RESUMO
Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein's interphase functions. KASH5 interacts with a dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein's recruitment to other cellular membranes. KASH5's N-terminal EF-hands are essential as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the transmembrane protein KASH5 is an activating adaptor for dynein and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.
Assuntos
Proteínas de Ciclo Celular , Dineínas do Citoplasma , Complexo Dinactina , Proteínas Associadas aos Microtúbulos , Animais , Cálcio/metabolismo , Dineínas do Citoplasma/genética , Dineínas do Citoplasma/metabolismo , Complexo Dinactina/genética , Complexo Dinactina/metabolismo , Mamíferos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
To complete mitosis, the bridge that links the two daughter cells needs to be cleaved. This step is carried out by the endosomal sorting complex required for transport (ESCRT) machinery. AKTIP, a protein discovered to be associated with telomeres and the nuclear membrane in interphase cells, shares sequence similarities with the ESCRT I component TSG101. Here we present evidence that during mitosis AKTIP is part of the ESCRT machinery at the midbody. AKTIP interacts with the ESCRT I subunit VPS28 and forms a circular supra-structure at the midbody, in close proximity with TSG101 and VPS28 and adjacent to the members of the ESCRT III module CHMP2A, CHMP4B and IST1. Mechanistically, the recruitment of AKTIP is dependent on MKLP1 and independent of CEP55. AKTIP and TSG101 are needed together for the recruitment of the ESCRT III subunit CHMP4B and in parallel for the recruitment of IST1. Alone, the reduction of AKTIP impinges on IST1 and causes multinucleation. Our data altogether reveal that AKTIP is a component of the ESCRT I module and functions in the recruitment of ESCRT III components required for abscission.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Mitose/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Citocinese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Transporte Proteico , Fuso Acromático/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
ING proteins interact with core histones through their plant homeodomains (PHDs) and with histone acetyltransferase (HAT) and histone deacetylase (HDAC) complexes to alter chromatin structure. Here we identify a lamin interaction domain (LID) found only in ING proteins, through which they bind to and colocalize with lamin A. Lamin knockout (LMNA(-/-)) cells show reduced levels of ING1 that mislocalize. Ectopic lamin A expression increases ING1 levels and re-targets it to the nucleus to act as an epigenetic regulator. ING1 lacking the LID does not interact with lamin A or affect apoptosis. In LMNA(-/-) cells, apoptosis is not affected by ING1. Mutation of lamin A results in several laminopathies, including Hutchinson-Gilford progeria syndrome (HGPS), a severe premature ageing disorder. HGPS cells have reduced ING1 levels that mislocalize. Expression of LID peptides to block lamin A-ING1 interaction induces phenotypes reminiscent of laminopathies including HGPS. These data show that targeting of ING1 to the nucleus by lamin A maintains ING1 levels and biological function. Known roles for ING proteins in regulating apoptosis and chromatin structure indicate that loss of lamin A-ING interaction may be an effector of lamin A loss, contributing to the HGPS phenotype.
Assuntos
Núcleo Celular/genética , Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lamina Tipo A/metabolismo , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/metabolismo , Mapeamento Cromossômico , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Humanos , Proteína 1 Inibidora do Crescimento , Peptídeos e Proteínas de Sinalização Intracelular/química , Rim/citologia , Lamina Tipo A/análise , Lamina Tipo A/genética , Camundongos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Progéria/genética , Progéria/metabolismo , Progéria/patologia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transfecção , Proteínas Supressoras de Tumor/químicaRESUMO
Anthracyclines are useful chemotherapeutic agents whose utility is limited by the development of irreversible cardiotoxicity. When tested, the pediatric population demonstrates an increased sensitivity to the cardiotoxicity of this class of agents, although the reasons for this increased sensitivity are unclear. The sarcoplasmic reticulum (SR) is a target for anthracycline cardiotoxicity in adults, but the effects of anthracycline on the SR in developing myocardium have not been examined. It may be possible to gain insight into the mechanisms of cardiotoxicity through a comparative approach. We compared the acute effects of doxorubicin, daunorubicin, and caffeine on contractile function in adult and neonatal rabbit myocardium. Frequency-dependent contractility, 90% relaxation times, and postrest potentiated contractions (a uniquely SR-dependent phenomenon) in adult myocardium were inhibited in a concentration- dependent manner. Neonatal myocardium, however, was resistant to the effects of these agents. The degree of contractile dysfunction was consistent with the difference in SR maturation between adult and developing myocardium. Anthracyclines exhibited effects similar to those of caffeine, an agent known to render the SR nonfunctional by the depletion of the releasable SR calcium pool. These results suggest that anthracyclines induce acute cardiac lesions through effects on the SR in adults, whereas cardiotoxic effects in the developing myocardium may proceed by a different mechanism.
