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1.
Science ; 383(6685): 870-876, 2024 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-38305685

RESUMEN

Microtubules are essential for intracellular organization and chromosome segregation. They are nucleated by the γ-tubulin ring complex (γTuRC). However, isolated vertebrate γTuRC adopts an open conformation that deviates from the microtubule structure, raising the question of the nucleation mechanism. In this study, we determined cryo-electron microscopy structures of human γTuRC bound to a nascent microtubule. Structural changes of the complex into a closed conformation ensure that γTuRC templates the 13-protofilament microtubules that exist in human cells. Closure is mediated by a latch that interacts with incorporating tubulin, making it part of the closing mechanism. Further rearrangements involve all γTuRC subunits and the removal of the actin-containing luminal bridge. Our proposed mechanism of microtubule nucleation by human γTuRC relies on large-scale structural changes that are likely the target of regulation in cells.


Asunto(s)
Microtúbulos , Tubulina (Proteína) , Humanos , Microscopía por Crioelectrón , Microtúbulos/química , Estructura Cuaternaria de Proteína , Tubulina (Proteína)/metabolismo
2.
Mol Biol Cell ; 35(1): ar12, 2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-37991893

RESUMEN

Chromosome segregation relies on the correct assembly of a bipolar spindle. Spindle pole self-organization requires dynein-dependent microtubule (MT) transport along other MTs. However, during M-phase RanGTP triggers MT nucleation and branching generating polarized arrays with nonastral organization in which MT minus ends are linked to the sides of other MTs. This raises the question of how branched-MT nucleation and dynein-mediated transport cooperate to organize the spindle poles. Here, we used RanGTP-dependent MT aster formation in Xenopus laevis (X. laevis) egg extract to study the interplay between these two seemingly conflicting organizing principles. Using temporally controlled perturbations of MT nucleation and dynein activity, we found that branched MTs are not static but instead dynamically redistribute over time as poles self-organize. Our experimental data together with computer simulations suggest a model where dynein together with dynactin and NuMA directly pulls and move branched MT minus ends toward other MT minus ends.


Asunto(s)
Dineínas , Huso Acromático , Animales , Dineínas/metabolismo , Xenopus laevis/metabolismo , Huso Acromático/metabolismo , Microtúbulos/metabolismo , Complejo Dinactina , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Xenopus/metabolismo
3.
J Cell Biol ; 222(10)2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37615667

RESUMEN

During mitosis, microtubules in the spindle turn over continuously. At spindle poles, where microtubule minus ends are concentrated, microtubule nucleation and depolymerization, the latter required for poleward microtubule flux, happen side by side. How these seemingly antagonistic processes of nucleation and depolymerization are coordinated is not understood. Here, we reconstitute this coordination in vitro combining different pole-localized activities. We find that the spindle pole-localized kinesin-13 KIF2A is a microtubule minus-end depolymerase, in contrast to its paralog MCAK. Due to its asymmetric activity, KIF2A still allows microtubule nucleation from the γ-tubulin ring complex (γTuRC), which serves as a protective cap shielding the minus end against KIF2A binding. Efficient γTuRC uncapping requires the combined action of KIF2A and a microtubule severing enzyme, leading to treadmilling of the uncapped microtubule driven by KIF2A. Together, these results provide insight into the molecular mechanisms by which a minimal protein module coordinates microtubule nucleation and depolymerization at spindle poles consistent with their role in poleward microtubule flux.


Asunto(s)
Cinesinas , Centro Organizador de los Microtúbulos , Cinesinas/genética , Microtúbulos , Mitosis , Polos del Huso , Humanos
4.
J Cell Sci ; 136(12)2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37357828

RESUMEN

Mitotic spindle assembly during cell division is a highly regulated process. Ran-GTP produced around chromosomes controls the activity of a multitude of spindle assembly factors by releasing them from inhibitory interaction with importins. A major consequence of Ran-GTP regulation is the local stimulation of branched microtubule nucleation around chromosomes, which is mediated by the augmin complex (composed of the eight subunits HAUS1-HAUS8), a process that is crucially important for correct spindle assembly. However, augmin is not known to be a direct target of the Ran-GTP pathway, raising the question of how its activity is controlled. Here, we present the in vitro reconstitution of Ran-GTP-regulated microtubule binding of the human augmin complex. We demonstrate that importins directly bind to augmin, which prevents augmin from binding to microtubules. Ran-GTP relieves this inhibition. Therefore, the augmin complex is a direct target of the Ran-GTP pathway, suggesting that branching microtubule nucleation is directly regulated by the Ran-GTP gradient around chromosomes in dividing cells.


Asunto(s)
Carioferinas , Huso Acromático , Humanos , Huso Acromático/metabolismo , Carioferinas/metabolismo , Microtúbulos/metabolismo , Transducción de Señal , Guanosina Trifosfato/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína de Unión al GTP ran/metabolismo , Proteínas de Ciclo Celular/metabolismo
5.
iScience ; 26(2): 106063, 2023 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-36852161

RESUMEN

Active filament networks can organize into various dynamic architectures driven by cross-linking motors. Densities and kinetic properties of motors and microtubules have been shown previously to determine active microtubule network self-organization, but the effects of other control parameters are less understood. Using computer simulations, we study here how microtubule lengths and crowding effects determine active network architecture and dynamics. We find that attractive interactions mimicking crowding effects or long microtubules both promote the formation of extensile nematic networks instead of asters. When microtubules are very long and the network is highly connected, a new isotropically motile network state resembling a "gliding mesh" is predicted. Using in vitro reconstitutions, we confirm the existence of this gliding mesh experimentally. These results provide a better understanding of how active microtubule network organization can be controlled, with implications for cell biology and active materials in general.

6.
Proc Natl Acad Sci U S A ; 119(33): e2206398119, 2022 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-35960844

RESUMEN

During cell division, cross-linking motors determine the architecture of the spindle, a dynamic microtubule network that segregates the chromosomes in eukaryotes. It is unclear how motors with opposite directionality coordinate to drive both contractile and extensile behaviors in the spindle. Particularly, the impact of different cross-linker designs on network self-organization is not understood, limiting our understanding of self-organizing structures in cells but also our ability to engineer new active materials. Here, we use experiment and theory to examine active microtubule networks driven by mixtures of motors with opposite directionality and different cross-linker design. We find that although the kinesin-14 HSET causes network contraction when dominant, it can also assist the opposing kinesin-5 KIF11 to generate extensile networks. This bifunctionality results from HSET's asymmetric design, distinct from symmetric KIF11. These findings expand the set of rules underlying patterning of active microtubule assemblies and allow a better understanding of motor cooperation in the spindle.


Asunto(s)
Cinesinas , Microtúbulos , Proteínas Oncogénicas , Huso Acromático , División Celular , Humanos , Cinesinas/química , Cinesinas/fisiología , Microtúbulos/química , Microtúbulos/fisiología , Proteínas Oncogénicas/química , Proteínas Oncogénicas/fisiología , Huso Acromático/química , Huso Acromático/fisiología
7.
Methods Mol Biol ; 2430: 315-336, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35476342

RESUMEN

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. How γTuRC nucleates microtubules, and how nucleation is regulated is not understood. To gain an understanding of γTuRC activity and regulation at the molecular level, it is important to measure quantitatively how γTuRC interacts with tubulin and potential regulators in space and time. Here, we describe a total internal reflection fluorescence microscopy-based assay on chemically functionalized glass slides for the in vitro study of surface immobilized purified γTuRC. The assay allows to measure microtubule nucleation by γTuRC in real time and at a single molecule level over a wide variety of assay conditions, in the absence and presence of potential regulators. This setup provides a previously unavailable opportunity for quantitative studies of the kinetics of microtubule nucleation by γTuRC.


Asunto(s)
Centrosoma , Proteínas Asociadas a Microtúbulos , Microscopía , Proteínas Asociadas a Microtúbulos/química , Centro Organizador de los Microtúbulos , Microtúbulos/química
8.
Proc Natl Acad Sci U S A ; 119(2)2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-34996871

RESUMEN

Microtubules (MTs) are polymers of αß-tubulin heterodimers that stochastically switch between growth and shrinkage phases. This dynamic instability is critically important for MT function. It is believed that GTP hydrolysis within the MT lattice is accompanied by destabilizing conformational changes and that MT stability depends on a transiently existing GTP cap at the growing MT end. Here, we use cryo-electron microscopy and total internal reflection fluorescence microscopy of GTP hydrolysis-deficient MTs assembled from mutant recombinant human tubulin to investigate the structure of a GTP-bound MT lattice. We find that the GTP-MT lattice of two mutants in which the catalytically active glutamate in α-tubulin was substituted by inactive amino acids (E254A and E254N) is remarkably plastic. Undecorated E254A and E254N MTs with 13 protofilaments both have an expanded lattice but display opposite protofilament twists, making these lattices distinct from the compacted lattice of wild-type GDP-MTs. End-binding proteins of the EB family have the ability to compact both mutant GTP lattices and to stabilize a negative twist, suggesting that they promote this transition also in the GTP cap of wild-type MTs, thereby contributing to the maturation of the MT structure. We also find that the MT seam appears to be stabilized in mutant GTP-MTs and destabilized in GDP-MTs, supporting the proposal that the seam plays an important role in MT stability. Together, these structures of catalytically inactive MTs add mechanistic insight into the GTP state of MTs, the stability of the GTP- and GDP-bound lattice, and our overall understanding of MT dynamic instability.


Asunto(s)
Microscopía por Crioelectrón , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Humanos , Hidrólisis , Cinesinas , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/genética , Proteínas Recombinantes , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestructura
9.
Life Sci Alliance ; 4(12)2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34580180

RESUMEN

During mitosis, the spindle undergoes morphological and dynamic changes. It reorganizes at the onset of the anaphase when the antiparallel bundler PRC1 accumulates and recruits central spindle proteins to the midzone. Little is known about how the dynamic properties of the central spindle change during its morphological changes in human cells. Using gene editing, we generated human cells that express from their endogenous locus fluorescent PRC1 and EB1 to quantify their native spindle distribution and binding/unbinding turnover. EB1 plus end tracking revealed a general slowdown of microtubule growth, whereas PRC1, similar to its yeast orthologue Ase1, binds increasingly strongly to compacting antiparallel microtubule overlaps. KIF4A and CLASP1 bind more dynamically to the central spindle, but also show slowing down turnover. These results show that the central spindle gradually becomes more stable during mitosis, in agreement with a recent "bundling, sliding, and compaction" model of antiparallel midzone bundle formation in the central spindle during late mitosis.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Edición Génica/métodos , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/genética , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/genética , Huso Acromático/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/genética , Línea Celular Transformada , Segregación Cromosómica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Unión Proteica/genética , Transfección/métodos
10.
J Mol Biol ; 432(23): 6168-6172, 2020 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-33068635

RESUMEN

The αß-tubulin heterodimer is the fundamental building block of microtubules, making it central to several cellular processes. Despite the apparent simplicity of heterodimerisation, the associated energetics and kinetics remain disputed, largely due to experimental challenges associated with quantifying affinities in the <µM range. We use mass photometry to observe tubulin monomers and heterodimers in solution simultaneously, thereby quantifying the αß-tubulin dissociation constant (8.48 ± 1.22 nM) and its tightening in the presence of GTP (3.69 ± 0.65 nM), at a dissociation rate >10-2 s-1. Our results demonstrate the capabilities of mass photometry for quantifying protein-protein interactions and clarify the energetics and kinetics of tubulin heterodimerisation.


Asunto(s)
Conformación Proteica , Mapas de Interacción de Proteínas/genética , Tubulina (Proteína)/genética , Animales , Guanosina Trifosfato/metabolismo , Cinética , Fotometría , Multimerización de Proteína/genética , Termodinámica , Tubulina (Proteína)/química
11.
Dev Cell ; 53(5): 603-617.e8, 2020 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-32433913

RESUMEN

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. The mechanism of its regulation is not understood. We purified human γTuRC and measured its nucleation properties in a total internal reflection fluorescence (TIRF) microscopy-based real-time nucleation assay. We find that γTuRC stably caps the minus ends of microtubules that it nucleates stochastically. Nucleation is inefficient compared with microtubule elongation. The 4 Å resolution cryoelectron microscopy (cryo-EM) structure of γTuRC, combined with crosslinking mass spectrometry analysis, reveals an asymmetric conformation with only part of the complex in a "closed" conformation matching the microtubule geometry. Actin in the core of the complex, and MZT2 at the outer perimeter of the closed part of γTuRC appear to stabilize the closed conformation. The opposite side of γTuRC is in an "open," nucleation-incompetent conformation, leading to a structural asymmetry explaining the low nucleation efficiency of purified human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.


Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Tubulina (Proteína)/química , Actinas/química , Actinas/metabolismo , Microscopía por Crioelectrón , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Microtúbulos/química , Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , Imagen Individual de Molécula , Tubulina (Proteína)/metabolismo
12.
Elife ; 92020 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-32053491

RESUMEN

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region.


Asunto(s)
Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Animales , Sitios de Unión , Humanos , Hidrólisis , Cinética , Tubulina (Proteína)/metabolismo
13.
Curr Biol ; 29(13): 2120-2130.e7, 2019 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-31231047

RESUMEN

In anaphase spindles, antiparallel microtubules associate to form tight midzone bundles, as required for functional spindle architecture and correct chromosome segregation. Several proteins selectively bind to these overlaps to control cytokinesis. How midzone bundles assemble is poorly understood. Here, using an in vitro reconstitution approach, we demonstrate that minimal midzone bundles can reliably self-organize in solution from dynamic microtubules, the microtubule crosslinker PRC1, and the motor protein KIF4A. The length of the central antiparallel overlaps in these microtubule bundles is similar to that observed in cells and is controlled by the PRC1/KIF4A ratio. Experiments and computer simulations demonstrate that minimal midzone bundle formation results from promoting antiparallel microtubule crosslinking, stopping microtubule plus-end dynamicity, and motor-driven midzone compaction and alignment. The robustness of this process suggests that a similar self-organization mechanism may contribute to the reorganization of the spindle architecture during the metaphase to anaphase transition in cells.


Asunto(s)
Anafase/fisiología , Escherichia coli/fisiología , Microtúbulos/metabolismo , Huso Acromático/metabolismo
14.
Phys Biol ; 16(4): 046004, 2019 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-31013252

RESUMEN

Active networks composed of filaments and motor proteins can self-organize into a variety of architectures. Computer simulations in two or three spatial dimensions and including or omitting steric interactions between filaments can be used to model active networks. Here we examine how these modelling choices affect the state space of network self-organization. We compare the networks generated by different models of a system of dynamic microtubules and microtubule-crosslinking motors. We find that a thin 3D model that includes steric interactions between filaments is the most versatile, capturing a variety of network states observed in recent experiments. In contrast, 2D models either with or without steric interactions which prohibit microtubule crossings can produce some, but not all, observed network states. Our results provide guidelines for the most appropriate choice of model for the study of different network types and elucidate mechanisms of active network organization.


Asunto(s)
Microtúbulos/química , Proteínas Motoras Moleculares/química , Simulación por Computador , Reactivos de Enlaces Cruzados/química , Citoesqueleto/metabolismo , Multimerización de Proteína , Transducción de Señal
15.
Structure ; 27(3): 497-506.e4, 2019 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-30661854

RESUMEN

Microtubules are cytoskeletal filaments of eukaryotic cells made of αß-tubulin heterodimers. Structural studies of non-microtubular tubulin rely mainly on molecules that prevent its self-assembly and are used as crystallization chaperones. Here we identified artificial proteins from an αRep library that are specific to α-tubulin. Turbidity experiments indicate that these αReps impede microtubule assembly in a dose-dependent manner and total internal reflection fluorescence microscopy further shows that they specifically block growth at the microtubule (-) end. Structural data indicate that they do so by targeting the α-tubulin longitudinal surface. Interestingly, in one of the complexes studied, the α subunit is in a conformation that is intermediate between the ones most commonly observed in X-ray structures of tubulin and those seen in the microtubule, emphasizing the plasticity of tubulin. These α-tubulin-specific αReps broaden the range of tools available for the mechanistic study of microtubule dynamics and its regulation.


Asunto(s)
Proteínas Recombinantes de Fusión/farmacología , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Moleculares , Biblioteca de Péptidos , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Secuencias Repetitivas de Aminoácido
16.
Cell ; 175(3): 796-808.e14, 2018 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-30340043

RESUMEN

During cell division, mitotic motors organize microtubules in the bipolar spindle into either polar arrays at the spindle poles or a "nematic" network of aligned microtubules at the spindle center. The reasons for the distinct self-organizing capacities of dynamic microtubules and different motors are not understood. Using in vitro reconstitution experiments and computer simulations, we show that the human mitotic motors kinesin-5 KIF11 and kinesin-14 HSET, despite opposite directionalities, can both organize dynamic microtubules into either polar or nematic networks. We show that in addition to the motor properties the natural asymmetry between microtubule plus- and minus-end growth critically contributes to the organizational potential of the motors. We identify two control parameters that capture system composition and kinetic properties and predict the outcome of microtubule network organization. These results elucidate a fundamental design principle of spindle bipolarity and establish general rules for active filament network organization.


Asunto(s)
Cinesinas/metabolismo , Microtúbulos/metabolismo , Simulación de Dinámica Molecular , Huso Acromático/metabolismo , Animales , Humanos , Cinesinas/química , Microtúbulos/química , Células Sf9 , Huso Acromático/química , Spodoptera
17.
Soft Matter ; 14(6): 901-909, 2018 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-29364311

RESUMEN

Microtubules and motor proteins form active filament networks that are critical for a variety of functions in living cells. Network topology and dynamics are the result of a self-organisation process that takes place within the boundaries of the cell. Previous biochemical in vitro studies with biomimetic systems consisting of purified motors and microtubules have demonstrated that confinement has an important effect on the outcome of the self-organisation process. However, the pathway of motor/microtubule self-organisation under confinement and its effects on network morphology are still poorly understood. Here, we have investigated how minus-end directed microtubule cross-linking kinesins organise microtubules inside polymer-stabilised microfluidic droplets of well-controlled size. We find that confinement can impose a novel pathway of microtubule aster formation proceeding via the constriction of an initially spherical motor/microtubule network. This mechanism illustrates the close relationship between confinement, network contraction, and aster formation. The spherical constriction pathway robustly produces single, well-centred asters with remarkable reproducibility across thousands of droplets. These results show that the additional constraint of well-defined confinement can improve the robustness of active network self-organisation, providing insight into the design principles of self-organising active networks in micro-scale confinement.

18.
Biophys J ; 113(9): 2055-2067, 2017 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-29117528

RESUMEN

Most kinesin motors move in only one direction along microtubules. Members of the kinesin-5 subfamily were initially described as unidirectional plus-end-directed motors and shown to produce piconewton forces. However, some fungal kinesin-5 motors are bidirectional. The force production of a bidirectional kinesin-5 has not yet been measured. Therefore, it remains unknown whether the mechanism of the unconventional minus-end-directed motility differs fundamentally from that of plus-end-directed stepping. Using force spectroscopy, we have measured here the forces that ensembles of purified budding yeast kinesin-5 Cin8 produce in microtubule gliding assays in both plus- and minus-end direction. Correlation analysis of pause forces demonstrated that individual Cin8 molecules produce additive forces in both directions of movement. In ensembles, Cin8 motors were able to produce single-motor forces up to a magnitude of ∼1.5 pN. Hence, these properties appear to be conserved within the kinesin-5 subfamily. Force production was largely independent of the directionality of movement, indicating similarities between the motility mechanisms for both directions. These results provide constraints for the development of models for the bidirectional motility mechanism of fission yeast kinesin-5 and provide insight into the function of this mitotic motor.


Asunto(s)
Cinesinas/metabolismo , Fenómenos Mecánicos , Movimiento , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Fenómenos Biomecánicos , Cinesinas/química , Microscopía de Fuerza Atómica , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
19.
Elife ; 62017 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-29120325

RESUMEN

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements ('ridge' and 'wedge') in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.


Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Multimerización de Proteína , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Microscopía Fluorescente , Modelos Moleculares , Unión Proteica , Conformación Proteica
20.
EMBO J ; 36(22): 3387-3404, 2017 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-29038173

RESUMEN

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus-ends in an EB-dependent manner or moving processively towards minus ends in an adaptor protein-dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal-D2 (BicD2) or the multifunctional regulator Lissencephaly-1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus-end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.


Asunto(s)
Movimiento Celular , Dineínas/metabolismo , Microtúbulos/metabolismo , Animales , Complejo Dinactina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Biológicos , Probabilidad , Sus scrofa
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