The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore–microtubule interface in kinetoplastids.

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The unconventional kinetoplastid kinetochore protein KKT4 tracks with dynamic microtubule tips

10.1101/216812 ◽

2017 ◽

Cited By ~ 1

Author(s):

Aida Llauró ◽

Hanako Hayashi ◽

Megan E. Bailey ◽

Alex Wilson ◽

Patryk Ludzia ◽

...

Keyword(s):

Chromosome Segregation ◽

Binding Activity ◽

Load Bearing ◽

Kinetochore Protein ◽

Microtubule Binding ◽

Binding Domains ◽

Primary Element ◽

Microtubule Binding Domain ◽

Spindle Microtubules

AbstractKinetochores are multiprotein machines that drive chromosome segregation in all eukaryotes by maintaining persistent, load-bearing linkages between the chromosomes and the tips of dynamic spindle microtubules. Kinetochores in commonly studied eukaryotes are assembled from widely conserved components like the Ndc80 complex that directly binds microtubules. However, in evolutionarily-divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here we show that a kinetochore protein from T. brucei called KKT4 binds directly to microtubules, diffuses along the microtubule lattice, and tracks with disassembling microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a minimal microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Laser trapping experiments show that KKT4 can maintain load-bearing attachments to both growing and shortening microtubule tips. Thus, despite its lack of similarity to other known microtubule-binding proteins, KKT4 has key functions required for harnessing microtubule dynamics to drive chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.

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1H, 13C and 15N resonance assignments for the microtubule-binding domain of the kinetoplastid kinetochore protein KKT4 from Trypanosoma brucei

Biomolecular NMR Assignments ◽

10.1007/s12104-020-09968-1 ◽

2020 ◽

Vol 14 (2) ◽

pp. 309-315 ◽

Cited By ~ 1

Author(s):

Patryk Ludzia ◽

Bungo Akiyoshi ◽

Christina Redfield

Keyword(s):

Trypanosoma Brucei ◽

Resonance Assignments ◽

Binding Domain ◽

Kinetochore Protein ◽

Microtubule Binding ◽

Microtubule Binding Domain

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Modulation of tau phosphorylation and intracellular localization by cellular stress

Biochemical Journal ◽

10.1042/bj3450263 ◽

2000 ◽

Vol 345 (2) ◽

pp. 263-270 ◽

Cited By ~ 21

Author(s):

Scott M. JENKINS ◽

Marcus ZINNERMAN ◽

Craig GARNER ◽

Gail V. W. JOHNSON

Keyword(s):

Osmotic Stress ◽

Protein Kinases ◽

Intracellular Localization ◽

Tau Phosphorylation ◽

Binding Domain ◽

Microtubule Binding ◽

Microtubule Binding Domain

Tau is a microtubule-associated protein that is functionally modulated by phosphorylation and hyperphosphorylated in several neurodegenerative diseases. Because phosphorylation regulates both normal and pathological tau functioning, it is of great interest to identify the signalling pathways and enzymes capable of modulating tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and localization in response to osmotic stress, which activates the stress-activated protein kinases (SAPKs), a family of proline-directed protein kinases shown to phosphorylate tau in vitro and hypothesized to phosphorylate tau in Alzheimer's disease. Immunoblot analysis with phosphorylation-dependent antibodies revealed that osmotic stress increased tau phosphorylation at the non-Ser/Thr-Pro sites Ser-262/356, within the microtubule-binding domain, as well as Ser/Thr-Pro sites outside of tau's microtubule-binding domain. Although all SAPKs examined were activated by osmotic stress, none of the endogenous SAPKs mediated the increase in tau phosphorylation. However, when transfected into SH-SY5Y cells, SAPK3, but not the other SAPKs examined, phosphorylated tau in situ in response to activation by osmotic stress. Osmotic-stress-induced tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and an increase in the amount of soluble tau. This stress-induced alteration in tau localization was only partially due to phosphorylation at Ser-262/356 by a staurosporine-sensitive, non-proline-directed, protein kinase. Taken together, these results suggest that osmotic stress activates at least two tau-directed protein kinases, one proline-directed and one non-proline-directed, that SAPK3 can phosphorylate tau on Ser/Thr-Pro residues in situ, and that Ser-262/356 phosphorylation only partially regulates tau localization in the cell.

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Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5552 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5552-5562 ◽

Cited By ~ 24

Author(s):

E Roulet ◽

M T Armentero ◽

G Krey ◽

B Corthésy ◽

C Dreyer ◽

...

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Xenopus Laevis ◽

Transcriptional Activation ◽

Binding Activity ◽

New Members ◽

Binding Domains ◽

Dna Binding Activity

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

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MTCL2 is a new Golgi-resident microtubule-regulating protein, essential for organizing asymmetric microtubule network

10.1101/2020.08.19.251967 ◽

2020 ◽

Author(s):

Risa Matsuoka ◽

Masateru Miki ◽

Sonoko Mizuno ◽

Yurina Ito ◽

Atsushi Suzuki

Keyword(s):

Coiled Coil ◽

Golgi Membrane ◽

Microtubule Network ◽

Microtubule Binding ◽

Microtubule Stabilization ◽

Binding Domains ◽

Microtubule Binding Domain ◽

Ribbon Structure ◽

Golgi Ribbon

AbstractThe Golgi apparatus plays important roles in organizing the asymmetric microtubule network essential for polarized vesicle transport. The Golgi-associated coiled-coil protein MTCL1 is crucially involved in Golgi functioning by interconnecting and stabilizing microtubules on the Golgi membrane through its N- and C-terminal microtubule-binding domains. Here, we report the presence of a mammalian paralog of MTCL1, named MTCL2, lacking the N-terminal microtubule-binding domain. MTCL2 localizes to the Golgi membrane through the N-terminal region and directly binds microtubules through the conserved C-terminal domain without promoting microtubule stabilization. Knockdown experiments demonstrated essential roles of MTCL2 in accumulating MTs around the Golgi and regulating the Golgi ribbon structure. In vitro wound healing assays further suggested a possible intriguing activity of MTCL2 in integrating the centrosomal and Golgi-associated microtubules around the Golgi ribbon, thus supporting directional migration. Altogether, the present results demonstrate that cells utilize two members of the MTCL protein family to differentially regulate the Golgi-associated microtubules for controlling cell polarity.

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Mechanism of action of rigosertib does not involve tubulin binding

10.1101/2019.12.12.874719 ◽

2019 ◽

Cited By ~ 2

Author(s):

Stacey J. Baker ◽

Stephen C. Cosenza ◽

Saikrishna Athuluri-Divakar ◽

M.V. Ramana Reddy ◽

Rodrigo Vasquez-Del Carpio ◽

...

Keyword(s):

Mechanism Of Action ◽

Human Tumor ◽

Binding Activity ◽

Phase Iii ◽

Clinical Grade ◽

Wild Type ◽

Binding Domains ◽

Tubulin Binding

SUMMARYRigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cells in vitro and in vivo and is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant β-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib. Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor. The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.

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Dynein, Dynactin, and Kinesin II's Interaction with Microtubules Is Regulated during Bidirectional Organelle Transport

The Journal of Cell Biology ◽

10.1083/jcb.151.1.155 ◽

2000 ◽

Vol 151 (1) ◽

pp. 155-166 ◽

Cited By ~ 39

Author(s):

Eric L. Reese ◽

Leah T. Haimo

Keyword(s):

Pigment Granule ◽

Cytoplasmic Dynein ◽

Binding Activity ◽

The State ◽

Organelle Transport ◽

Microtubule Binding ◽

Pigment Granules ◽

Microtubule Motors

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

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Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80

eLife ◽

10.7554/elife.26866 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 17

Author(s):

David M Kern ◽

Julie K Monda ◽

Kuan-Chung Su ◽

Elizabeth M Wilson-Kubalek ◽

Iain M Cheeseman

Keyword(s):

Chromosome Segregation ◽

Human Cells ◽

Cross Linking ◽

Microtubule Binding ◽

Binding Domains ◽

Anaphase Onset ◽

Ndc80 Complex ◽

Outstanding Question ◽

Dynamic Microtubule

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

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Short Stop provides an essential link between F-actin and microtubules during axon extension

Development ◽

10.1242/dev.129.5.1195 ◽

2002 ◽

Vol 129 (5) ◽

pp. 1195-1204 ◽

Cited By ~ 8

Author(s):

Seungbok Lee ◽

Peter A. Kolodziej

Keyword(s):

Cultured Cells ◽

Binding Motif ◽

Cellular Motility ◽

Connecting Rod ◽

Neuronal Morphogenesis ◽

Microtubule Binding ◽

Axon Extension ◽

Binding Domains ◽

Microtubule Binding Domain ◽

Underlying Mechanisms

Coordination of F-actin and microtubule dynamics is important for cellular motility and morphogenesis, but little is known about underlying mechanisms. short stop (shot) encodes an evolutionarily conserved, neuronally expressed family of rod-like proteins required for sensory and motor axon extension in Drosophila melanogaster. We identify Shot isoforms that contain N-terminal F-actin and C-terminal microtubule-binding domains, and that crosslink F-actin and microtubules in cultured cells. The F-actin- and microtubule-binding domains of Shot are required in the same molecule for axon extension, though the length of the connecting rod domain can be dramatically reduced without affecting activity. Shot therefore functions as a cytoskeletal crosslinker in axon extension, rather than mediating independent interactions with F-actin and microtubules. A Ca2+-binding motif located adjacent to the microtubule-binding domain is also required for axon extension, suggesting that intracellular Ca2+ release may regulate Shot activity. These results suggest that Shot coordinates regulated interactions between F-actin and microtubules that are crucial for neuronal morphogenesis.

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