RESUMEN
Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa , Proteínas Adaptadoras Transductoras de Señales , Complejo Dinactina , Dineínas , Proteínas Asociadas a Microtúbulos , Proteínas del Tejido Nervioso , Microscopía por Crioelectrón , Complejo Dinactina/química , Complejo Dinactina/genética , Complejo Dinactina/metabolismo , Dineínas/química , Dineínas/genética , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Unión Proteica , Humanos , Células HeLa , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Repeticiones WD40 , Mapeo de Interacción de ProteínasRESUMEN
The microtubule motor dynein is critical for the assembly and positioning of mitotic spindles. In Caenorhabditis elegans, these dynein functions have been extensively studied in the early embryo but remain poorly explored in other developmental contexts. Here, we use a hypomorphic dynein mutant to investigate the motor's contribution to asymmetric stem cell-like divisions in the larval epidermis. Live imaging of seam cell divisions that precede formation of the seam syncytium shows that mutant cells properly assemble but frequently misorient their spindle. Misoriented divisions misplace daughter cells from the seam cell row, generate anucleate compartments due to aberrant cytokinesis, and disrupt asymmetric cell fate inheritance. Consequently, the seam becomes disorganized and populated with extra cells that have lost seam identity, leading to fatal epidermal rupture. We show that dynein orients the spindle through the cortical GOA-1Gα-LIN-5NuMA pathway by directing the migration of prophase centrosomes along the anterior-posterior axis. Spindle misorientation in the dynein mutant can be partially rescued by elongating cells, implying that dynein-dependent force generation and cell shape jointly promote correct asymmetric division of epithelial stem cells.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Dineínas/genética , Dineínas/metabolismo , Mitosis , Centrosoma/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Huso Acromático/metabolismo , Profase , Epidermis/metabolismoRESUMEN
Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 "hot-wiring" approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility.
Asunto(s)
Proteínas de Caenorhabditis elegans , Dineínas , Animales , Dineínas/metabolismo , Transporte Biológico , Cilios/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Flagelos/metabolismoRESUMEN
Cytokinesis is the last step of cell division, when one cell physically divides into two cells. Cytokinesis is driven by an equatorial contractile ring and signals from antiparallel microtubule bundles (the central spindle) that form between the two masses of segregating chromosomes. Bundling of central spindle microtubules is essential for cytokinesis in cultured cells. Using a temperature-sensitive mutant of SPD-1, the homolog of the microtubule bundler PRC1, we demonstrate that SPD-1 is required for robust cytokinesis in the Caenorhabditis elegans early embryo. SPD-1 inhibition results in broadening of the contractile ring, creating an elongated intercellular bridge between sister cells at the last stages of ring constriction that fails to seal. Moreover, depleting anillin/ANI-1 in SPD-1-inhibited cells results in myosin loss from the contractile ring during the second half of furrow ingression, which in turn results in furrow regression and cytokinesis failure. Our results thus reveal a mechanism involving the joint action of anillin and PRC1, which operates during the later stages of furrow ingression to ensure continued functioning of the contractile ring until cytokinesis is complete.
Asunto(s)
Proteínas de Caenorhabditis elegans , Citocinesis , Animales , Proteínas Contráctiles/genética , Miosinas , Microtúbulos , Caenorhabditis elegans , Proteínas de Microfilamentos , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
The microtubule minus-end-directed motility of cytoplasmic dynein 1 (dynein), arguably the most complex and versatile cytoskeletal motor, is harnessed for diverse functions, such as long-range organelle transport in neuronal axons and spindle assembly in dividing cells. The versatility of dynein raises a number of intriguing questions, including how is dynein recruited to its diverse cargo, how is recruitment coupled to activation of the motor, how is motility regulated to meet different requirements for force production and how does dynein coordinate its activity with that of other microtubule-associated proteins (MAPs) present on the same cargo. Here, these questions will be discussed in the context of dynein at the kinetochore, the supramolecular protein structure that connects segregating chromosomes to spindle microtubules in dividing cells. As the first kinetochore-localized MAP described, dynein has intrigued cell biologists for more than three decades. The first part of this Review summarizes current knowledge about how kinetochore dynein contributes to efficient and accurate spindle assembly, and the second part describes the underlying molecular mechanisms and highlights emerging commonalities with dynein regulation at other subcellular sites.
Asunto(s)
Dineínas , Cinetocoros , Proteínas Asociadas a Microtúbulos/genética , Dineínas Citoplasmáticas/genética , AxonesRESUMEN
A landmark event in the transition from interphase to mitosis in metazoans is nuclear envelope breakdown (NEBD). Important mitotic events occur prior to NEBD, including condensation of replicated chromosomes and assembly of kinetochores to rapidly engage spindle microtubules. Here, we show that nuclear-enriched protein phosphatase 4 (PP4) ensures robust assembly of the microtubule-coupling outer kinetochore prior to NEBD. In the absence of PP4, chromosomes exhibit extended monopolar orientation after NEBD and subsequently mis-segregate. A secondary consequence of diminished outer kinetochore assembly is defective sister chromatid resolution. After NEBD, a cytoplasmic activity compensates for PP4 loss, leading to outer kinetochore assembly and recovery of chromosomes from monopolar orientation to significant bi-orientation. The Ndc80-Ska microtubule-binding module of the outer kinetochore is required for this recovery. PP4 associates with the inner kinetochore protein CENP-C; however, disrupting the PP4-CENP-C interaction does not perturb chromosome segregation. These results establish that PP4-dependent outer kinetochore assembly prior to NEBD is critical for timely and proper engagement of chromosomes with spindle microtubules.
Asunto(s)
Cinetocoros , Microtúbulos , Membrana Nuclear , Fosfoproteínas Fosfatasas , Segregación Cromosómica , Cinetocoros/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Mitosis , Fosfoproteínas Fosfatasas/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Membrana Nuclear/metabolismo , AnimalesRESUMEN
Cytokinesis requires the constriction of an actomyosin-based contractile ring and involves multiple F-actin crosslinkers. We show that partial depletion of the C. elegans cytokinetic formin generates contractile rings with low F-actin levels that constrict but are structurally fragile, and we use this background to investigate the roles of the crosslinkers plastin/PLST-1 and ß-heavy-spectrin/SMA-1 during ring constriction. We show that the removal of PLST-1 or SMA-1 has opposite effects on the structural integrity of fragile rings. PLST-1 loss reduces cortical tension that resists ring constriction and makes fragile rings less prone to ruptures and regressions, whereas SMA-1 loss exacerbates structural defects, leading to frequent ruptures and cytokinesis failure. Fragile rings without SMA-1 or containing a shorter SMA-1, repeatedly rupture at the same site, and SMA-1::GFP accumulates at repair sites in fragile rings and in rings cut by laser microsurgery. These results establish that ß-heavy-spectrin stabilizes the constricting ring and reveals the importance of ß-heavy-spectrin size for network connectivity at low F-actin density.
Asunto(s)
Citoesqueleto de Actina , Citocinesis , Espectrina , Actinas , Actomiosina , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/metabolismo , Forminas , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Espectrina/metabolismoRESUMEN
The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3's interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3's RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3-bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3's leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3-bound dynein and kinesin-1 participate in bidirectional organelle transport.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Dineínas Citoplasmáticas , Cinesinas , Proteínas del Tejido Nervioso , Orgánulos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Humanos , Cinesinas/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Orgánulos/metabolismo , Células Receptoras Sensoriales/metabolismoRESUMEN
The dynein-2 motor complex drives retrograde intraflagellar transport (IFT), playing a pivotal role in the assembly and functions of cilia. However, the mechanisms that regulate dynein-2 motility remain poorly understood. Here, we identify the Caenorhabditis elegans WDR60 homologue, WDR-60, and dissect the roles of this intermediate chain using genome editing and live imaging of endogenous dynein-2/IFT components. We find that loss of WDR-60 impairs dynein-2 recruitment to cilia and its incorporation onto anterograde IFT trains, reducing retrograde motor availability at the ciliary tip. Consistent with this, we show that fewer dynein-2 motors power WDR-60-deficient retrograde IFT trains, which move at reduced velocities and fail to exit cilia, accumulating on the distal side of the transition zone. Remarkably, disrupting the transition zone's NPHP module almost fully restores ciliary exit of underpowered retrograde trains in wdr-60 mutants. This work establishes WDR-60 as a major contributor to IFT, and the NPHP module as a roadblock to dynein-2 passage through the transition zone.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Cilios/metabolismo , Proteínas del Citoesqueleto/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas del Citoesqueleto/química , Dineínas/química , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Mutación/genética , Dominios Proteicos , Células Receptoras Sensoriales/metabolismoRESUMEN
Cytokinesis, the process that partitions the mother cell into two daughter cells, requires the assembly and constriction of an equatorial actomyosin network. Different types of non-motor F-actin crosslinkers localize to the network, but their functional contribution remains poorly understood. Here, we describe a synergy between the small rigid crosslinker plastin and the large flexible crosslinker spectrin in the C. elegans one-cell embryo. In contrast to single inhibitions, co-inhibition of plastin and the ßH-spectrin (SMA-1) results in cytokinesis failure due to progressive disorganization and eventual collapse of the equatorial actomyosin network. Cortical localization dynamics of non-muscle myosin II in co-inhibited embryos mimic those observed after drug-induced F-actin depolymerization, suggesting that the combined action of plastin and spectrin stabilizes F-actin in the contractile ring. An in silico model predicts that spectrin is more efficient than plastin at stabilizing the ring and that ring formation is relatively insensitive to ßH-spectrin length, which is confirmed in vivo with a sma-1 mutant that lacks 11 of its 29 spectrin repeats. Our findings provide the first evidence that spectrin contributes to cytokinesis and highlight the importance of crosslinker interplay for actomyosin network integrity.
Asunto(s)
Actomiosina , Citocinesis , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Glicoproteínas de Membrana , Proteínas de Microfilamentos , Espectrina/genéticaRESUMEN
The microtubule motor cytoplasmic dynein 1 (dynein) and its essential activator dynactin have conserved roles in spindle assembly and positioning during female meiosis and mitosis, but their contribution to male meiosis remains poorly understood. Here, we characterize the G33S mutation in the C. elegans dynactin subunit DNC-1, which corresponds to G59S in human p150Glued that causes motor neuron disease. In spermatocytes, dnc-1(G33S) delays spindle assembly and penetrantly inhibits anaphase spindle elongation in meiosis I, which prevents the segregation of homologous chromosomes. By contrast, chromosomes segregate without errors in the early dnc-1(G33S) embryo. Deletion of the DNC-1 N-terminus shows that defective meiosis in dnc-1(G33S) spermatocytes is not due to the inability of DNC-1 to interact with microtubules. Instead, our results suggest that the DNC-1(G33S) protein, which is aggregation prone in vitro, is less stable in spermatocytes than the early embryo, resulting in different phenotypic severity in the two dividing tissues. Thus, the dnc-1(G33S) mutant reveals that dynein-dynactin drive meiotic chromosome segregation in spermatocytes and illustrates that the extent to which protein misfolding leads to loss of function can vary significantly between cell types.
Asunto(s)
Segregación Cromosómica , Complejo Dinactina/metabolismo , Dineínas/metabolismo , Espermatocitos/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Cromosomas , Dineínas Citoplasmáticas/metabolismo , Complejo Dinactina/genética , Femenino , Humanos , Masculino , Meiosis , Mitosis , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Mutación , Huso Acromático/metabolismoRESUMEN
Centromeres, the chromosomal loci that ensure chromosome segregation by directing kinetochore assembly, are typically marked by the histone CENP-A. A study in CENP-A-deficient insects finds that virtually any chromosomal region with low nucleosome turnover can assemble kinetochores, highlighting the extraordinary plasticity of holocentromeres.
Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Animales , Centrómero/metabolismo , Proteína A Centromérica/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Insectos/genética , Cinetocoros/metabolismoRESUMEN
Cytokinesis is the last step of cell division that physically partitions the mother cell into two daughter cells. Cytokinesis requires the assembly and constriction of a contractile ring, a circumferential array of filamentous actin (F-actin), non-muscle myosin II motors (myosin), and actin-binding proteins that forms at the cell equator. Cytokinesis is accompanied by long-range cortical flows from regions of relaxation toward regions of compression. In the C. elegans one-cell embryo, it has been suggested that anterior-directed cortical flows are the main driver of contractile ring assembly. Here, we use embryos co-expressing motor-dead and wild-type myosin to show that cortical flows can be severely reduced without major effects on contractile ring assembly and timely completion of cytokinesis. Fluorescence recovery after photobleaching in the ingressing furrow reveals that myosin recruitment kinetics are also unaffected by the absence of cortical flows. We find that myosin cooperates with the F-actin crosslinker plastin to align and compact F-actin bundles at the cell equator, and that this cross-talk is essential for cytokinesis. Our results thus argue against the idea that cortical flows are a major determinant of contractile ring assembly. Instead, we propose that contractile ring assembly requires localized concerted action of motor-competent myosin and plastin at the cell equator.
RESUMEN
The spindle checkpoint protects against aneuploidy by ensuring that dividing cells only proceed with chromosome segregation once all kinetochores are stably attached to spindle microtubules. The checkpoint protein MAD1 localizes to the corona, a structural expansion of the kinetochore forming in the absence of microtubule attachment, but molecular mechanism or functional significance of this localization remains unknown. Recent results now show that cyclin B1 recruits MAD1 to the corona and that this MAD1 pool is required for robust checkpoint signaling.
Asunto(s)
Cinetocoros , Puntos de Control de la Fase M del Ciclo Celular , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , Microtúbulos , Huso Acromático/genéticaRESUMEN
The kinetochore is at the heart of chromosome segregation in mitosis and meiosis. Rather than a static linker complex for chromatin and spindle microtubules, it is highly dynamic in composition, size, and shape. While known for decades that it can expand and grow a fibrous meshwork known as the corona, it was until recently unclear what constitutes this 'crown' and what its relevance is for kinetochore function. Here, we highlight recent discoveries in fibrous corona biology, and place them in the context of the processes that orchestrate high-fidelity chromosome segregation.
Asunto(s)
Segregación Cromosómica , Cinetocoros/metabolismo , Animales , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Microtúbulos/metabolismo , Modelos Biológicos , Transducción de SeñalRESUMEN
Cytokinesis in animal cells requires the assembly and constriction of a contractile actomyosin ring. Non-muscle myosin II is essential for cytokinesis, but the role of its motor activity remains unclear. Here, we examine cytokinesis in C. elegans embryos expressing non-muscle myosin motor mutants generated by genome editing. Two non-muscle motor-dead myosins capable of binding F-actin do not support cytokinesis in the one-cell embryo, and two partially motor-impaired myosins delay cytokinesis and render rings more sensitive to reduced myosin levels. Further analysis of myosin mutants suggests that it is myosin motor activity, and not the ability of myosin to crosslink F-actin, that drives the alignment and compaction of F-actin bundles during contractile ring assembly, and that myosin motor activity sets the pace of contractile ring constriction. We conclude that myosin motor activity is required at all stages of cytokinesis. Finally, characterization of the corresponding motor mutations in C. elegans major muscle myosin shows that motor activity is required for muscle contraction but is dispensable for F-actin organization in adult muscles.This article has an associated 'The people behind the papers' interview.
Asunto(s)
Citocinesis , Miosina Tipo II/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Animales , Plaquetas/metabolismo , Caenorhabditis elegans , Fase de Segmentación del Huevo/metabolismo , Edición Génica , Proteínas Fluorescentes Verdes/metabolismo , Homocigoto , Humanos , Ratones , Músculos/metabolismo , Mutación , Miosinas/metabolismo , Fosforilación , Interferencia de ARNRESUMEN
All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.
Asunto(s)
Dineínas Citoplasmáticas/genética , Dineínas Citoplasmáticas/metabolismo , Dineínas/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Secuencia Conservada , Complejo Dinactina , Dineínas/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Unión Proteica/fisiología , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Huso AcromáticoRESUMEN
Cytokinesis completes cell division by constriction of an actomyosin contractile ring that separates the two daughter cells. Here we use the early Caenorhabditis elegans embryo to explore how the actin filament network in the ring and the surrounding cortex is regulated by the single cytokinesis formin CYK-1 and the ARP2/3 complex, which nucleate nonbranched and branched filaments, respectively. We show that CYK-1 and the ARP2/3 complex are the predominant F-actin nucleators responsible for generating distinct cortical F-actin architectures and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and acute inhibition of CYK-1 after furrow ingression slows ring constriction rate, suggesting that CYK-1 activity is required throughout ring constriction. Surprisingly, although the ARP2/3 complex does not localize in the contractile ring, depletion of the ARP2 subunit or treatment with ARP2/3 complex inhibitor delays contractile ring formation and constriction. We present evidence that the delays are due to an excess in formin-nucleated cortical F-actin, suggesting that the ARP2/3 complex negatively regulates CYK-1 activity. We conclude that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Citocinesis , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Caenorhabditis elegans/embriología , Polaridad Celular , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , CinéticaRESUMEN
The kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, although dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into micrometer-scale filaments, and we identify the ROD-1 ß-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ's capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that reversible RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.
Asunto(s)
Caenorhabditis elegans/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Animales , Caenorhabditis elegans/embriología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dineínas/metabolismo , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMEN
Cytoplasmic dynein 1 (dynein) is the predominant microtubule minus end-directed motor in animals and participates in a wide range of cellular processes, including membrane trafficking, nuclear migration, and cell division. Dynein's functional diversity depends on co-factors that regulate its subcellular localization, interaction with cargo, and motor activity. The ubiquitous co-factor nuclear distribution gene E (NudE) is implicated in many of dynein's functions, and mutations in NudE cause the brain developmental disease microcephaly. To identify genetic interactors of the Caenorhabditis elegans NudE homolog nud-2, we performed a genome-wide RNAi screen with the null allele nud-2(ok949), which compromises dynein function but leaves animals viable and fertile. Using bacterial feeding to deliver dsRNAs in a 96-well liquid format and a semi-automated fluorescence microscopy approach for counting parents and progeny, we screened 19762 bacterial clones and identified 38 genes whose inhibition caused enhanced lethality in nud-2(ok949) relative to the nud-2(+) control. Further study of these genes, many of which participate in cell division, promises to provide insight into the function and regulation of dynein.