Suchergebnisse

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. ; The authors acknowledge the support of the BioSciences Screening i3S Scientific Platform. Funding for this project was provided by the European Research Council under the European Union's Seventh Framework Programme (ERC grant agreement n o ERC-2013-StG-338410-DYNEINOME to R.G.), by the European Molecular Biology Organization (EMBO Installation Grant 2545 to R.G.), by the Fundacao para a Ciencia e a Tecnologia (IF/01015/2013/CP1157/CT0006 to R.G. and SFRH/BD/103495/2014 to H. R.), and by 'Norte-01-0145-FEDER-000029-Advancing cancer research: from basic knowledge to application', supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (FEDER)

In mitosis, themolecular motor dynein is recruited to kinetochores by the Rod-Zw10-Zwilch complex (RZZ) and Spindly to control spindle assembly checkpoint (SAC) signaling and microtubule attachment. How the ubiquitous dynein co-factors Lis1 and NudE contribute to these functions remains poorly understood. Here, we show that the C. elegans NudE homolog NUD-2 is dispensable for dynein- and LIS-1-dependent mitotic spindle assembly in the zygote. This facilitates functional characterization of kinetochore-localized NUD-2, which is recruited by the CENP-F-like proteins HCP-1 and HCP-2 independently of RZZ- Spindly and dynein-LIS-1. Kinetochore dynein levels are reduced in ¿nud-2 embryos, and, as occurs upon RZZ inhibition, loss of NUD-2 delays the formation of load-bearing kinetochore-microtubule attachments and causes chromatin bridges in anaphase. Survival of ¿nud-2 embryos requires a functional SAC, and kinetochores without NUD-2 recruit an excess of SAC proteins. Consistent with this, SAC signaling in early ¿nud-2 embryos extends mitotic duration and prevents high rates of chromosome mis-segregation. Our results reveal that both NUD-2 and RZZ-Spindly are essential for dynein function at kinetochores, and that the gain in SAC strength during early embryonic development is relevant under conditions that mildly perturb mitosis. ; This project was funded by the European Research Council under the European Union's Seventh Framework Programme (ERC grant agreement no. ERC-2013-StG-338410-DYNEINOME to R.G.), and the European Union's Horizon 2020 Research and Innovation Programme (ERC grant agreement no. ERC-2014-StG-640553-ACTOMYO to A.X.C.). Additional funding was fromthe European Molecular Biology Organization (Installation Grant; R.G.), from the Fundação para a Ciência e a Tecnologia(IF/01015/2013/CP1157/CT0006; R.G.), and from the project 'Norte-01-0145-FEDER-000029, Advancing cancer research: from basic knowledge to application', supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (FEDER). Deposited in PMC for immediate release.

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-bind-ing 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. ; This work was financed by the Fundo Europeu de Desenvolvimento Regional (FEDER) through the Norte Portugal Regional Operational Programme (NORTE 2020), Portugal 2020 (RG); by the Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project NORTE-01-0145-FEDER-030507 (RG); by FCT fellowships IF/01015/2013/CP1157/CT0006 (RG) and SFRH/ BPD/101898/2014 (DJB); by the European Research Council under the European Union's Seventh Framework Programme, ERC grant agreement no. ERC-2013-StG-338410-DYNEINOME (RG), and by a start-up package of the University of Colorado (BV). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The deposited article is a post-print version and has peer review. This deposit is composed by the main article, and it hasn't any supplementary materials associated. There is no public supplementary material available. ; The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres. ; Wellcome Trust grants: ( 073915, 095021, 077707, 092076); NIH grants: (R01-GM082989, R37 GM32238, GM088313, GM074150, R01GM088371, 5R01GM61169, GM083988, R01GM088716, R37GM024364, R01GM074728, R01 GM063045, R01 GM098500, GM059618, GM69429, GM083988, Core Grant CA06927); Government of Japan- Funding Program for Next Generation World-Leading Researchers grant: (LS122); EMBO Installation Grant; ERC grants: (241548 [MitoSys], 258068 [Systems Microscopy], 281198, BioSynCen, KINSIGN, PRECISE); Cancer Research UK; NIH/NIGMS grant: (R01-50412, R01-50412); Oklahoma Center for Advancement of Science and Technology; McCasland Foundation; Japanese Society for the Promotion of Science; Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT); FCT grants: (BIA-BCM/100557/2008, BIAPRO/100537/2008, PTDC/SAU-GMG/099704/2008, PTDC/SAU-ONC/112917/2009, COMPETE-FEDER, ON2); Netherlands Organisation for Scientific Research grants: (NWO-Vici-016.130.661, NWO-Vici-91812610); Dutch Cancer Society grants: (UU2009-4311, UU2011-5134); Spanish Ministry of Economy grants: (CSD2007-015, SAF2010-25157); EU grant: (Marie Curie ITN "Nucleosome 4D"); BBSRC grant: (BB/I021353/1, BB/H013024); Kazusa DNA Research Institute Foundation; CCSRI grant: (700824), NSF grant: (award number 1024973); Swiss National Foundation; University of Geneva; Foundation Louis-Jeantet; March of Dimes grant: (6-FY10-294); Science Foundation Ireland grant: (12/IA/1370); Commonwealth of Pennsylvania. ; info:eu-repo/semantics/publishedVersion