RESUMO
The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. Here, we show that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule-associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex.
Assuntos
Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Motivos de Aminoácidos/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Células HeLa , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Mitose/genética , Mitose/fisiologia , Orientação/fisiologiaRESUMO
Astrin is a mitotic spindle-associated protein required for the correct alignment of all chromosomes at the metaphase plate. Astrin depletion delays chromosome alignment and causes the loss of normal spindle architecture and sister chromatid cohesion before anaphase onset. Here we describe an astrin complex containing kinastrin/SKAP, a novel kinetochore and mitotic spindle protein, and three minor interaction partners: dynein light chain, Plk1, and Sgo2. Kinastrin is the major astrin-interacting protein in mitotic cells, and is required for astrin targeting to microtubule plus ends proximal to the plus tip tracking protein EB1. Cells overexpressing or depleted of kinastrin mislocalize astrin and show the same mitotic defects as astrin-depleted cells. Importantly, astrin fails to localize to and track microtubule plus ends in cells depleted of or overexpressing kinastrin. These findings suggest that microtubule plus end targeting of astrin is required for normal spindle architecture and chromosome alignment, and that perturbations of this pathway result in delayed mitosis and nonphysiological separase activation.
