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- Nat Cell Biol 11(9):1045 (2009)
- Ambition, surprise and delight: necessary lessons
- Nat Cell Biol 11(9):1046 (2009)
I want to purchase this article Register now Price: US$32 In order to purchase this article you must be a registered user. I want to subscribe to Nature Cell Biology Select this option to purchase a personal subscription to Nature Cell Biology. - SAD kinase keeps centrosomes lonely
- Nat Cell Biol 11(9):1047-1048 (2009)
I want to purchase this article Register now Price: US$18 In order to purchase this article you must be a registered user. I want to subscribe to Nature Cell Biology Select this option to purchase a personal subscription to Nature Cell Biology. - RISC hitches onto endosome trafficking
- Nat Cell Biol 11(9):1049-1051 (2009)
I want to purchase this article Register now Price: US$18 In order to purchase this article you must be a registered user. I want to subscribe to Nature Cell Biology Select this option to purchase a personal subscription to Nature Cell Biology. - Steroid hormone pulsing drives cyclic gene expression
- Nat Cell Biol 11(9):1051-1053 (2009)
I want to purchase this article Register now Price: US$18 In order to purchase this article you must be a registered user. I want to subscribe to Nature Cell Biology Select this option to purchase a personal subscription to Nature Cell Biology. - Aurora A moonlights in neurite extension
- Nat Cell Biol 11(9):1053-1054 (2009)
I want to purchase this article Register now Price: US$18 In order to purchase this article you must be a registered user. I want to subscribe to Nature Cell Biology Select this option to purchase a personal subscription to Nature Cell Biology. - Research highlights
- Nat Cell Biol 11(9):1055 (2009)
I want to purchase this article Register now Price: US$32 In order to purchase this article you must be a registered user. I want to subscribe to Nature Cell Biology Select this option to purchase a personal subscription to Nature Cell Biology. - An essential role of the aPKC–Aurora A–NDEL1 pathway in neurite elongation by modulation of microtubule dynamics
- Nat Cell Biol 11(9):1057-1068 (2009)
Orchestrated remodelling of the cytoskeketon is prominent during neurite extension. In contrast with the extensive characterization of actin filament regulation, little is known about the dynamics of microtubules during neurite extension. Here we identify an atypical protein kinase C (aPKC)–Aurora A–NDEL1 pathway that is crucial for the regulation of microtubule organization during neurite extension. aPKC phosphorylates Aurora A at Thr 287 (T287), which augments interaction with TPX2 and facilitates activation of Aurora A at the neurite hillock, followed by phosphorylation of NDEL1 at S251 and recruitment. Suppression of aPKC, Aurora A or TPX2, or disruption of Ndel1, results in severe impairment of neurite extension. Analysis of microtubule dynamics with a microtubule plus-end marker revealed that suppression of the aPKC–Aurora A–NDEL1 pathway resulted in a significant decrease in the frequency of microtubule emanation from the microtubule organizing centre (M! TOC), suggesting that Aurora A acts downstream of aPKC. These findings demonstrate a surprising role of aPKC–Aurora A–NDEL1 pathway in microtubule remodelling during neurite extension. - Golgi-derived CLASP-dependent microtubules control Golgi organization and polarized trafficking in motile cells
- Nat Cell Biol 11(9):1069-1080 (2009)
Microtubules are indispensable for Golgi complex assembly and maintenance, which are integral parts of cytoplasm organization during interphase in mammalian cells. Here, we show that two discrete microtubule subsets drive two distinct, yet simultaneous, stages of Golgi assembly. In addition to the radial centrosomal microtubule array, which positions the Golgi in the centre of the cell, we have identified a role for microtubules that form at the Golgi membranes in a manner dependent on the microtubule regulators CLASPs. These Golgi-derived microtubules draw Golgi ministacks together in tangential fashion and are crucial for establishing continuity and proper morphology of the Golgi complex. We propose that specialized functions of these two microtubule arrays arise from their specific geometries. Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs, suggesting that correct organization of the Golgi complex! by microtubules is essential for cell polarization and motility. - SADB phosphorylation of -tubulin regulates centrosome duplication
Alvarado-Kristensson M Rodríguez MJ Silió V Valpuesta JM Carrera AC - Nat Cell Biol 11(9):1081-1092 (2009)
Symmetrical cell division requires duplication of DNA and protein content to generate two daughter cells. Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood. We describe an alternative splice form of SadB encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action through phosphorylation of -tubulin on Ser 131, as expression of a phosphomimetic Ser 131-to-Asp -tubulin mutant alone increased centrosome numbers, whereas non-phosphorylatable Ala 131--tubulin impaired centrosome duplication. We propose that SADB kinase activity controls centrosome homeostasis by regulating phosphorylation of -tubulin. - Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription
- Nat Cell Biol 11(9):1093-1102 (2009)
Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian horm! one release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation. - Fused sister kinetochores initiate the reductional division in meiosis I
- Nat Cell Biol 11(9):1103-1108 (2009)
During meiosis I the genome is reduced to the haploid content by a coordinated reductional division event. Homologous chromosomes align, recombine and segregate while the sister chromatids co-orient and move to the same pole1, 2. Several data suggest that sister kinetochores co-orient early in metaphase I and that sister chromatid cohesion (which requires Rec8 and Shugoshin) supports monopolar orientation. Nevertheless, it is unclear how the sister kinetochores function as single unit during this period. A gene (monopolin)3 with a co-orienting role was identified in Saccharomyces cerevisiae; however, it does not have the same function in fission yeast4 and no similar genes have been found in other species. Here we pursue this issue using knockdown mutants of the core kinetochore protein MIS12 (minichromosome instability 12). MIS12 binds to base of the NDC80 complex, which in turn binds directly to microtubules5, 6, 7. In maize plants with systemically reduced levels of! MIS12, a visible MIS12–NDC80 bridge between sister kinetochores at meiosis I is broken. Kinetochores separate and orient randomly in metaphase I, causing chromosomes to stall in anaphase due to normal cohesion, marked by Shugoshin, between the chromatids. The data establish that sister kinetochores in meiosis I are fused by a shared microtubule-binding face and that this direct linkage is required for reductional division. - A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast
- Nat Cell Biol 11(9):1109-1115 (2009)
Kinetochores are large multiprotein complexes that mediate chromosome segregation in all eukaryotes by dynamically connecting specialized chromosome regions, termed centromeres, to the plus-ends of spindle microtubules1, 2. Even the relatively simple kinetochores of the budding yeast Saccharomyces cerevisiae consist of more than 80 proteins, making analysis of their respective roles a daunting task3. Here, we have developed a system that allows us to artificially recruit proteins to DNA sequences and determine whether they can provide any aspect of kinetochore function in vivo. We show that artificial recruitment of the microtubule-binding Dam1 complex to a plasmid lacking any centromere DNA is sufficient to confer mitotic stabilization. The Dam1-based artificial kinetochores are able to attach, bi-orient and segregate mini-chromosomes on the mitotic spindle, and they bypass the requirement for essential DNA-binding components of natural kinetochores. Thus, we have bui! lt a simplified chromosome segregation system by directly recruiting a microtubule force-transducing component to DNA. - Recruiting a microtubule-binding complex to DNA directs chromosome segregation in budding yeast
- Nat Cell Biol 11(9):1116-1120 (2009)
Accurate chromosome segregation depends on the kinetochore, which is the complex of proteins that link microtubules to centromeric DNA1. The kinetochore of the budding yeast Saccharomyces cerevisiae consists of more than 80 proteins assembled on a 125-bp region of DNA1. We studied the assembly and function of kinetochore components by fusing individual kinetochore proteins to the lactose repressor (LacI) and testing their ability to improve segregation of a plasmid carrying tandem repeats of the lactose operator (LacO). Targeting Ask1, a member of the Dam1–DASH microtubule-binding complex, creates a synthetic kinetochore that performs many functions of a natural kinetochore: it can replace an endogenous kinetochore on a chromosome, bi-orient sister kinetochores at metaphase during the mitotic cycle, segregate sister chromatids, and repair errors in chromosome attachment. We show the synthetic kinetochore functions do not depend on the DNA-binding components of the na! tural kinetochore but do require other kinetochore proteins. We conclude that tethering a single kinetochore protein to DNA triggers assembly of the complex structure that directs mitotic chromosome segregation. - Organizer restriction through modulation of Bozozok stability by the E3 ubiquitin ligase Lnx-like
- Nat Cell Biol 11(9):1121-1127 (2009)
The organizer anchors the primary embryonic axis, and balance between dorsal (organizer) and ventral domains is fundamental to body patterning. LNX (ligand of Numb protein-X) is a RING finger and four PDZ domain-containing E3 ubiquitin ligase1, 2. LNX serves as a binding platform and may have a role in cell fate determination, but its in vivo functions are unknown1, 2, 3, 4, 5. Here we show that Lnx-l (Lnx-like) functions as a critical regulator of dorso-ventral axis formation in zebrafish. Depletion of Lnx-l using specific antisense morpholinos (MOs) caused strong embryonic dorsalization. We identified Bozozok (Boz, also known as Dharma or Nieuwkoid) as a binding partner and substrate of Lnx-l. Boz is a homeodomain-containing transcriptional repressor induced by canonical Wnt signalling that is critical for dorsal organizer formation6, 7, 8, 9, 10, 11, 12. Lnx-l induced K48-linked polyubiquitylation of Boz, leading to its proteasomal degradation in human 293T cells an! d in zebrafish embryos. Dorsalization induced by Boz overexpression was suppressed by raising the level of Lnx-l, but Lnx-l failed to counteract dorsalization caused by mutant Boz lacking a critical motif for Lnx-l binding. Furthermore, dorsalization induced by depletion of Lnx-l was alleviated by attenuation of Boz expression. We conclude that Lnx-l modulates Boz activity to prevent the invasion of ventral regions of the embryo by organizer tissue. These studies introduce a ubiquitin ligase, Lnx-l, as a balancing modulator of axial patterning in the zebrafish embryo. - Axin determines cell fate by controlling the p53 activation threshold after DNA damage
- Nat Cell Biol 11(9):1128-1134 (2009)
Cells can undergo either cell-cycle arrest or apoptosis after genotoxic stress, based on p53 activity1, 2, 3, 4, 5, 6. Here we show that cellular fate commitment depends on Axin forming distinct complexes with Pirh2, Tip60, HIPK2 and p53. In cells treated with sublethal doses of ultra-violet (UV) radiation or doxorubicin (Dox), Pirh2 abrogates Axin-induced p53 phosphorylation at Ser 46 catalysed by HIPK2, by competing with HIPK2 for binding to Axin. However, on lethal treatment, Tip60 interacts with Axin and abrogates Pirh2–Axin binding, forming an Axin–Tip60–HIPK2–p53 complex that allows maximal p53 activation to trigger apoptosis. We also provide evidence that the ATM/ATR pathway mediates the Axin–Tip60 complex assembly. An axin mutation promotes carcinogenesis in AxinFu/+ (Axin-Fused) mice, consistent with a dominant-negative role for AxinFu in p53 activation. Thus, Axin is a critical determinant in p53-dependent tumour suppression in which Pirh2 and Tip60! have different roles in triggering cell-cycle arrest or apoptosis depending on the severity of genotoxic stress. - p53 isoforms 133p53 and p53 are endogenous regulators of replicative cellular senescence
- Nat Cell Biol 11(9):1135-1142 (2009)
The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumour progression in vivo1, 2, 3. We show that the human p53 isoforms 133p53 and p534 function in an endogenous regulatory mechanism for p53-mediated replicative senescence. Induced p53 and diminished 133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA5, 6, 7, 8, 9, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA (short interfering RNA)-mediated knockdown of endogenous 133p53 induced cellular senescence, which was attributed to the regulation of p21WAF1 and other p53 transcriptional target genes. In overexpression experiments, whereas p53 cooperated with full-length p53 to accelerate cellular senescence, 133p53 repressed miR-34a e! xpression and extended the cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (that is, elevated p53 and reduced 133p53) was observed in vivo in colon adenomas with senescent phenotypes10, 11. The increased 133p53 and decreased p53 isoform expression found in colon carcinoma may signal an escape from the senescence barrier during the progression from adenoma to carcinoma. - Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity
- Nat Cell Biol 11(9):1143-1149 (2009)
In animals, P-bodies or GW-bodies appear to cause the congregation of proteins involved in microRNA (miRNA)-mediated post-transcriptional silencing. The localization of P-bodies does not overlap with that of known organelles and are thus considered independent of lipid bilayers. Nonetheless, an miRNA effector protein, argonaute 2 (AGO2), was initially identified as membrane-associated, and some miRNAs have been found in secreted vesicles (exosomes) that derive from endo-lysosomal compartments called multivesicular bodies (MVBs). Proteins can be sorted in a ubiquitin-dependent manner into MVBs by three heteromeric subcomplexes, collectively termed ESCRT (endosomal sorting complex required for transport), to be further secreted in exosomes and/or degraded by the lysosome. Here we show that GW-bodies containing GW182 and AGO2, two main components of the RNA-induced silencing complex (RISC), are distinct from P-bodies due to their congregation with endosomes and MVBs. More! over, miRNAs and miRNA-repressible mRNAs are enriched at these cellular membranes, suggesting that endosomes and/or MVBs are sites of miRNA-loaded RISC (miRISC) accumulation and, possibly, action. We further show that purified exosome-like vesicles secreted by MVBs are considerably enriched in GW182, but not P-body components, AGO2 or miRNA-repressible mRNA. Moreover, cells depleted of some ESCRT components show compromised miRNA-mediated gene silencing and over-accumulate GW182, which associates with ubiquitylated proteins. Therefore, GW182, possibly in association with a fraction of miRNA-loaded AGO2, is sorted into MVBs for secretion and/or lysosomal degradation. We propose that this process promotes continuous assembly or disassembly of membrane-associated miRISCs, which is possibly required for miRNA loading or target recognition and subsequent silencing. - Silencing by small RNAs is linked to endosomal trafficking
- Nat Cell Biol 11(9):1150-1156 (2009)
Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs1, 2. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies3. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum4. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport)6 depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in ! loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm. - Zcchc11-dependent uridylation of microRNA directs cytokine expression
- Nat Cell Biol 11(9):1157-1163 (2009)
Mounting an effective host immune response without incurring inflammatory injury requires the precise regulation of cytokine expression1, 2. To achieve this, cytokine mRNAs are post-transcriptionally regulated by diverse RNA-binding proteins and microRNAs (miRNAs) targeting their 3' untranslated regions (UTRs)3, 4. Zcchc11 (zinc-finger, CCHC domain-containing protein 11) contains RNA-interacting motifs5, and has been implicated in signalling pathways involved in cytokine expression6. The nature of the Zcchc11 protein and how it influences cytokine expression are unknown. Here we show that Zcchc11 directs cytokine expression by uridylating cytokine-targeting miRNAs. Zcchc11 is a ribonucleotidyltransferase with a preference for uridine and is essential for maintaining the poly(A) tail length and stability of transcripts for interleukin-6 (IL-6) and other specific cytokines. The miR-26 family of miRNAs targets IL-6, and the addition of terminal uridines to the miR-26 3' e! nd abrogates IL-6 repression. Whereas 78% of miR-26a sequences in control cells contained 1–3 uridines on their 3' ends, less than 0.1% did so in Zcchc11-knockdown cells. Thus, Zcchc11 fine tunes IL-6 production by uridylating miR-26a, which we propose is an enzymatic modification of the terminal nucleotide sequence of mature miRNA as a means to regulate gene expression. - Identification of chromosome sequence motifs that mediate meiotic pairing and synapsis in C. elegans
- Nat Cell Biol 11(9):1163 (2009)
Introduction In the version of this article initially published online and in print, the labelling of Fig 1e was incorrect. The correct version of this figure is shown below. This error has been corrected in the HTML and PDF version of the article.
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