulin mutants can incorporate into the microtubule network and support viability of yeast growth. This perspective on tubulinopathy mutations draws on previous studies and additional data to provide a fresh perspective on how TUBA1A mutations disrupt neurodevelopment.
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http://dx.doi.org/10.1002/cm.21567DOI Listing
March 2020

Help or hindrance: how do microtubule-based forces contribute to genome damage and repair?

Curr Genet 2020 Apr 9;66(2):303-311. Epub 2019 Sep 9.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA.

Forces generated by molecular motors and the cytoskeleton move the nucleus and genome during many cellular processes, including cell migration and division. How these forces impact the genome, and whether cells regulate cytoskeletal forces to preserve genome integrity is unclear. We recently demonstrated that, in budding yeast, mutants that stabilize the microtubule cytoskeleton cause excessive movement of the mitotic spindle and nucleus. We found that increased nuclear movement results in DNA damage and increased time to repair the damage through homology-directed repair. Our results indicate that nuclear movement impairs DNA repair through increased tension on chromosomes and nuclear deformation. However, the previous studies have shown genome mobility, driven by cytoskeleton-based forces, aids in homology-directed DNA repair. This sets up an apparent paradox, where genome mobility may prevent or promote DNA repair. Hence, this review explores how the genome is affected by nuclear movement and how genome mobility could aid or hinder homology-directed repair.
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http://dx.doi.org/10.1007/s00294-019-01033-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7061087PMC
April 2020

Astral microtubule forces alter nuclear organization and inhibit DNA repair in budding yeast.

Mol Biol Cell 2019 07 8;30(16):2000-2013. Epub 2019 May 8.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045.

Dividing cells must balance the maintenance of genome integrity with the generation of cytoskeletal forces that control chromosome position. In this study, we investigate how forces on astral microtubules impact the genome during cell division by using live-cell imaging of the cytoskeleton, chromatin, and DNA damage repair in budding yeast. Our results demonstrate that dynein-dependent forces on astral microtubules are propagated through the spindle during nuclear migration and when in excess can increase the frequency of double-stranded breaks (DSBs). Under these conditions, we find that homology-directed repair of DSBs is delayed, indicating antagonism between nuclear migration and the mechanism of homology-directed repair. These effects are partially rescued by mutants that weaken pericentric cohesion or mutants that decrease constriction on the nucleus as it moves through the bud neck. We propose that minimizing nuclear movement aids in finding a donor strand for homologous recombination.
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http://dx.doi.org/10.1091/mbc.E18-12-0808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727761PMC
July 2019

A unified model for microtubule rescue.

Mol Biol Cell 2019 03 23;30(6):753-765. Epub 2019 Jan 23.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045.

How microtubules transition from depolymerization to polymerization, known as rescue, is poorly understood. Here we examine two models for rescue: 1) an "end-driven" model in which the depolymerizing end stochastically switches to a stable state; and 2) a "lattice-driven" model in which rescue sites are integrated into the microtubule before depolymerization. We test these models using a combination of computational simulations and in vitro experiments with purified tubulin. Our findings support the "lattice-driven" model by identifying repeated rescue sites in microtubules. In addition, we discover an important role for divalent cations in determining the frequency and location of rescue sites. We use "wash-in" experiments to show that divalent cations inhibit rescue during depolymerization, but not during polymerization. We propose a unified model in which rescues are driven by embedded rescue sites in microtubules, but the activity of these sites is influenced by changes in the depolymerizing ends.
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http://dx.doi.org/10.1091/mbc.E18-08-0541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589779PMC
March 2019

TUBA1A mutations identified in lissencephaly patients dominantly disrupt neuronal migration and impair dynein activity.

Hum Mol Genet 2019 04;28(8):1227-1243

Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA.

The microtubule cytoskeleton supports diverse cellular morphogenesis and migration processes during brain development. Mutations in tubulin genes are associated with severe human brain malformations known as 'tubulinopathies'; however, it is not understood how molecular-level changes in microtubule subunits lead to brain malformations. In this study, we demonstrate that missense mutations affecting arginine at position 402 (R402) of TUBA1A α-tubulin selectively impair dynein motor activity and severely and dominantly disrupt cortical neuronal migration. TUBA1A is the most commonly affected tubulin gene in tubulinopathy patients, and mutations altering R402 account for 30% of all reported TUBA1A mutations. We show for the first time that ectopic expression of TUBA1A-R402C and TUBA1A-R402H patient alleles is sufficient to dominantly disrupt cortical neuronal migration in the developing mouse brain, strongly supporting a causal role in the pathology of brain malformation. To isolate the precise molecular impact of R402 mutations, we generated analogous R402C and R402H mutations in budding yeast α-tubulin, which exhibit a simplified microtubule cytoskeleton. We find that R402 mutant tubulins assemble into microtubules that support normal kinesin motor activity but fail to support the activity of dynein motors. Importantly, the level of dynein impairment scales with the expression level of the mutant in the cell, suggesting a 'poisoning' mechanism in which R402 mutant α-tubulin acts dominantly by populating microtubules with defective binding sites for dynein. Based on our results, we propose a new model for the molecular pathology of tubulinopathies that may also extend to other tubulin-related neuropathies.
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http://dx.doi.org/10.1093/hmg/ddy416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452179PMC
April 2019

Regulation of microtubule dynamic instability by the carboxy-terminal tail of β-tubulin.

Life Sci Alliance 2018 May 19;1(2). Epub 2018 Apr 19.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA.

Dynamic instability is an intrinsic property of microtubules; however, we do not understand what domains of αβ-tubulins regulate this activity or how these regulate microtubule networks in cells. Here, we define a role for the negatively charged carboxy-terminal tail (CTT) domain of β-tubulin in regulating dynamic instability. By combining in vitro studies with purified mammalian tubulin and in vivo studies with tubulin mutants in budding yeast, we demonstrate that β-tubulin CTT inhibits microtubule stability and regulates the structure and stability of microtubule plus ends. Tubulin that lacks β-tubulin CTT polymerizes faster and depolymerizes slower in vitro and forms microtubules that are more prone to catastrophe. The ends of these microtubules exhibit a more blunted morphology and rapidly switch to disassembly after tubulin depletion. In addition, we show that β-tubulin CTT is required for magnesium cations to promote depolymerization. We propose that β-tubulin CTT regulates the assembly of stable microtubule ends and provides a tunable mechanism to coordinate dynamic instability with ionic strength in the cell.
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http://dx.doi.org/10.26508/lsa.201800054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6022761PMC
May 2018

The -Tubulin gene in Brain Development: A Key Ingredient in the Neuronal Isotype Blend.

J Dev Biol 2017 Sep 19;5(3). Epub 2017 Sep 19.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, MS8108, 12801 E 17th Ave, Aurora, CO 80045, USA.

Microtubules are dynamic cytoskeletal polymers that mediate numerous, essential functions such as axon and dendrite growth and neuron migration throughout brain development. In recent years, sequencing has revealed dominant mutations that disrupt the tubulin protein building blocks of microtubules. These tubulin mutations lead to a spectrum of devastating brain malformations, complex neurological and physical phenotypes, and even fatality. The most common tubulin gene mutated is the α-tubulin gene , which is the most prevalent α-tubulin gene expressed in post-mitotic neurons. The normal role of TUBA1A during neuronal maturation, and how mutations alter its function to produce the phenotypes observed in patients, remains unclear. This review synthesizes current knowledge of TUBA1A function and expression during brain development, and the brain malformations caused by mutations in .
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http://dx.doi.org/10.3390/jdb5030008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648057PMC
September 2017

Dynein is regulated by the stability of its microtubule track.

J Cell Biol 2017 07 1;216(7):2047-2058. Epub 2017 Jun 1.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO

How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, we discover that spindle movement is regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. We find that dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150 Our results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. We propose that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning.
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http://dx.doi.org/10.1083/jcb.201611105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5496616PMC
July 2017

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast.

J Vis Exp 2017 04 20(122). Epub 2017 Apr 20.

Department of Cell and Developmental Biology, University of Colorado School of Medicine;

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.
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http://dx.doi.org/10.3791/55610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462104PMC
April 2017

Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells.

Nat Commun 2016 08 3;7:12426. Epub 2016 Aug 3.

Department of Cell and Developmental Biology, School of Medicine, Anschutz Medical Campus, University of Colorado Denver, Aurora, Colorado 80045, USA.

Coordinated polarization of epithelial cells is a key step during morphogenesis that leads to the formation of an apical lumen. Rab11 and its interacting protein FIP5 are necessary for the targeting of apical endosomes to the midbody and apical membrane initiation site (AMIS) during lumenogenesis. However, the machinery that mediates AMIS establishment and FIP5-endosome targeting remains unknown. Here we identify a FIP5-interacting protein, Cingulin, which localizes to the AMIS and functions as a tether mediating FIP5-endosome targeting. We analysed the machinery mediating AMIS recruitment to the midbody and determined that both branched actin and microtubules are required for establishing the site of the nascent lumen. We demonstrate that the Rac1-WAVE/Scar complex mediates Cingulin recruitment to the AMIS by inducing branched actin formation, and that Cingulin directly binds to microtubule C-terminal tails through electrostatic interactions. We propose a new mechanism for apical endosome targeting and AMIS formation around the midbody during epithelial lumenogenesis.
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http://dx.doi.org/10.1038/ncomms12426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976216PMC
August 2016

The negatively charged carboxy-terminal tail of β-tubulin promotes proper chromosome segregation.

Mol Biol Cell 2016 06 6;27(11):1786-96. Epub 2016 Apr 6.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045

Despite the broadly conserved role of microtubules in chromosome segregation, we have a limited understanding of how molecular features of tubulin proteins contribute to the underlying mechanisms. Here we investigate the negatively charged carboxy-terminal tail domains (CTTs) of α- and β-tubulins, using a series of mutants that alter or ablate CTTs in budding yeast. We find that ablating β-CTT causes elevated rates of chromosome loss and cell cycle delay. Complementary live-cell imaging and electron tomography show that β-CTT is necessary to properly position kinetochores and organize microtubules within the assembling spindle. We identify a minimal region of negatively charged amino acids that is necessary and sufficient for proper chromosome segregation and provide evidence that this function may be conserved across species. Our results provide the first in vivo evidence of a specific role for tubulin CTTs in chromosome segregation. We propose that β-CTT promotes the ordered segregation of chromosomes by stabilizing the spindle and contributing to forces that move chromosomes toward the spindle poles.
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http://dx.doi.org/10.1091/mbc.E15-05-0300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884069PMC
June 2016

Novel α-tubulin mutation disrupts neural development and tubulin proteostasis.

Dev Biol 2016 Jan 30;409(2):406-19. Epub 2015 Nov 30.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA. Electronic address:

Mutations in the microtubule cytoskeleton are linked to cognitive and locomotor defects during development, and neurodegeneration in adults. How these mutations impact microtubules, and how this alters function at the level of neurons is an important area of investigation. Using a forward genetic screen in mice, we identified a missense mutation in Tuba1a α-tubulin that disrupts cortical and motor neuron development. Homozygous mutant mice exhibit cortical dysgenesis reminiscent of human tubulinopathies. Motor neurons fail to innervate target muscles in the limbs and show synapse defects at proximal targets. To directly examine effects on tubulin function, we created analogous mutations in the α-tubulin isotypes in budding yeast. These mutations sensitize yeast cells to microtubule stresses including depolymerizing drugs and low temperatures. Furthermore, we find that mutant α-tubulin is depleted from the cell lysate and from microtubules, thereby altering ratios of α-tubulin isotypes. Tubulin-binding cofactors suppress the effects of the mutation, indicating an important role for these cofactors in regulating the quality of the α-tubulin pool. Together, our results give new insights into the functions of Tuba1a, mechanisms for regulating tubulin proteostasis, and how compromising these may lead to neural defects.
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http://dx.doi.org/10.1016/j.ydbio.2015.11.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4724489PMC
January 2016

Tubulin cofactors and Arl2 are cage-like chaperones that regulate the soluble αβ-tubulin pool for microtubule dynamics.

Elife 2015 Jul 24;4. Epub 2015 Jul 24.

Department of Molecular Cellular Biology, University of California, Davis, Davis, United States.

Microtubule dynamics and polarity stem from the polymerization of αβ-tubulin heterodimers. Five conserved tubulin cofactors/chaperones and the Arl2 GTPase regulate α- and β-tubulin assembly into heterodimers and maintain the soluble tubulin pool in the cytoplasm, but their physical mechanisms are unknown. Here, we reconstitute a core tubulin chaperone consisting of tubulin cofactors TBCD, TBCE, and Arl2, and reveal a cage-like structure for regulating αβ-tubulin. Biochemical assays and electron microscopy structures of multiple intermediates show the sequential binding of αβ-tubulin dimer followed by tubulin cofactor TBCC onto this chaperone, forming a ternary complex in which Arl2 GTP hydrolysis is activated to alter αβ-tubulin conformation. A GTP-state locked Arl2 mutant inhibits ternary complex dissociation in vitro and causes severe defects in microtubule dynamics in vivo. Our studies suggest a revised paradigm for tubulin cofactors and Arl2 functions as a catalytic chaperone that regulates soluble αβ-tubulin assembly and maintenance to support microtubule dynamics.
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http://dx.doi.org/10.7554/eLife.08811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574351PMC
July 2015

Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails.

Curr Biol 2014 Jun 15;24(12):1295-1303. Epub 2014 May 15.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045, USA. Electronic address:

Background: Microtubules (MTs) support diverse transport and force generation processes in cells. Both α- and β-tubulin proteins possess carboxy-terminal tail regions (CTTs) that are negatively charged, intrinsically disordered, and project from the MT surface where they interact with motors and other proteins. Although CTTs are presumed to play important roles in MT networks, these roles have not been determined in vivo.

Results: We examined the function of CTTs in vivo by using a systematic collection of mutants in budding yeast. We find that CTTs are not essential; however, loss of either α- or β-CTT sensitizes cells to MT-destabilizing drugs. β-CTT, but not α-CTT, regulates MT dynamics by increasing frequencies of catastrophe and rescue events. In addition, β-CTT is critical for the assembly of the mitotic spindle and its elongation during anaphase. We use genome-wide genetic interaction screens to identify roles for α- and β-CTTs, including a specific role for β-CTT in supporting kinesin-5/Cin8. Our genetic screens also identified novel interactions with pathways not related to canonical MT functions.

Conclusions: We conclude that α- and β-CTTs play important and largely discrete roles in MT networks. β-CTT promotes MT dynamics. β-CTT also regulates force generation in the mitotic spindle by supporting kinesin-5/Cin8 and dampening dynein. Our genetic screens identify links between α- and β-CTT and additional cellular pathways and suggest novel functions.
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http://dx.doi.org/10.1016/j.cub.2014.03.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4070440PMC
June 2014

Stopped in its tracks: negative regulation of the dynein motor by the yeast protein She1.

Authors:
Jeffrey K Moore

Bioessays 2013 Aug 13;35(8):677-82. Epub 2013 May 13.

Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO, USA.

How do cells direct the microtubule motor protein dynein to move cellular components to the right place at the right time? Recent studies in budding yeast shed light on a new mechanism for directing dynein, involving the protein She1. She1 restricts where and when dynein moves the nucleus and mitotic spindle. Experiments with purified proteins show that She1 binds to microtubules and inhibits dynein by stalling the motor on its track. Here I describe what we have learned so far about She1, based on a combination of genetic, cell biology, and biophysical approaches. These findings set the stage for further interrogation of the She1 mechanism, and raise the question of whether similar mechanisms exist in other species.
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http://dx.doi.org/10.1002/bies.201300016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3893767PMC
August 2013

Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

PLoS One 2013 5;8(4):e59453. Epub 2013 Apr 5.

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA.

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059453PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3618186PMC
October 2013

Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning.

Mol Biol Cell 2011 Aug 1;22(15):2690-701. Epub 2011 Jun 1.

Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110, USA.

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.
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http://dx.doi.org/10.1091/mbc.E11-01-0075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145545PMC
August 2011

The spindle position checkpoint is coordinated by the Elm1 kinase.

J Cell Biol 2010 Nov;191(3):493-503

Department of Cell Biology and Physiology, Washington University, St. Louis, MO 63110, USA.

How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother-daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization.
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http://dx.doi.org/10.1083/jcb.201006092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003319PMC
November 2010

Coordinating mitosis with cell polarity: Molecular motors at the cell cortex.

Semin Cell Dev Biol 2010 May 28;21(3):283-9. Epub 2010 Jan 28.

Dept of Cell Biology and Physiology, Washington University, St Louis, MO 63110, United States.

In many cell divisions, the position of the spindle apparatus is coordinated with polarity signals at the cell cortex so that copies of the genome are delivered to regions of the cell that are designated for differential inheritance by the two progeny. To coordinate spindle position with cell polarity, the spindle interfaces with elements on the cortex, where molecular motors often produce the forces that power displacement. Here we describe the molecular pathways by which cortical motors translocate the spindle in budding yeast, where the mechanisms are understood relatively well, and we compare these pathways to spindle positioning processes in metazoan systems, where the molecular details are less well understood.
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http://dx.doi.org/10.1016/j.semcdb.2010.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2844471PMC
May 2010

The spindle position checkpoint requires positional feedback from cytoplasmic microtubules.

Curr Biol 2009 Dec 12;19(23):2026-30. Epub 2009 Nov 12.

Department of Cell Biology and Physiology, Washington University in St. Louis, St. Louis, MO 63110, USA.

The objective of mitosis is to provide a copy of the genome to each progeny of a cell division. This requires the separation of duplicate chromatids by the spindle apparatus and the delivery of one set of chromosomes to each of the daughter cells. In budding yeast, the fidelity of chromosome delivery depends on the spindle position checkpoint, which prolongs mitosis until one end of the anaphase spindle arrives in the bud. Here we tested the hypothesis that the activity of the spindle position checkpoint depends on persistent interactions between cytoplasmic microtubules and the mother-bud neck, the future site of cytokinesis. We used laser ablation to disrupt microtubule interactions with the bud neck, and we found that loss of microtubules from the neck leads to mitotic exit in a majority of checkpoint-activated cells. Our findings suggest that cytoplasmic microtubules are used to monitor the location of the spindle in the dividing cell and, in the event of positioning errors, relay a signal to inhibit mitotic exit until the spindle is appropriately positioned.
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http://dx.doi.org/10.1016/j.cub.2009.10.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805762PMC
December 2009

Function of dynein in budding yeast: mitotic spindle positioning in a polarized cell.

Cell Motil Cytoskeleton 2009 Aug;66(8):546-55

Department of Cell Biology and Physiology, Washington University, St Louis, Missouri, USA.

Cytoplasmic dynein is a microtubule motor that powers minus-end-directed motility in a variety of biological settings. The budding yeast, Saccharomyces cerevisiae, has been a useful system for the study of dynein, due to its molecular genetics and cell biology capabilities, coupled with the conservation of dynein-pathway proteins. In this review we discuss how budding yeast use dynein to manipulate the position of the mitotic spindle and the nucleus during cell division, using cytoplasmic microtubules, and we describe our current understanding of the genes required for dynein function. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
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http://dx.doi.org/10.1002/cm.20364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2746759PMC
August 2009

Neurodegeneration mutations in dynactin impair dynein-dependent nuclear migration.

Proc Natl Acad Sci U S A 2009 Mar 11;106(13):5147-52. Epub 2009 Mar 11.

Department of Cell Biology and Physiology, Washington University, Saint Louis, MO 63110, USA.

Neurodegenerative disease in humans and mice can be caused by mutations affecting the microtubule motor dynein or its biochemical regulator, dynactin, a multiprotein complex required for dynein function (1-4). A single amino acid change, G59S, in the conserved cytoskeletal-associated protein glycine-rich (CAP-Gly) domain of the p150(glued) subunit of dynactin can cause motor neuron degeneration in humans and mice, which resembles ALS (2, 5-8). The molecular mechanism by which G59S impairs the function of dynein is not understood. Also, the relevance of the CAP-Gly domain for dynein motility has not been demonstrated in vivo. Here, we generate a mutant that is analogous to G59S in budding yeast, and show that this mutation produces a highly specific phenotype related to dynein function. The effect of the point mutation is identical to that of complete loss of the CAP-Gly domain. Our results demonstrate that the CAP-Gly domain has a critical role in the initiation and persistence of dynein-dependent movement of the mitotic spindle and nucleus, but it is otherwise dispensable for dynein-based movement. The need for this function appears to be context-dependent, and we speculate that CAP-Gly activity may only be necessary when dynein needs to overcome high force thresholds to produce movement.
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http://dx.doi.org/10.1073/pnas.0810828106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2664072PMC
March 2009

Dynactin function in mitotic spindle positioning.

Traffic 2008 Apr 22;9(4):510-27. Epub 2008 Jan 22.

Department of Cell Biology and Physiology, Washington University, Saint Louis, MO 63110, USA.

Dynactin is a multisubunit protein complex necessary for dynein function. Here, we investigated the function of dynactin in budding yeast. Loss of dynactin impaired movement and positioning of the mitotic spindle, similar to loss of dynein. Dynactin subunits required for function included p150(Glued), dynamitin, actin-related protein (Arp) 1 and p24. Arp10 and capping protein were dispensable, even in combination. All dynactin subunits tested localized to dynamic plus ends of cytoplasmic microtubules, to stationary foci on the cell cortex and to spindle pole bodies. The number of molecules of dynactin in those locations was small, less than five. In the absence of dynactin, dynein accumulated at plus ends and did not appear at the cell cortex, consistent with a role for dynactin in offloading dynein from the plus end to the cortex. Dynein at the plus end was necessary for dynactin plus-end targeting. p150(Glued) was the only dynactin subunit sufficient for plus-end targeting. Interactions among the subunits support a molecular model that resembles the current model for brain dynactin in many respects; however, three subunits at the pointed end of brain dynactin appear to be absent from yeast.
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http://dx.doi.org/10.1111/j.1600-0854.2008.00710.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2367371PMC
April 2008

Vehicle-controlled, double-blind, randomized study of imiquimod 5% cream applied 3 days per week in one or two courses of treatment for actinic keratoses on the head.

J Am Acad Dermatol 2007 Aug 18;57(2):265-8. Epub 2007 May 18.

Wake Forest University School of Medicine, Department of Dermatology, Medical Center Blvd, Winston-Salem, NC 27157, USA.

Background: A shorter dosing regimen of imiquimod for the treatment of actinic keratosis may be effective, with long-term clinical benefits.

Objective: Imiquimod in one or two shorter courses of treatment was evaluated.

Methods: Patients with actinic keratosis lesions on the head applied imiquimod or vehicle cream 3x/wk for 4 weeks (course 1). Patients with remaining lesions received another course of treatment. Complete and partial clearance rates were evaluated after course 1, after course 2 (overall), and 1 year later.

Results: Complete clearance rates were 26.8% (course 1) and 53.7% (overall). Partial clearance rates were 36.6% (course 1) and 61.0% (overall). One-year follow-up recurrence rates were 39% (imiquimod) and 57% (vehicle).

Limitations: Blinded investigators may have been biased toward patients treated with imiquimod identified by treatment site reactions.

Conclusion: Imiquimod 3x/wk in one or two courses of treatment appears to be effective for the treatment of actinic keratoses on the head, providing long-term clinical benefits. Some recurrences do occur, so long-term follow-up is recommended.
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http://dx.doi.org/10.1016/j.jaad.2007.01.047DOI Listing
August 2007

Retinal breaks observed during pars plana vitrectomy.

Am J Ophthalmol 2007 Jul 23;144(1):32-36. Epub 2007 May 23.

Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida 33101, USA.

Purpose: To quantitate the frequency and features of retinal breaks discovered at the time of vitrectomy and to evaluate the outcomes with prophylactic treatment.

Design: A consecutive, single-surgeon, retrospective, observational case series from a two-year period.

Methods: Medical records were reviewed for all patients who underwent primary, standard, three-port pars plana vitrectomy (PPV) between January 1, 2000, and December 31, 2001. Intraoperative findings recorded included the number, location, and categorization of retinal breaks and their method of management. Postoperative features recorded included the presence or absence of a retinal detachment (RD).

Results: There were 65 retinal breaks found in 48 (11.6%) of 415 eyes and included 30 (7.2%) eyes with definite breaks, nine (2.2%) with suspicious breaks, and nine (2.2%) with probably preexisting breaks. Breaks that were described as being large (n = 5) were more commonly associated with the right-hand sclerotomy (P = .041), although other categories of breaks were not. After surgery, the overall incidence of RD was 2.2% (nine of 415 eyes). The rate of RD among the 48 eyes with retinal breaks (of any category) was also 2.1% (one eye). All RDs in this series occurred more than three months after initial vitrectomy and, accordingly, were probably unrelated to retinal breaks that occurred during surgery.

Conclusions: Recognition of retinal breaks and intraoperative treatment with retinopexy and air-fluid exchange during vitrectomy reduces the postoperative risk of RD to that among eyes without observed intraoperative retinal breaks.
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http://dx.doi.org/10.1016/j.ajo.2007.03.048DOI Listing
July 2007

The cyclin-dependent kinase Cdc28p regulates multiple aspects of Kar9p function in yeast.

Mol Biol Cell 2007 Apr 24;18(4):1187-202. Epub 2007 Jan 24.

Department of Biology, University of Rochester, Rochester, NY 14627, USA.

During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.
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http://dx.doi.org/10.1091/mbc.e06-04-0360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1838993PMC
April 2007