Publications by authors named "Richard J McKenney"

31 Publications

New insights into the mechanism of dynein motor regulation by lissencephaly-1.

Elife 2020 07 21;9. Epub 2020 Jul 21.

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

Lissencephaly ('smooth brain') is a severe brain disease associated with numerous symptoms, including cognitive impairment, and shortened lifespan. The main causative gene of this disease - lissencephaly-1 (LIS1) - has been a focus of intense scrutiny since its first identification almost 30 years ago. LIS1 is a critical regulator of the microtubule motor cytoplasmic dynein, which transports numerous cargoes throughout the cell, and is a key effector of nuclear and neuronal transport during brain development. Here, we review the role of LIS1 in cellular dynein function and discuss recent key findings that have revealed a new mechanism by which this molecule influences dynein-mediated transport. In addition to reconciling prior observations with this new model for LIS1 function, we also discuss phylogenetic data that suggest that LIS1 may have coevolved with an autoinhibitory mode of cytoplasmic dynein regulation.
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http://dx.doi.org/10.7554/eLife.59737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373426PMC
July 2020

Absence of SCAPER causes male infertility in humans and by modulating microtubule dynamics during meiosis.

J Med Genet 2020 Jun 11. Epub 2020 Jun 11.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Background: Mutation () have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies.

Methods: Human clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of () ( orthologue) CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy.

Results: We show that patients homozygous for a mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells Interestingly, a null mutants for the ubiquitously expressed gene are viable and female fertile but male sterile. We further show that male sterility in null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs.

Conclusions: Our results show that null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and meiosis.
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http://dx.doi.org/10.1136/jmedgenet-2020-106946DOI Listing
June 2020

LIS1 cracks open dynein.

Nat Cell Biol 2020 05;22(5):515-517

Department of Molecular and Cellular Biology, University of California - Davis, Davis, CA, USA.

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http://dx.doi.org/10.1038/s41556-020-0500-5DOI Listing
May 2020

A Combinatorial MAP Code Dictates Polarized Microtubule Transport.

Dev Cell 2020 04 27;53(1):60-72.e4. Epub 2020 Feb 27.

Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616, USA. Electronic address:

Many eukaryotic cells distribute their intracellular components asymmetrically through regulated active transport driven by molecular motors along microtubule tracks. While intrinsic and extrinsic regulation of motor activity exists, what governs the overall distribution of activated motor-cargo complexes within cells remains unclear. Here, we utilize in vitro reconstitution of purified motor proteins and non-enzymatic microtubule-associated proteins (MAPs) to demonstrate that MAPs exhibit distinct influences on the motility of the three main classes of transport motors: kinesin-1, kinesin-3, and cytoplasmic dynein. Further, we dissect how combinations of MAPs affect motors and unveil MAP9 as a positive modulator of kinesin-3 motility. From these data, we propose a general "MAP code" that has the capacity to strongly bias directed movement along microtubules and helps elucidate the intricate intracellular sorting observed in highly polarized cells such as neurons.
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http://dx.doi.org/10.1016/j.devcel.2020.01.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181406PMC
April 2020

The kinesin-5 tail domain directly modulates the mechanochemical cycle of the motor domain for anti-parallel microtubule sliding.

Elife 2020 Jan 20;9. Epub 2020 Jan 20.

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

Kinesin-5 motors organize mitotic spindles by sliding apart microtubules. They are homotetramers with dimeric motor and tail domains at both ends of a bipolar minifilament. Here, we describe a regulatory mechanism involving direct binding between tail and motor domains and its fundamental role in microtubule sliding. Kinesin-5 tails decrease microtubule-stimulated ATP-hydrolysis by specifically engaging motor domains in the nucleotide-free or ADP states. Cryo-EM reveals that tail binding stabilizes an open motor domain ATP-active site. Full-length motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering. The tail is critical for motors to zipper together two microtubules by generating substantial sliding forces. The tail is essential for mitotic spindle localization, which becomes severely reduced in tail-deleted motors. Our studies suggest a revised microtubule-sliding model, in which kinesin-5 tails stabilize motor domains in the microtubule-bound state by slowing ATP-binding, resulting in high-force production at both homotetramer ends.
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http://dx.doi.org/10.7554/eLife.51131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015671PMC
January 2020

The tail wags the motor.

Nat Chem Biol 2019 11;15(11):1033-1034

Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA, USA.

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http://dx.doi.org/10.1038/s41589-019-0367-6DOI Listing
November 2019

Microtubules gate tau condensation to spatially regulate microtubule functions.

Nat Cell Biol 2019 09 2;21(9):1078-1085. Epub 2019 Sep 2.

Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA, USA.

Tau is an abundant microtubule-associated protein in neurons. Tau aggregation into insoluble fibrils is a hallmark of Alzheimer's disease and other types of dementia, yet the physiological state of tau molecules within cells remains unclear. Using single-molecule imaging, we directly observe that the microtubule lattice regulates reversible tau self-association, leading to localized, dynamic condensation of tau molecules on the microtubule surface. Tau condensates form selectively permissible barriers, spatially regulating the activity of microtubule-severing enzymes and the movement of molecular motors through their boundaries. We propose that reversible self-association of tau molecules, gated by the microtubule lattice, is an important mechanism of the biological functions of tau, and that oligomerization of tau is a common property shared between the physiological and disease-associated forms of the molecule.
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http://dx.doi.org/10.1038/s41556-019-0375-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748660PMC
September 2019

Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors.

Proc Natl Acad Sci U S A 2019 09 27;116(37):18429-18434. Epub 2019 Aug 27.

Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, 980-0845 Miyagi, Japan;

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations in are associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for how mutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found that mutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into the homolog revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.
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http://dx.doi.org/10.1073/pnas.1905690116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744892PMC
September 2019

Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH.

J Biol Chem 2019 05 16;294(22):8779-8790. Epub 2019 Apr 16.

From the Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, California 94143,

Tau, a member of the MAP2/tau family of microtubule-associated proteins, stabilizes and organizes axonal microtubules in healthy neurons. In neurodegenerative tauopathies, tau dissociates from microtubules and forms neurotoxic extracellular aggregates. MAP2/tau family proteins are characterized by three to five conserved, intrinsically disordered repeat regions that mediate electrostatic interactions with the microtubule surface. Here, we used molecular dynamics, microtubule-binding experiments, and live-cell microscopy, revealing that highly-conserved histidine residues near the C terminus of each microtubule-binding repeat are pH sensors that can modulate tau-microtubule interaction strength within the physiological intracellular pH range. We observed that at low pH (<7.5), these histidines are positively charged and interact with phenylalanine residues in a hydrophobic cleft between adjacent tubulin dimers. At higher pH (>7.5), tau deprotonation decreased binding to microtubules both and in cells. Electrostatic and hydrophobic characteristics of histidine were both required for tau-microtubule binding, as substitutions with constitutively and positively charged nonaromatic lysine or uncharged alanine greatly reduced or abolished tau-microtubule binding. Consistent with these findings, tau-microtubule binding was reduced in a cancer cell model with increased intracellular pH but was rapidly restored by decreasing the pH to normal levels. These results add detailed insights into the intracellular regulation of tau activity that may be relevant in both normal and pathological conditions.
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http://dx.doi.org/10.1074/jbc.RA118.007004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6552421PMC
May 2019

Cdt1 stabilizes kinetochore-microtubule attachments via an Aurora B kinase-dependent mechanism.

J Cell Biol 2018 10 28;217(10):3446-3463. Epub 2018 Aug 28.

Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL

Robust kinetochore-microtubule (kMT) attachment is critical for accurate chromosome segregation. G2/M-specific depletion of human Cdt1 that localizes to kinetochores in an Ndc80 complex-dependent manner leads to abnormal kMT attachments and mitotic arrest. This indicates an independent mitotic role for Cdt1 in addition to its prototypic function in DNA replication origin licensing. Here, we show that Cdt1 directly binds to microtubules (MTs). Endogenous or transiently expressed Cdt1 localizes to both mitotic spindle MTs and kinetochores. Deletion mapping of Cdt1 revealed that the regions comprising the middle and C-terminal winged-helix domains but lacking the N-terminal unstructured region were required for efficient MT binding. Mitotic kinase Aurora B interacts with and phosphorylates Cdt1. Aurora B-phosphomimetic Cdt1 exhibited attenuated MT binding, and its cellular expression induced defective kMT attachments with a concomitant delay in mitotic progression. Thus we provide mechanistic insight into how Cdt1 affects overall kMT stability in an Aurora B kinase phosphorylation-dependent manner; which is envisioned to augment the MT-binding of the Ndc80 complex.
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http://dx.doi.org/10.1083/jcb.201705127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168275PMC
October 2018

Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin.

Dev Cell 2018 07;46(2):204-218.e7

Department of Biology, Drexel University, Philadelphia, PA 19104, USA. Electronic address:

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that, during entry into dendrites, axonally destined cargos move with a retrograde bias toward the cell body, while dendritically destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor cargo further into dendrites. In neurons and in vitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysine-rich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axons-dendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.
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http://dx.doi.org/10.1016/j.devcel.2018.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396981PMC
July 2018

Magnetic Cytoskeleton Affinity Purification of Microtubule Motors Conjugated to Quantum Dots.

Bioconjug Chem 2018 07 3;29(7):2278-2286. Epub 2018 Jul 3.

Department of Molecular Physiology and Biophysics , University of Vermont , Burlington , Vermont 05405 , United States.

We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 μg protein, with yield of ∼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452869PMC
July 2018

Author Correction: Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility.

Nat Struct Mol Biol 2018 04;25(4):355

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.

In the version of this article initially published online, an incorrect accession code, EMD-5NW4, was introduced on page 1 of the article PDF, in section 'BICD2N mediates the association of two dynein dimers with a single dynactin'. This has been corrected to PDB 5NW4. The error has been corrected in the PDF and HTML versions of this article.
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http://dx.doi.org/10.1038/s41594-018-0043-7DOI Listing
April 2018

Antagonism between the dynein and Ndc80 complexes at kinetochores controls the stability of kinetochore-microtubule attachments during mitosis.

J Biol Chem 2018 04 23;293(16):5755-5765. Epub 2018 Feb 23.

From the Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and

Chromosome alignment and segregation during mitosis require kinetochore-microtubule (kMT) attachments that are mediated by the molecular motor dynein and the kMT-binding complex Ndc80. The Rod-ZW10-Zwilch (RZZ) complex is central to this coordination as it has an important role in dynein recruitment and has recently been reported to have a key function in the regulation of stable kMT attachments in besides its role in activating the spindle assembly checkpoint (SAC). However, the mechanism by which these protein complexes control kMT attachments to drive chromosome motility during early mitosis is still unclear. Here, using total internal reflection fluorescence microscopy, we observed that higher concentrations of Ndc80 inhibited dynein binding to MTs, providing evidence that Ndc80 and dynein antagonize each other's function. High-resolution microscopy and siRNA-mediated functional disruption revealed that severe defects in chromosome alignment induced by depletion of dynein or the dynein adapter Spindly are rescued by codepletion of the RZZ component Rod in human cells. Interestingly, rescue of the chromosome alignment defects was independent of Rod function in SAC activation and was accompanied by a remarkable restoration of stable kMT attachments. Furthermore, the chromosome alignment rescue depended on the plus-end-directed motility of centromere protein E (CENP-E) because cells codepleted of CENP-E, Rod, and dynein could not establish stable kMT attachments or align their chromosomes properly. Our findings support the idea that dynein may control the function of the Ndc80 complex in stabilizing kMT attachments directly by interfering with Ndc80-MT binding or indirectly by controlling the Rod-mediated inhibition of Ndc80.
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http://dx.doi.org/10.1074/jbc.RA117.001699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912454PMC
April 2018

Cryo-electron tomography reveals that dynactin recruits a team of dyneins for processive motility.

Nat Struct Mol Biol 2018 03 7;25(3):203-207. Epub 2018 Feb 7.

Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA.

Cytoplasmic dynein is a protein complex that transports molecular cargo along microtubules (MTs), playing a key role in the intracellular trafficking network. Vertebrate dynein's movement becomes strikingly enhanced upon interacting with dynactin and a cargo adaptor such as BicaudalD2. However, the mechanisms responsible for increased transport activity are not well understood, largely owing to limited structural information. We used cryo-electron tomography (cryo-ET) to visualize the 3D structure of the MT-bound dynein-dynactin complex from Mus musculus and show that the dynactin-cargo adaptor complex binds two dimeric dyneins. This configuration imposes spatial and conformational constraints on both dynein dimers, positioning the four motor domains in proximity to one another and oriented toward the MT minus end. We propose that grouping multiple dyneins onto a single dynactin scaffold promotes collective force production, increased processivity, and unidirectional movement, suggesting mechanistic parallels to axonemal dynein. These findings provide structural insights into a previously unknown mechanism for dynein regulation.
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http://dx.doi.org/10.1038/s41594-018-0027-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969528PMC
March 2018

Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization.

Dev Cell 2018 01;44(2):233-247.e4

Department of Molecular and Cellular Biology, University of California - Davis, Davis, CA 95616, USA. Electronic address:

Cytoplasmic dynein-1 is a minus-end-directed motor protein that transports cargo over long distances and organizes the intracellular microtubule (MT) network. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, resulting in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, suggesting that minus-end accumulations of motors produce network-scale contractile stresses. Our data provide a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the cytoskeleton, for example, during mitotic spindle assembly.
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http://dx.doi.org/10.1016/j.devcel.2017.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082141PMC
January 2018

Differential effects of the dynein-regulatory factor Lissencephaly-1 on processive dynein-dynactin motility.

J Biol Chem 2017 07 2;292(29):12245-12255. Epub 2017 Jun 2.

From the Department of Molecular and Cellular Biology, University of California-Davis, Davis, California 95616 and

Cytoplasmic dynein is the primary minus-end-directed microtubule motor protein in animal cells, performing a wide range of motile activities, including transport of vesicular cargos, mRNAs, viruses, and proteins. Lissencephaly-1 (LIS1) is a highly conserved dynein-regulatory factor that binds directly to the dynein motor domain, uncoupling the enzymatic and mechanical cycles of the motor and stalling dynein on the microtubule track. Dynactin, another ubiquitous dynein-regulatory factor, releases dynein from an autoinhibited state, leading to a dramatic increase in fast, processive dynein motility. How these opposing activities are integrated to control dynein motility is unknown. Here, we used fluorescence single-molecule microscopy to study the interaction of LIS1 with the processive dynein-dynactin-BicD2N (DDB) complex. Surprisingly, in contrast to the prevailing model for LIS1 function established in the context of dynein alone, we found that binding of LIS1 to DDB does not strongly disrupt processive motility. Motile DDB complexes bound up to two LIS1 dimers, and mutational analysis suggested that LIS1 binds directly to the dynein motor domains during DDB movement. Interestingly, LIS1 enhanced DDB velocity in a concentration-dependent manner, in contrast to observations of the effect of LIS1 on the motility of isolated dynein. Thus, LIS1 exerts concentration-dependent effects on dynein motility and can synergize with dynactin to enhance processive dynein movement. Our results suggest that the effect of LIS1 on dynein motility depends on both LIS1 concentration and the presence of other regulatory factors such as dynactin and may provide new insights into the mechanism of LIS1 haploinsufficiency in the neurodevelopmental disorder lissencephaly.
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http://dx.doi.org/10.1074/jbc.M117.790048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519373PMC
July 2017

Phosphorylation of β-Tubulin by the Down Syndrome Kinase, Minibrain/DYRK1a, Regulates Microtubule Dynamics and Dendrite Morphogenesis.

Neuron 2016 05 21;90(3):551-63. Epub 2016 Apr 21.

Department of Physiology and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address:

Dendritic arborization patterns are consistent anatomical correlates of genetic disorders such as Down syndrome (DS) and autism spectrum disorders (ASDs). In a screen for abnormal dendrite development, we identified Minibrain (MNB)/DYRK1a, a kinase implicated in DS and ASDs, as a regulator of the microtubule cytoskeleton. We show that MNB is necessary to establish the length and cytoskeletal composition of terminal dendrites by controlling microtubule growth. Altering MNB levels disrupts dendrite morphology and perturbs neuronal electrophysiological activity, resulting in larval mechanosensation defects. Using in vivo and in vitro approaches, we uncover a molecular pathway whereby direct phosphorylation of β-tubulin by MNB inhibits tubulin polymerization, a function that is conserved for mammalian DYRK1a. Our results demonstrate that phosphoregulation of microtubule dynamics by MNB/DYRK1a is critical for dendritic patterning and neuronal function, revealing a previously unidentified mode of posttranslational microtubule regulation in neurons and uncovering a conserved pathway for a DS- and ASD-associated kinase.
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http://dx.doi.org/10.1016/j.neuron.2016.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4860041PMC
May 2016

Tyrosination of α-tubulin controls the initiation of processive dynein-dynactin motility.

EMBO J 2016 06 11;35(11):1175-85. Epub 2016 Mar 11.

Department of Cellular and Molecular Pharmacology, the Howard Hughes Medical Institute University of California, San Francisco, CA, USA

Post-translational modifications (PTMs) of α/β-tubulin are believed to regulate interactions with microtubule-binding proteins. A well-characterized PTM involves in the removal and re-ligation of the C-terminal tyrosine on α-tubulin, but the purpose of this tyrosination-detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule-binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α-tubulin tyrosine facilitates initial motor-tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C-terminal α-tubulin tyrosine on dynein-dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein-driven motility in cells.
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http://dx.doi.org/10.15252/embj.201593071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888239PMC
June 2016

Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes.

Science 2014 Jul 19;345(6194):337-41. Epub 2014 Jun 19.

Department of Cellular and Molecular Pharmacology and the Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA.

Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.
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http://dx.doi.org/10.1126/science.1254198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224444PMC
July 2014

Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains.

Elife 2013 Sep 3;2:e00943. Epub 2013 Sep 3.

Department Cellular and Molecular Pharmacology , Howard Hughes Medical Institute, University of California, San Francisco , San Francisco , United States.

Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins. DOI:http://dx.doi.org/10.7554/eLife.00943.001.
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http://dx.doi.org/10.7554/eLife.00943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762337PMC
September 2013

Multiple modes of cytoplasmic dynein regulation.

Nat Cell Biol 2012 Feb 29;14(3):224-30. Epub 2012 Feb 29.

Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA.

In performing its multiple cellular functions, the cytoplasmic dynein motor is subject to complex regulation involving allosteric mechanisms within the dynein complex, as well as numerous extramolecular interactions controlling subcellular targeting and motor activity. Recent work has distinguished high- and low-load regulatory modes for cytoplasmic dynein, which, combined with a diversity of targeting mechanisms, accounts for a very broad range of functions.
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http://dx.doi.org/10.1038/ncb2420DOI Listing
February 2012

Mechanical stochastic tug-of-war models cannot explain bidirectional lipid-droplet transport.

Proc Natl Acad Sci U S A 2011 Nov 14;108(47):18960-5. Epub 2011 Nov 14.

Department of Neurobiology, Physiology and Behavior, University of California, Davis, CA 95616, USA.

Intracellular transport via the microtubule motors kinesin and dynein plays an important role in maintaining cell structure and function. Often, multiple kinesin or dynein motors move the same cargo. Their collective function depends critically on the single motors' detachment kinetics under load, which we experimentally measure here. This experimental constraint--combined with other experimentally determined parameters--is then incorporated into theoretical stochastic and mean-field models. Comparison of modeling results and in vitro data shows good agreement for the stochastic, but not mean-field, model. Many cargos in vivo move bidirectionally, frequently reversing course. Because both kinesin and dynein are present on the cargos, one popular hypothesis explaining the frequent reversals is that the opposite-polarity motors engage in unregulated stochastic tugs-of-war. Then, the cargos' motion can be explained entirely by the outcome of these opposite-motor competitions. Here, we use fully calibrated stochastic and mean-field models to test the tug-of-war hypothesis. Neither model agrees well with our in vivo data, suggesting that, in addition to inevitable tugs-of-war between opposite motors, there is an additional level of regulation not included in the models.
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http://dx.doi.org/10.1073/pnas.1107841108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223464PMC
November 2011

High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport.

J Cell Biol 2011 Oct;195(2):193-201

Department of Pathology and Cell Biology, Columbia University, New York, NY 10027, USA.

The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities. We used a novel approach to overcome these challenges, combining acute in vivo inhibition with automated high temporal and spatial resolution particle tracking. Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs. Acute LIS1 inhibition or LIS1 RNA interference had little effect on lysosomes/late endosomes but severely inhibited axonal transport of large, but not small, vesicular structures. Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo. Our data also reveal a specific but cell type-restricted role for LIS1 in large vesicular transport and provide the first quantitative support for a general role for LIS1 in high-load dynein functions.
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http://dx.doi.org/10.1083/jcb.201104076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198168PMC
October 2011

Mutually exclusive cytoplasmic dynein regulation by NudE-Lis1 and dynactin.

J Biol Chem 2011 Nov 12;286(45):39615-22. Epub 2011 Sep 12.

Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA.

Cytoplasmic dynein is responsible for a wide range of cellular roles. How this single motor protein performs so many functions has remained a major outstanding question for many years. Part of the answer is thought to lie in the diversity of dynein regulators, but how the effects of these factors are coordinated in vivo remains unexplored. We previously found NudE to bind dynein through its light chain 8 (LC8) and intermediate chain (IC) subunits (1), the latter of which also mediates the dynein-dynactin interaction (2). We report here that NudE and dynactin bind to a common region within the IC, and compete for this site. We find LC8 to bind to a novel sequence within NudE, without detectably affecting the dynein-NudE interaction. We further find that commonly used dynein inhibitory reagents have broad effects on the interaction of dynein with its regulatory factors. Together these results reveal an unanticipated mechanism for preventing dual regulation of individual dynein molecules, and identify the IC as a nexus for regulatory interactions within the dynein complex.
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http://dx.doi.org/10.1074/jbc.M111.289017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234784PMC
November 2011

LIS1 and NudE induce a persistent dynein force-producing state.

Cell 2010 Apr;141(2):304-14

Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.

Cytoplasmic dynein is responsible for many aspects of cellular and subcellular movement. LIS1, NudE, and NudEL are dynein interactors initially implicated in brain developmental disease but now known to be required in cell migration, nuclear, centrosomal, and microtubule transport, mitosis, and growth cone motility. Identification of a specific role for these proteins in cytoplasmic dynein motor regulation has remained elusive. We find that NudE stably recruits LIS1 to the dynein holoenzyme molecule, where LIS1 interacts with the motor domain during the prepowerstroke state of the dynein crossbridge cycle. NudE abrogates dynein force production, whereas LIS1 alone or with NudE induces a persistent-force dynein state that improves ensemble function of multiple dyneins for transport under high-load conditions. These results likely explain the requirement for LIS1 and NudE in the transport of nuclei, centrosomes, chromosomes, and the microtubule cytoskeleton as well as the particular sensitivity of migrating neurons to reduced LIS1 expression.
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http://dx.doi.org/10.1016/j.cell.2010.02.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881166PMC
April 2010

NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores.

J Cell Biol 2007 Aug 6;178(4):583-94. Epub 2007 Aug 6.

Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA.

NudE and NudEL are related proteins that interact with cytoplasmic dynein and LIS1. Their functional relationship and involvement in LIS1 and dynein regulation are not completely understood. We find that NudE and NudEL each localize to mitotic kinetochores before dynein, dynactin, ZW10, and LIS1 and exhibit additional temporal and spatial differences in distribution from the motor protein. Inhibition of NudE and NudEL caused metaphase arrest with misoriented chromosomes and defective microtubule attachment. Dynein and dynactin were both displaced from kinetochores by the injection of an anti-NudE/NudEL antibody. Dynein but not dynactin interacted with NudE surprisingly through the dynein intermediate and light chains but not the motor domain. Together, these results identify a common function for NudE and NudEL in mitotic progression and identify an alternative mechanism for dynein recruitment to and regulation at kinetochores.
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http://dx.doi.org/10.1083/jcb.200610112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064466PMC
August 2007

Expression patterns of LIS1, dynein and their interaction partners dynactin, NudE, NudEL and NudC in human gliomas suggest roles in invasion and proliferation.

Acta Neuropathol 2007 May 13;113(5):591-9. Epub 2007 Jan 13.

Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, 812-8582 Fukuoka, Japan.

Diffusely infiltrating gliomas are the most common type of primary intracranial neoplasm in humans. One of the major obstacles to the effective treatment of these tumors is their highly infiltrative growth. However, mechanisms controlling their migration and proliferation are poorly understood. Glioma cells resemble neural progenitors, and we hypothesize that gliomas recapitulate the capacity of migration and proliferation of progenitors that takes place during brain development. Based on recent evidence implicating cytoplasmic dynein and its regulatory proteins in neural progenitor migration and division, we conducted immunohistochemical evaluation of surgically resected human glioma samples for the presence and distribution of these proteins. We examined expression of LIS1, the gene responsible for type I lissencephaly, cytoplasmic dynein and the dynein- and LIS1-interacting factors dynactin, NudE/NudEL and NudC, which play significant roles in neural progenitor cell behavior. We found that each of these proteins is expressed in all histological types and grades of human neuroectodermal tumors examined. Immunohistochemical analysis revealed that the levels of expression varied from cell to cell within each tumor, ranging from very high to undetectable. This stands in contrast to the low levels of diffuse staining seen in non-neoplastic brain tissue. Of particular interest, we noted tumor cells infiltrating the white matter and tumor cells undergoing cell division amongst the cells with notably high expression levels. These findings are compatible with the idea that LIS1 and its interacting proteins play a role in glioma migration and proliferation analogous to their role during brain development.
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http://dx.doi.org/10.1007/s00401-006-0180-7DOI Listing
May 2007