Proteins that are essential for embryo production, cell division and early embryonic events frequently reused later in embryogenesis, during organismal development or the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method uses expression of fusion between GFP-targeting nanobody SOCS-box containing ubiquitin ligase adaptor to target GFP-tagged proteins degradation. When combined with endogenous locus...

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies injury. Here, we report novel pathways regeneration, identified by extending our previous function-based screen using C. elegans mechanosensory neuron axotomy model. We identify an unexpected role nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, regeneration. NMAT-2 inhibits regrowth via...

The Ndc80 outer kinetochore complex plays a critical role in kinetochore-microtubule attachment, yet our understanding of the mechanism by which this interacts with spindle microtubules for timely and accurate chromosome segregation remains limited. Here we address issue using an ndc80 mutant (ndc80-NH12) from fission yeast that contains point mutation within ubiquitous internal loop. This is normal assembly bipolar formation defective proper end-on attachment to microtubule, alignment...

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by plus-end–directed motor kinesin-5 that generates outward force to establish bipolarity. Its inhibition leads emergence of monopolar spindles with arrest. Intriguingly, simultaneous inactivation minus-end–directed kinesin-14 restores bipolarity in systems. Here we show fission yeast, three independent pathways contribute absence kinesin-5/Cut7 and kinesin-14/Pkl1. One...

The capacity of an axon to regenerate is regulated by its external environment and cell-intrinsic factors. Studies in a variety organisms suggest that alterations axonal microtubule (MT) dynamics have potent effects on regeneration. We review recent findings the regulation MT during regeneration, focusing nematode Caenorhabditis elegans. In C. elegans dual leucine zipper kinase (DLK) promotes whereas exchange factor for Arf6 (EFA-6) inhibits Both DLK EFA-6 respond injury control regeneration...

mRNA decay factors regulate turnover by recruiting non-translating mRNAs and targeting them for translational repression degradation. How pathways cellular function in vivo with specificity is poorly understood. Here, we show that C. elegans factors, including the repressors CAR-1/LSM14 CGH-1/DDX6, decapping enzymes DCAP-1/DCP1, neurons to differentially axon development, maintenance, regrowth following injury. In neuronal cell bodies, CAR-1 fully colocalizes CGH-1 partially DCAP-1,...

Upon establishment of proper kinetochore-microtubule attachment, the spindle assembly checkpoint (SAC) must be silenced to allow anaphase onset in which sister chromatids segregate equally two daughter cells. However, how attachment leads timely remains elusive. Furthermore, molecular mechanisms chromosome movement during A remain unclear. In this study, we show that fission yeast Alp7/TACC protein recruits a complex consisting kinesin-8 (Klp5-Klp6) and phosphatase 1 (PP1) kinetochore upon...

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role bipolar assembly. However, it remains elusive how proteins are recruited centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within domain required for localisation. Critically, these sequences essential functions of Alp7, including proper formation progression. Moreover, identified...

The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment a cohort SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, component outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as platform for Bub3 localization upon its phosphorylation by Mph1/MPS1. Ndc80 protein, major...

Temperature-sensitive (ts) mutants provide powerful tools for investigation of cellular functions 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 pcp1, and can be suited any genes involved other processes.

ABSTRACT Microtubules (MTs) are dynamic components of the cytoskeleton and play essential roles in morphogenesis maintenance tissue cell integrity. Despite recent advances understanding MT ultrastructure, organization, growth control, how cells regulate organization at cortex remains poorly understood. The EFA-6/EFA6 proteins recently identified membrane-associated that inhibit cortical dynamics. Here, combining visualization endogenously tagged C. elegans EFA-6 with genetic screening, we...

Abstract Proteins essential for embryo production, cell division, and early embryonic events are frequently re-utilized later in embryogenesis, during organismal development, or the adult. Examining protein function across these different biological contexts requires tissue-specific perturbation. Here, we describe a method that utilizes expression of fusion between GFP-targeting nanobody SOCS-box containing ubiquitin ligase adaptor to target GFP tagged proteins degradation. When combined...

The spindle pole body (SPB) in fungi is the equivalent of animal centrosome. A number previous studies have identified many, if not all, components SPB. SPB structural platform for microtubule nucleation and plays important roles, both mitosis meiosis. absolutely essential cell survival its abnormalities give rise to aberrant division morphogenesis. Therefore, it crucial understand how organises itself functions individual are regulated. We report here a procedure generate temperature...

The spindle pole body (SPB) in fungi is the equivalent of animal centrosome. A number previous studies have identified many, if not all, components SPB. SPB structural platform for microtubule nucleation and plays important roles, both mitosis meiosis. absolutely essential cell survival its abnormalities give rise to aberrant division morphogenesis. Therefore, it crucial understand how organises itself functions individual are regulated. We report here a procedure generate temperature...

Abstract Temperature-sensitive (ts) mutants provide powerful tools, thereby investigating cellular functions of essential genes. We report here a simple procedure to generate ts mutations using error-prone PCR in pcp1 that encodes spindle pole body (SPB) component Schizosaccharomyces pombe . This manipulation is not restricted analysis Pcp1, and can be suited any genes involved other processes.