RESUMO
Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Anáfase , Animais , Fenômenos Biomecânicos , Proteínas de Ciclo Celular/fisiologia , Difusão , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Modelos Teóricos , Miosinas/química , Pinças Ópticas , Ratos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/fisiologia , Estresse Mecânico , Miosinas Ventriculares/químicaRESUMO
The diversity of dynein's functions in mammalian cells is a manifestation of both the existence of multiple dynein heavy chain isoforms and an extensive set of associated protein subunits. In this study, we have identified and characterized a novel subunit of the mammalian cytoplasmic dynein 2 complex. The sequence similarity between this 33-kDa subunit and the light intermediate chains (LICs) of cytoplasmic dynein 1 suggests that this protein is a dynein 2 LIC (D2LIC). D2LIC contains a P-loop motif near its NH(2) terminus, and it shares a short region of similarity to the yeast GTPases Spg1p and Tem1p. The D2LIC subunit interacts specifically with DHC2 (or cDhc1b) in both reciprocal immunoprecipitations and sedimentation assays. The expression of D2LIC also mirrors that of DHC2 in a variety of tissues. D2LIC colocalizes with DHC2 at the Golgi apparatus throughout the cell cycle. On brefeldin A-induced Golgi fragmentation, a fraction of D2LIC redistributes to the cytoplasm, leaving behind a subset of D2LIC that is localized around the centrosome. Our results suggest that D2LIC is a bona fide subunit of cytoplasmic dynein 2 that may play a role in maintaining Golgi organization by binding cytoplasmic dynein 2 to its Golgi-associated cargo.
Assuntos
Dineínas/genética , Dineínas/metabolismo , Complexo de Golgi/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Sequência de Bases , Brefeldina A/farmacologia , Células COS , Linhagem Celular , Dineínas/química , Humanos , Isoenzimas , Lectinas de Ligação a Manose/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Nocodazol/farmacologia , Subunidades Proteicas , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Distribuição Tecidual , Tubulina (Proteína)/metabolismoRESUMO
Firm attachments between kinetochores and dynamic spindle microtubules (MTs) are important for accurate chromosome segregation. Centromere protein F (CENP-F) has been shown to include two MT-binding domains, so it may participate in this key mitotic process. Here, we show that the N-terminal MT-binding domain of CENP-F prefers curled oligomers of tubulin relative to MT walls by approximately fivefold, suggesting that it may contribute to the firm bonds between kinetochores and the flared plus ends of dynamic MTs. A polypeptide from CENP-F's C terminus also bound MTs, and either protein fragment diffused on a stable MT wall. They also followed the ends of dynamic MTs as they shortened. When either fragment was coupled to a microbead, the force it could transduce from a shortening MT averaged 3-5 pN but could exceed 10 pN, identifying CENP-F as a highly effective coupler to shortening MTs.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/genética , Cinetocoros/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Sítios de Ligação , Bovinos , Linhagem Celular Tumoral , Humanos , Mitose/genética , Polimerização , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Tubulin can polymerize in two distinct arrangements: "B-lattices," in which the alpha-tubulins of one protofilament lie next to alpha-tubulins in the neighboring protofilaments, or the "A" configuration, where alpha-tubulins lie beside beta-tubulins. Microtubules (MTs) in flagellar axonemes and those assembled from pure tubulin in vitro display only B-lattices, but recent work shows that A-lattices are found when tubulin co-polymerizes in vitro with an allele of end-binding protein 1 that lacks C-terminal sequences. This observation suggests that cytoplasmic MTs, which form in the presence of this "tip-associating protein," may have A-lattices. To test this hypothesis, we have decorated interphase MTs in 3T3 cells with monomeric motor domains from the kinesin-like protein Eg5. These MTs show only B-lattices, as confirmed by visual inspection of electron cryo-tomograms and power spectra of single projection views, imaged at higher electron dose. This result is significant because 13 protofilament MTs with B-lattices must include a "seam," one lateral domain where adjacent dimers are in the A-configuration. It follows that cytoplasmic MTs are not cylindrically symmetric; they have two distinct faces, which may influence the binding patterns of functionally significant MT-interacting proteins.
Assuntos
Citoplasma/ultraestrutura , Microtúbulos/ultraestrutura , Tubulina (Proteína)/ultraestrutura , Células 3T3 , Animais , Células Cultivadas , CamundongosRESUMO
Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5(+) and klp6(+) genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end-directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.