RESUMO
Proteins that are essential for embryo production, cell division and early embryonic events are frequently reused later in embryogenesis, during organismal development or in the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that uses expression of a fusion between a GFP-targeting nanobody and a SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins for degradation. When combined with endogenous locus GFP tagging by CRISPR-Cas9 or with rescue of a null mutant with a GFP fusion, this approach enables routine and efficient tissue-specific protein ablation. We show that this approach works in multiple tissues - the epidermis, intestine, body wall muscle, ciliated sensory neurons and touch receptor neurons - where it recapitulates expected loss-of-function mutant phenotypes. The transgene toolkit and the strain set described here will complement existing approaches to enable routine analysis of the tissue-specific roles of C. elegans proteins.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Genes de Helmintos , Técnicas Genéticas , Proteínas de Fluorescência Verde/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Mutação , Proteólise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Distribuição TecidualRESUMO
Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions of essential genes. We report here asimple procedure to generate ts mutations using error-prone PCR within pcp1 that encodes aspindle pole body (SPB) component in Schizosaccharomyces pombe. This manipulation is not restricted to pcp1, and can be suited to any essential genes involved in other processes.
Assuntos
Genes Fúngicos , Mutação , Reação em Cadeia da Polimerase/métodos , Schizosaccharomyces/genética , Corpos Polares do Fuso/metabolismo , Temperatura , Proteínas de Ciclo Celular , Proteínas Nucleares/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Upon establishment of proper kinetochore-microtubule attachment, the spindle assembly checkpoint (SAC) must be silenced to allow onset of anaphase, which is when sister chromatids segregate equally to two daughter cells. However, how proper kinetochore-microtubule attachment leads to timely anaphase onset remains elusive. Furthermore, the molecular mechanisms of chromosome movement during anaphase A remain unclear. In this study, we show that the fission yeast Alp7/TACC protein recruits a protein complex consisting of the kinesin-8 (Klp5-Klp6) and protein phosphatase 1 (PP1) to the kinetochore upon kinetochore-microtubule attachment. Accumulation of this complex at the kinetochore, on the one hand, facilitates SAC inactivation through PP1, and, on the other hand, accelerates polewards chromosome movement driven by the Klp5-Klp6 motor. We identified an alp7 mutant that had specific defects in binding to the Klp5-Klp6-PP1 complex but with normal localisation to the microtubule and kinetochore. Consistent with our proposition, this mutant shows delayed anaphase onset and decelerated chromosome movement during anaphase A. We propose that the recruitment of kinesin-8-PP1 to the kinetochore through Alp7/TACC interaction plays a crucial role in regulation of timely mitotic progression and chromosome movement during anaphase A.
Assuntos
Anáfase/genética , Cinesinas/genética , Proteínas Associadas aos Microtúbulos/genética , Proteína Fosfatase 1/genética , Proteínas de Schizosaccharomyces pombe/genética , Segregação de Cromossomos/genética , Cinesinas/metabolismo , Cinetocoros , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/genética , Complexos Multiproteicos , Proteínas Nucleares , Ligação Proteica , Proteína Fosfatase 1/metabolismo , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Mis-regulation (e.g. overproduction) of the human Ndc80/Hec1 outer kinetochore protein has been associated with aneuploidy and tumourigenesis, but the genetic basis and underlying mechanisms of this phenomenon remain poorly understood. Recent studies have identified the ubiquitous Ndc80 internal loop as a protein-protein interaction platform. Binding partners include the Ska complex, the replication licensing factor Cdt1, the Dam1 complex, TACC-TOG microtubule-associated proteins (MAPs) and kinesin motors. We review the field and propose that the overproduction of Ndc80 may unfavourably absorb these interactors through the internal loop domain and lead to a change in the equilibrium of MAPs and motors in the cells. This sequestration will disrupt microtubule dynamics and the proper segregation of chromosomes in mitosis, leading to aneuploid formation. Further investigation of Ndc80 internal loop-MAPs interactions will bring new insights into their roles in kinetochore-microtubule attachment and tumourigenesis.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Animais , Carcinogênese/metabolismo , Proteínas do Citoesqueleto , Expressão Gênica , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Proteínas Nucleares/química , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de ProteínasRESUMO
Microtubules (MTs) are dynamic components of the cytoskeleton and play essential roles in morphogenesis and maintenance of tissue and cell integrity. Despite recent advances in understanding MT ultrastructure, organization, and growth control, how cells regulate MT organization at the cell cortex remains poorly understood. The EFA-6/EFA6 proteins are recently identified membrane-associated proteins that inhibit cortical MT dynamics. Here, combining visualization of endogenously tagged C. elegans EFA-6 with genetic screening, we uncovered tubulin-dependent regulation of EFA-6 patterning. In the mature epidermal epithelium, EFA-6 forms punctate foci in specific regions of the apical cortex, dependent on its intrinsically disordered region (IDR). We further show the EFA-6 IDR is sufficient to form biomolecular condensates in vitro. In screens for mutants with altered GFP::EFA-6 localization, we identified a novel gain-of-function (gf) mutation in an α-tubulin tba-1 that induces ectopic EFA-6 foci in multiple cell types. tba-1(gf) animals exhibit temperature-sensitive embryonic lethality, which is partially suppressed by efa-6(lf), indicating the interaction between tubulins and EFA-6 is important for normal development. TBA-1(gf) shows reduced incorporation into filamentous MTs but has otherwise mild effects on cellular MT organization. The ability of TBA-1(gf) to trigger ectopic EFA-6 foci formation requires ß-tubulin TBB-2 and the chaperon EVL-20/Arl2. The tba-1(gf)-induced EFA-6 foci display slower turnover, contain the MT-associated protein TAC-1/TACC, and require the EFA-6 MTED. Our results reveal a novel crosstalk between cellular tubulins and cortical MT regulators in vivo.
RESUMO
mRNA decay factors regulate mRNA turnover by recruiting non-translating mRNAs and targeting them for translational repression and mRNA degradation. How mRNA decay pathways regulate cellular function in vivo with specificity is poorly understood. Here, we show that C. elegans mRNA decay factors, including the translational repressors CAR-1/LSM14 and CGH-1/DDX6, and the decapping enzymes DCAP-1/DCP1, function in neurons to differentially regulate axon development, maintenance, and regrowth following injury. In neuronal cell bodies, CAR-1 fully colocalizes with CGH-1 and partially colocalizes with DCAP-1, suggesting that mRNA decay components form at least two types of cytoplasmic granules. Following axon injury in adult neurons, loss of CAR-1 or CGH-1 results in increased axon regrowth and growth cone formation, whereas loss of DCAP-1 or DCAP-2 results in reduced regrowth. To determine how CAR-1 inhibits regrowth, we analyzed mRNAs bound to pan-neuronally expressed GFP::CAR-1 using a crosslinking and immunoprecipitation-based approach. Among the putative mRNA targets of CAR-1, we characterized the roles of micu-1, a regulator of the mitochondrial calcium uniporter MCU-1, in axon injury. We show that loss of car-1 results increased MICU-1 protein levels, and that enhanced axon regrowth in car-1 mutants is dependent on micu-1 and mcu-1. Moreover, axon injury induces transient calcium influx into axonal mitochondria, dependent on MCU-1. In car-1 loss-of-function mutants and in micu-1 overexpressing animals, the axonal mitochondrial calcium influx is more sustained, which likely underlies enhanced axon regrowth. Our data uncover a novel pathway that controls axon regrowth through axonal mitochondrial calcium uptake.
Assuntos
Axônios/fisiologia , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Cálcio/metabolismo , Endorribonucleases/genética , Regeneração Nervosa/genética , RNA Nucleotidiltransferases/genética , Proteínas de Ligação a RNA/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endorribonucleases/metabolismo , Dinâmica Mitocondrial , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Caenorhabditis elegans provides an amenable system to explore whether newly composed ribosomes are required to progress through development. Despite the complex pattern of tissues that are formed during embryonic development, we found that null homozygotes lacking any of the five different ribosomal proteins (RPs) can produce fully functional first-stage larvae, with similar developmental competence seen upon complete deletion of the multi-copy ribosomal RNA locus. These animals, relying on maternal but not zygotic contribution of ribosomal components, are capable of completing embryogenesis. In the absence of new ribosomal components, the resulting animals are arrested before progression from the first larval stage and fail in two assays for postembryonic plasticity of neuronal structure. Mosaic analyses of larvae that are a mixture of ribosome-competent and non-competent cells suggest a global regulatory mechanism in which ribosomal insufficiency in a subset of cells triggers organism-wide growth arrest.
Assuntos
Caenorhabditis elegans/embriologia , Desenvolvimento Embrionário , Organogênese , Ribossomos/metabolismo , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Mosaicismo , Mutação/genética , Fenótipo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição Gênica , Zigoto/metabolismoRESUMO
Development of a neuron critically depends on the organization of its cytoskeleton. Cytoskeletal components, such as tubulins and actins, have the remarkable ability to organize themselves into filaments and networks to support specialized and compartmentalized functions. Alterations in cytoskeletal proteins have long been associated with a variety of neurodevelopmental disorders. This review focuses on recent findings, primarily from forward genetic screens in Caenorhabditis elegans that illustrate how different tubulin protein isotypes can play distinct roles in neuronal development and function. Additionally, we discuss studies revealing new regulators of the actin cytoskeleton, and highlight recent technological advances in in vivo imaging and functional dissection of the neuronal cytoskeleton.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Sistema Nervoso/citologia , Sistema Nervoso/crescimento & desenvolvimento , Neurônios/metabolismo , Animais , Caenorhabditis elegans , HumanosRESUMO
The PIWI-interacting RNA (piRNA) pathway has long been thought to function solely in the germline, but evidence for its functions in somatic cells is emerging. Here we report an unexpected role for the piRNA pathway in Caenorhabditis elegans sensory axon regeneration after injury. Loss of function in a subset of components of the piRNA pathway results in enhanced axon regrowth. Two essential piRNA factors, PRDE-1 and PRG-1/PIWI, inhibit axon regeneration in a gonad-independent and cell-autonomous manner. By smFISH analysis we find that prde-1 transcripts are present in neurons, as well as germ cells. The piRNA pathway inhibits axon regrowth independent of nuclear transcriptional silencing but dependent on the slicer domain of PRG-1/PIWI, suggesting that post-transcriptional gene silencing is involved. Our results reveal the neuronal piRNA pathway as a novel intrinsic repressor of axon regeneration.
Assuntos
Proteínas Argonautas/metabolismo , Axônios/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , RNA Interferente Pequeno/metabolismo , Regeneração , Animais , Caenorhabditis elegans , Células Germinativas/metabolismo , Transdução de SinaisRESUMO
The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.
Assuntos
Axônios/metabolismo , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , NAD/metabolismo , Regeneração Nervosa/genética , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Axônios/ultraestrutura , Axotomia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Testes Genéticos , Repetição Kelch , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Anotação de Sequência Molecular , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismoRESUMO
Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end-directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end-directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.
Assuntos
Segregação de Cromossomos/fisiologia , Cinesinas/fisiologia , Fuso Acromático/fisiologia , Anáfase , Catepsina A/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Mitose , Ligação Proteica , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático/metabolismo , Corpos Polares do Fuso/metabolismoRESUMO
The capacity of an axon to regenerate is regulated by its external environment and by cell-intrinsic factors. Studies in a variety of organisms suggest that alterations in axonal microtubule (MT) dynamics have potent effects on axon regeneration. We review recent findings on the regulation of MT dynamics during axon regeneration, focusing on the nematode Caenorhabditis elegans. In C. elegans the dual leucine zipper kinase (DLK) promotes axon regeneration, whereas the exchange factor for Arf6 (EFA-6) inhibits axon regeneration. Both DLK and EFA-6 respond to injury and control axon regeneration in part via MT dynamics. How the DLK and EFA-6 pathways are related is a topic of active investigation, as is the mechanism by which EFA-6 responds to axonal injury. We evaluate potential candidates, such as the MT affinity-regulating kinase PAR-1/MARK, in regulation of EFA-6 and axonal MT dynamics in regeneration.
RESUMO
The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1.
Assuntos
Cinetocoros/enzimologia , Proteínas Quinases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Motivos de Aminoácidos , Pontos de Checagem da Fase M do Ciclo Celular , Mitose , Proteínas Quinases/química , Transporte Proteico , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Moduladores de Tubulina/farmacologiaRESUMO
The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Corpos Polares do Fuso/metabolismo , Sequência de Aminoácidos , Antígenos/metabolismo , Proteínas de Ciclo Celular , Sequência Conservada , Proteínas Associadas aos Microtúbulos/química , Mitose , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Transporte Proteico , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/químicaRESUMO
Our understanding of the structure and function of kinetochores has advanced dramatically over the past 10 years, yet how the plus end of spindle microtubules interacts with the kinetochore and establishes amphitelic attachment for proper sister chromatid segregation remains unresolved. However, several recent reports from different organisms have shed new light on this issue. A key player in microtubule-kinetochore interaction is the conserved Ndc80 outer kinetochore complex. In both yeast and human cells in particular, a ubiquitous internal 'loop' found in the Ndc80 molecule interrupting its C-terminal coiled-coil domain plays critical roles in protein-protein interaction, by recruiting microtubule-binding proteins to ensure proper kinetochore-microtubule attachment. In this commentary, we summarise the recent progress made and discuss the evolutionary significance of this loop's role in microtubule dynamics at the kinetochore for accurate chromosome segregation.
RESUMO
The Ndc80 outer kinetochore complex plays a critical role in kinetochore-microtubule attachment, yet our understanding of the mechanism by which this complex interacts with spindle microtubules for timely and accurate chromosome segregation remains limited. Here we address this issue using an ndc80 mutant (ndc80-NH12) from fission yeast that contains a point mutation within a ubiquitous internal loop. This mutant is normal for assembly of the Ndc80 complex and bipolar spindle formation yet defective in proper end-on attachment to the spindle microtubule, with chromosome alignment defects and missegregation happening later during mitosis. We find that ndc80-NH12 exhibits impaired localization of the microtubule-associated protein complex Alp7/transforming acidic coiled coil (TACC)-Alp14/tumor-overexpressed gene (TOG) to the mitotic kinetochore. Consistently, wild-type Ndc80 binds these two proteins, whereas the Ndc80-NH12 mutant protein displays a substantial reduction of interaction. Crucially, forced targeting of Alp7-Alp14 to the outer kinetochore rescues ndc80-NH12-mutant phenotypes. The loop was previously shown to bind Dis1/TOG, by which it ensures initial chromosome capture during early mitosis. Strikingly, ndc80-NH12 is normal in Dis1 localization. Genetic results indicate that the loop recruits Dis1/TOG and Alp7/TACC-Alp14/TOG independently. Our work therefore establishes that the Ndc80 loop plays sequential roles in spindle-kinetochore attachment by connecting the Ndc80 complex to Dis1/TOG and Alp7/TACC-Alp14/TOG.