163 results on '"Christine E. Holt"'
Search Results
2. On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons
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Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, Julie Qiaojin Lin, Roberta Cagnetta, Asha Dwivedy, Janaina de Freitas Nascimento, Francesca W. van Tartwijk, Florian Ströhl, Jean-Michel Cioni, Julia Schaeffer, Mark Carrington, Clemens F. Kaminski, Hosung Jung, William A. Harris, and Christine E. Holt more...
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Biology (General) ,QH301-705.5 - Abstract
Summary: Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons. : Local protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching. Keywords: axon, mRNA, local translation, ribosome, ribosomal proteins, Rps4x, axonal protein synthesis, ribosome remodeling, axon branching, neural wiring more...
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- 2019
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Catalog
3. miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA
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Anaïs Bellon, Archana Iyer, Simone Bridi, Flora C.Y. Lee, Cesaré Ovando-Vázquez, Eloina Corradi, Sara Longhi, Michela Roccuzzo, Stephanie Strohbuecker, Sindhu Naik, Peter Sarkies, Eric Miska, Cei Abreu-Goodger, Christine E. Holt, and Marie-Laure Baudet more...
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miRNAs ,local translation ,growth cone ,axon guidance ,brain wiring ,miR-182 ,Slit2 ,cofilin ,Biology (General) ,QH301-705.5 - Abstract
During brain wiring, cue-induced axon behaviors such as directional steering and branching are aided by localized mRNA translation. Different guidance cues elicit translation of subsets of mRNAs that differentially regulate the cytoskeleton, yet little is understood about how specific mRNAs are selected for translation. MicroRNAs (miRNAs) are critical translational regulators that act through a sequence-specific mechanism. Here, we investigate the local role of miRNAs in mRNA-specific translation during pathfinding of Xenopus laevis retinal ganglion cell (RGC) axons. Among a rich repertoire of axonal miRNAs, miR-182 is identified as the most abundant. Loss of miR-182 causes RGC axon targeting defects in vivo and impairs Slit2-induced growth cone (GC) repulsion. We find that miR-182 targets cofilin-1 mRNA, silencing its translation, and Slit2 rapidly relieves the repression without causing miR-182 degradation. Our data support a model whereby miR-182 reversibly gates the selection of transcripts for fast translation depending on the extrinsic cue. more...
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- 2017
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4. Growth Cone Tctp Is Dynamically Regulated by Guidance Cues
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Cláudio Gouveia Roque and Christine E. Holt
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Tctp ,mTORC ,Netrin-1 ,Ephrin-A1 ,axon guidance ,retinal ganglion cell ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Translationally controlled tumor protein (Tctp) contributes to retinal circuitry formation by promoting axon growth and guidance, but it remains unknown to what extent axonal Tctp specifically influences axon development programs. Various genome-wide profiling studies have ranked tctp transcripts among the most enriched in the axonal compartment of distinct neuronal populations, including embryonic retinal ganglion cells (RGCs), suggesting its expression can be regulated locally and that this may be important during development. Here, we report that growth cone Tctp levels change rapidly in response to Netrin-1 and Ephrin-A1, two guidance cues encountered by navigating RGC growth cones. This regulation is opposite in effect, as we observed protein synthesis- and mTORC1-dependent increases in growth cone Tctp levels after acute treatment with Netrin-1, but a decline upon exposure to Ephrin-A1, an inhibitor of mTORC1. Live imaging with translation reporters further showed that Netrin-1-induced synthesis of Tctp in growth cones is driven by a short 3′untranslated region (3′UTR) tctp mRNA isoform. However, acute inhibition of de novo Tctp synthesis in axons did not perturb the advance of retinal projections through the optic tract in vivo, indicating that locally produced Tctp is not necessary for normal axon growth and guidance. more...
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- 2018
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5. Cue-Polarized Transport of β-actin mRNA Depends on 3′UTR and Microtubules in Live Growth Cones
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Kin-Mei Leung, Bo Lu, Hovy Ho-Wai Wong, Julie Qiaojin Lin, Benita Turner-Bridger, and Christine E. Holt
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axon navigation ,growth cone ,mRNA trafficking ,β-actin ,3′UTR ,local protein synthesis ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Guidance cues trigger fast responses in axonal growth cones such as directional turning and collapse that require local protein synthesis. An attractive cue-gradient, such as Netrin-1, triggers de novo synthesis of β-actin localized to the near-side compartment of the growth cone that promotes F-actin assembly and attractive steering. How this precise spatial asymmetry in mRNA translation arises across the small expanse of the growth cone is poorly understood. Pre-localized mRNAs in the vicinity of activated receptors could be selectively translated and/or new mRNAs could be trafficked into the area. Here we have performed live imaging of fluorescent-tagged β-actin mRNA to investigate mRNA trafficking dynamics in Xenopus retinal ganglion cell (RGC) axons and growth cones in response to Netrin-1. A Netrin-1 gradient was found to elicit the transport of β-actin mRNA granules to the near-side of growth cones within a 4–7 min window. This polarized mRNA trafficking depended on the 3′ untranslated region (UTR) since mRNA-Δ3′UTR mutant failed to exhibit cue-induced localization. Global application of Netrin-1 significantly increased the anterograde movement of β-actin mRNA along axons and also promoted microtubule-dependent mRNA excursions from the central domain of the growth cone into the periphery (filopodia and lamellipodia). Dual channel imaging revealed β-actin mRNA riding behind the microtubule plus-end tracking protein, EB1, in movements along dynamic microtubules into filopodia. The mRNA-EB1 movements were unchanged by a Netrin-1 gradient indicating the dynamic microtubules themselves do not underlie the cue-induced polarity of RNA movement. Finally, fast-moving elongated “worm-like” trains of Cy3-RNA, distinct from mitochondria, were seen transporting RNA along axons in vitro and in vivo suggesting the existence of a novel transport organelle. Overall, the results provide evidence that the axonal trafficking of β-actin mRNA can be regulated by the guidance cue Netrin-1 to transduce the polarity of an extracellular stimulus and that the 3′UTR is essential for this cue-induced regulation. more...
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- 2018
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6. ESCRT-II controls retinal axon growth by regulating DCC receptor levels and local protein synthesis
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Filip A. Konopacki, Hovy Ho-Wai Wong, Asha Dwivedy, Anaïs Bellon, Michael D. Blower, and Christine E. Holt
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endocytosis ,escrt ,dcc ,netrin-1 ,axon guidance ,protein synthesis ,Biology (General) ,QH301-705.5 - Abstract
Endocytosis and local protein synthesis (LPS) act coordinately to mediate the chemotropic responses of axons, but the link between these two processes is poorly understood. The endosomal sorting complex required for transport (ESCRT) is a key regulator of cargo sorting in the endocytic pathway, and here we have investigated the role of ESCRT-II, a critical ESCRT component, in Xenopus retinal ganglion cell (RGC) axons. We show that ESCRT-II is present in RGC axonal growth cones (GCs) where it co-localizes with endocytic vesicle GTPases and, unexpectedly, with the Netrin-1 receptor, deleted in colorectal cancer (DCC). ESCRT-II knockdown (KD) decreases endocytosis and, strikingly, reduces DCC in GCs and leads to axon growth and guidance defects. ESCRT-II-depleted axons fail to turn in response to a Netrin-1 gradient in vitro and many axons fail to exit the eye in vivo. These defects, similar to Netrin-1/DCC loss-of-function phenotypes, can be rescued in whole (in vitro) or in part (in vivo) by expressing DCC. In addition, ESCRT-II KD impairs LPS in GCs and live imaging reveals that ESCRT-II transports mRNAs in axons. Collectively, our results show that the ESCRT-II-mediated endocytic pathway regulates both DCC and LPS in the axonal compartment and suggest that ESCRT-II aids gradient sensing in GCs by coupling endocytosis to LPS. more...
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- 2016
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7. Mutation of the ALS/FTD-associated RNA-binding protein FUS alters axonal cytoskeletal organisation
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Francesca W. van Tartwijk, Lucia C.S. Wunderlich, Ioanna Mela, Stanislaw Makarchuk, Maximilian A.H Jakobs, Seema Qamar, Kristian Franze, Gabriele S. Kaminski Schierle, Peter H. St George-Hyslop, Julie Qiaojin Lin, Christine E. Holt, and Clemens F. Kaminski more...
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SummaryAberrant condensation and localisation of the RNA-binding protein fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is also associated with cytoskeletal defects, genetically and through observations of compromised axonal transport. Here, we asked whether compromised axonal cytoskeletal organisation is an early feature of FUS-associated ALS/FTD. We used an ALS-associated mutant FUS(P525L) and the FTD-mimic hypomethylated FUS, FUS(16R), to investigate the common and distinct cytoskeletal changes found in these two reportedXenopusmodels. Combining a novel atomic force microscopy (AFM)-based approach forin vitrocytoskeletal characterisation andin vivoaxonal branching analysis, we found that mutant FUS reduced actin density in the dynamically remodelling growth cone, and reduced axonal branch complexity. We furthermore found evidence of an axon looping defect for FUS(P525L). Therefore, we show that compromised actin remodelling is potentially an important early event in FUS-associated pathogenesis.Abstract Figure more...
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- 2022
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8. mRNA transport, translation, and decay in adult mammalian central nervous system axons
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Jane Jung, Jiyeon Ohk, Hyeyoung Kim, Christine E. Holt, Hyun Jung Park, and Hosung Jung
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General Neuroscience - Abstract
Localized mRNA translation regulates synapse function and axon maintenance, but how compartment-specific mRNA repertoires are regulated is largely unknown. We developed an axonal transcriptome capture method that allows deep sequencing of metabolically labeled mRNAs from retinal ganglion cell axon terminals in mouse. Comparing axonal-to-somal transcriptomes and axonal translatome-to-transcriptome enables genome-wide visualization of mRNA transport and translation and unveils potential regulators tuned to each process. FMRP and TDP-43 stand out as key regulators of transport, and experiments in Fmr1 knockout mice validate FMRP's role in the axonal transportation of synapse-related mRNAs. Pulse-and-chase experiments enable genome-wide assessment of mRNA stability in axons and reveal a strong coupling between mRNA translation and decay. Measuring the absolute mRNA abundance per axon terminal shows that the adult axonal transcriptome is stably maintained by persistent transport. Our datasets provide a rich resource for unique insights into RNA-based mechanisms in maintaining presynaptic structure and function in vivo. more...
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- 2022
9. Receptor-Ribosome Coupling: A Link Between Extrinsic Signals and mRNA Translation in Neuronal Compartments
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Max Koppers and Christine E. Holt
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Neurons ,General Neuroscience ,Protein Biosynthesis ,RNA, Messenger ,Ribosomes ,Axons - Abstract
Axons receive extracellular signals that help to guide growth and synapse formation during development and to maintain neuronal function and survival during maturity. These signals relay information via cell surface receptors that can initiate local intracellular signaling at the site of binding, including local messenger RNA (mRNA) translation. Direct coupling of translational machinery to receptors provides an attractive way to activate this local mRNA translation and change the local proteome with high spatiotemporal resolution. Here, we first discuss the increasing evidence that different external stimuli trigger translation of specific subsets of mRNAs in axons via receptors and thus play a prominent role in various processes in both developing and mature neurons. We then discuss the receptor-mediated molecular mechanisms that regulate local mRNA translation with a focus on direct receptor-ribosome coupling. We advance the idea that receptor-ribosome coupling provides several advantages over other translational regulation mechanisms and is a common mechanism in cell communication. more...
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- 2022
10. Molecular control of local translation in axon development and maintenance
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Max Koppers, Christine E. Holt, Jean-Michel Cioni, Koppers, Max [0000-0002-7751-1082], Holt, Christine [0000-0003-2829-121X], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Cell ,Synaptogenesis ,Biology ,Axonal Transport ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Protein biosynthesis ,Animals ,MRNA transport ,RNA, Messenger ,Axon ,Cytoskeleton ,General Neuroscience ,RNA-Binding Proteins ,Translation (biology) ,Molecular control ,Axons ,Protein Transport ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,Protein Biosynthesis ,Neuroscience ,030217 neurology & neurosurgery - Abstract
The tips of axons are often far away from the cell soma where most proteins are synthesized. Recent work has revealed that axonal mRNA transport and localised translation are key regulatory mechanisms that allow these distant outposts of the cell to respond rapidly to extrinsic factors and maintain axonal homeostasis. Here, we review recent evidence pointing to an increasingly broad role for local protein synthesis in controlling axon shape, synaptogenesis and axon survival by regulating diverse cellular processes such as vesicle trafficking, cytoskeletal remodelling and mitochondrial integrity. We further highlight current research on the regulatory mechanisms that coordinate the localization and translation of functionally linked mRNAs in axons. more...
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- 2018
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11. The DCC receptor and its endosome associated role for regulating local protein synthesis
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Lucia C. Wunderlich, Francesca van Tartwijk, Julie Q. Lin, Max Koppers, Max Barysevich, Stanislaw Makarchuk, Maximilian A. Jakobs, Christine E. Holt, and Clemens F. Kaminski
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Biophysics - Published
- 2022
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12. Filopodyan: An open-source pipeline for the analysis of filopodia
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Richard Butler, Jennifer L. Gallop, Christine E. Holt, Vasja Urbančič, Manuel Peter, Frederick J. Livesey, Julia Mason, Benjamin Richier, Gallop, Jennifer [0000-0002-9978-1382], Urbancic, Vasja [0000-0001-8955-5923], Butler, Richard [0000-0002-3885-1332], Livesey, Frederick [0000-0001-6128-3372], Holt, Christine [0000-0003-2829-121X], and Apollo - University of Cambridge Repository more...
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0301 basic medicine ,Cell type ,Embryo, Nonmammalian ,animal structures ,macromolecular substances ,Biology ,Molecular heterogeneity ,Interactive editing ,Cell Line ,Tools ,User-Computer Interface ,Xenopus laevis ,03 medical and health sciences ,0302 clinical medicine ,Image Processing, Computer-Assisted ,Animals ,Humans ,Pseudopodia ,Growth cone ,Research Articles ,Actin ,030304 developmental biology ,0303 health sciences ,integumentary system ,Neuronal Growth ,Cell Biology ,Phenotype ,Cell biology ,Drosophila melanogaster ,030104 developmental biology ,Open source ,Microscopy, Fluorescence ,nervous system ,embryonic structures ,Filopodia ,030217 neurology & neurosurgery ,Filopodia formation - Abstract
Urbančič et al. developed an open-source platform called Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia at their tips and bases concurrently with their morphological and dynamic properties. This customizable tool therefore enables researchers to determine the relationship between protein localization and filopodium behavior., Filopodia have important sensory and mechanical roles in motile cells. The recruitment of actin regulators, such as ENA/VASP proteins, to sites of protrusion underlies diverse molecular mechanisms of filopodia formation and extension. We developed Filopodyan (filopodia dynamics analysis) in Fiji and R to measure fluorescence in filopodia and at their tips and bases concurrently with their morphological and dynamic properties. Filopodyan supports high-throughput phenotype characterization as well as detailed interactive editing of filopodia reconstructions through an intuitive graphical user interface. Our highly customizable pipeline is widely applicable, capable of detecting filopodia in four different cell types in vitro and in vivo. We use Filopodyan to quantify the recruitment of ENA and VASP preceding filopodia formation in neuronal growth cones, and uncover a molecular heterogeneity whereby different filopodia display markedly different responses to changes in the accumulation of ENA and VASP fluorescence in their tips over time. more...
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- 2017
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13. The structure and global distribution of the endoplasmic reticulum network is actively regulated by lysosomes
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David Holcman, Charles N. Christensen, Christine E. Holt, Lukas C. Kapitein, Pierre Parutto, Francesca W. van Tartwijk, Gabriele Kaminski Schierle, Edward Avezov, Julie Qiaojin Lin, Wilco Nijenhuis, Alan Tunnacliffe, Clemens F. Kaminski, Marcus Fantham, Meng Lu, Lu, Meng [0000-0001-9311-2666], van Tartwijk, Francesca [0000-0002-9795-2571], Lin, Qiaojin [0000-0002-2669-6478], Christensen, Charles [0000-0002-5355-1063], Avezov, Edward [0000-0002-2894-0585], Holt, Christine [0000-0003-2829-121X], Kaminski Schierle, Gabriele [0000-0002-1843-2202], Kaminski, Clemens [0000-0002-5194-0962], and Apollo - University of Cambridge Repository more...
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Chemistry ,Endoplasmic reticulum ,Cell ,Metabolic change ,Nutritional status ,3101 Biochemistry and Cell Biology ,Cell biology ,medicine.anatomical_structure ,Tubule ,Global distribution ,Lysosome ,medicine ,Causal link ,health care economics and organizations ,31 Biological Sciences - Abstract
The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains, sheets and interconnected tubules. These domains undergo dynamic reshaping, in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling within minutes are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status. The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven re-positioning of lysosomes, which leads to both a redistribution of the ER tubules and its global morphology. Lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development. more...
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- 2020
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14. A Protocol for Single-Molecule Translation Imaging in Xenopus Retinal Ganglion Cells
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Julie Qiaojin Lin, Clemens F. Kaminski, Hovy Ho-Wai Wong, Florian Ströhl, Francesca W. van Tartwijk, and Christine E. Holt
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biology ,Chemistry ,Xenopus ,Translation (biology) ,biology.organism_classification ,Retinal ganglion ,Cell biology - Published
- 2020
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15. Axon micro-dissection and transcriptome profiling reveals the in vivo RNA content of fully differentiated myelinated motor axons
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Christine E. Holt, Joaquina Farias, José R. Sotelo-Silveira, José R. Sotelo, Farías Joaquina, IIBCE, Holt C. E., Sotelo Sosa José Roberto, IIBCE, and Sotelo Silveira José Roberto, IIBCE
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Motor neuron ,Biology ,Article ,Axon ,Muscular Atrophy, Spinal ,Transcriptome ,Axonal mRNAs ,03 medical and health sciences ,Peripheral Nervous System ,medicine ,Animals ,Humans ,RNA, Messenger ,RNA-Seq ,Amyotrophic lateral sclerosis ,Molecular Biology ,Microdissection ,030304 developmental biology ,Motor Neurons ,0303 health sciences ,Gene Expression Profiling ,Amyotrophic Lateral Sclerosis ,030302 biochemistry & molecular biology ,Cell Differentiation ,medicine.disease ,Spinal muscular atrophies ,Axons ,Cell biology ,medicine.anatomical_structure ,nervous system ,Axoplasm ,Peripheral nervous system ,Local translation ,RNA ,mRNA localization - Abstract
Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in the maintenance of the axoplasm in a steady state. Recent studies have begun to identify the mRNAs localized in axons, which could be translated locally under different conditions. Despite that by now hundreds or thousands of mRNAs have been shown to be localized into the axonal compartment of cultured neurons in vitro, knowledge of which mRNAs are localized in mature myelinated axons is quite limited. With the purpose of characterizing the transcriptome of mature myelinated motor axons of peripheral nervous systems, we modified the axon microdissection method devised by Koenig, enabling the isolation of the axoplasm RNA to perform RNA-seq analysis. The transcriptome analysis indicates that the number of RNAs detected in mature axons is lower in comparison with in vitro data, depleted of glial markers, and enriched in neuronal markers. The mature myelinated axons are enriched for mRNAs related to cytoskeleton, translation, and oxidative phosphorylation. Moreover, it was possible to define core genes present in axons when comparing our data with transcriptomic data of axons grown in different conditions. This work provides evidence that axon microdissection is a valuable method to obtain genome-wide data from mature and myelinated axons of the peripheral nervous system, and could be especially useful for the study of axonal involvement in neurodegenerative pathologies of motor neurons such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophies (SMA). more...
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- 2020
16. Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons
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John G. Flanagan, Toshiaki Shigeoka, Lucia C. S. Wunderlich, Max Koppers, William A. Harris, Christine E. Holt, Maximilian Ah Jakobs, Clemens F. Kaminski, Julie Qiaojin Lin, Asha Dwivedy, Michael S Minett, Anaïs Bellon, Sixian Zhao, Pedro Vallejo-Ramirez, Roberta Cagnetta, Koppers, Max [0000-0002-7751-1082], Wunderlich, Lucia Cs [0000-0001-7200-1713], Vallejo-Ramirez, Pedro [0000-0002-7879-6761], Qiaojin Lin, Julie [0000-0002-2669-6478], Jakobs, Maximilian Ah [0000-0002-0879-7937], Kaminski, Clemens F [0000-0002-5194-0962], Harris, William A [0000-0002-9995-8096], Holt, Christine E [0000-0003-2829-121X], Apollo - University of Cambridge Repository, Wunderlich, Lucia CS [0000-0001-7200-1713], and Jakobs, Maximilian AH [0000-0002-0879-7937] more...
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0301 basic medicine ,Retinal Ganglion Cells ,Proteome ,Xenopus ,RNA-binding protein ,Stimulation ,Interactome ,Ribosome ,Xenopus laevis ,0302 clinical medicine ,retinal ganglion cell ,Biology (General) ,Receptor ,axon ,biology ,Chemistry ,General Neuroscience ,RNA-Binding Proteins ,Translation (biology) ,General Medicine ,Cell biology ,medicine.anatomical_structure ,Retinal ganglion cell ,Medicine ,Research Article ,Human ,Signal Transduction ,QH301-705.5 ,mRNA ,Science ,Receptors, Cell Surface ,General Biochemistry, Genetics and Molecular Biology ,guidance receptor ,03 medical and health sciences ,medicine ,Animals ,RNA, Messenger ,local protein synthesis ,General Immunology and Microbiology ,biology.organism_classification ,Axons ,030104 developmental biology ,Protein Biosynthesis ,Ribosomes ,030217 neurology & neurosurgery ,Developmental Biology ,Neuroscience - Abstract
Extrinsic cues trigger the local translation of specific mRNAs in growing axons via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a mechanism linking signals to local translation but it is not known how broadly this mechanism operates, nor whether it can selectively regulate mRNA translation. We report that receptor-ribosome coupling is employed by multiple guidance cue receptors and this interaction is mRNA-dependent. We find that different receptors associate with distinct sets of mRNAs and RNA-binding proteins. Cue stimulation of growing Xenopus retinal ganglion cell axons induces rapid dissociation of ribosomes from receptors and the selective translation of receptor-specific mRNAs. Further, we show that receptor-ribosome dissociation and cue-induced selective translation are inhibited by co-exposure to translation-repressive cues, suggesting a novel mode of signal integration. Our findings reveal receptor-specific interactomes and suggest a generalizable model for cue-selective control of the local proteome. more...
- Published
- 2019
17. Decision letter: Axon TRAP reveals learning-associated alterations in cortical axonal mRNAs in the lateral amygdala
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Christine E. Holt and Pablo E. Castillo
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Trap (computing) ,medicine.anatomical_structure ,medicine ,Axon ,Biology ,Amygdala ,Neuroscience - Published
- 2019
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18. Author response: Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons
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Christine E. Holt, John G. Flanagan, William A. Harris, Maximilian Ah Jakobs, Max Koppers, Anaïs Bellon, Julie Qiaojin Lin, Roberta Cagnetta, Clemens F. Kaminski, Michael S Minett, Toshiaki Shigeoka, Lucia C. S. Wunderlich, Sixian Zhao, Asha Dwivedy, and Pedro Vallejo-Ramirez more...
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Translation (biology) ,Biology ,Receptor ,Neuroscience ,Interactome - Published
- 2019
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19. Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons
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Christine E. Holt, Max Koppers, Roberta Cagnetta, Anaïs Bellon, Sixian Zhao, Michael S Minett, John G. Flanagan, Toshiaki Shigeoka, Lucia C. S. Wunderlich, and Clemens F. Kaminski
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Coupling (electronics) ,Cell surface receptor ,Chemistry ,Mechanism (biology) ,Translation (biology) ,Stimulation ,Receptor ,Ribosome ,Interactome ,Cell biology - Abstract
During neuronal wiring, extrinsic cues trigger the local translation of specific mRNAs in axons via cell surface receptors. The coupling of ribosomes to receptors has been proposed as a mechanism linking signals to local translation but it is not known how broadly this mechanism operates, nor whether it can selectively regulate mRNA translation. We report that receptor-ribosome coupling is employed by multiple guidance cue receptors and this interaction is mRNA-dependent. We find that different receptors bind to distinct sets of mRNAs and RNA-binding proteins. Cue stimulation induces rapid dissociation of ribosomes from receptors and the selective translation of receptor-specific mRNAs in retinal axon growth cones. Further, we show that receptor-ribosome dissociation and cue-induced selective translation are inhibited by simultaneous exposure to translation-repressive cues, suggesting a novel mode of signal integration. Our findings reveal receptor-specific interactomes and provide a general model for the rapid, localized and selective control of cue-induced translation. more...
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- 2019
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20. Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain
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Amelia J Thompson, Eva K Pillai, Ivan B Dimov, Sarah K Foster, Christine E Holt, Kristian Franze, Thompson, Amelia J [0000-0002-3912-3652], Franze, Kristian [0000-0002-8425-7297], and Apollo - University of Cambridge Repository more...
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musculoskeletal diseases ,Retinal Ganglion Cells ,animal structures ,Embryo, Nonmammalian ,QH301-705.5 ,Science ,durotaxis ,Mitosis ,Cell Count ,macromolecular substances ,developmental biology ,stiffness ,Xenopus laevis ,physics of living systems ,Animals ,Optic Tract ,Biology (General) ,mechanotransduction ,atomic force microscopy ,axon guidance ,technology, industry, and agriculture ,Brain ,xenopus ,Axons ,Biomechanical Phenomena ,body regions ,Cell Body ,Medicine ,mechanics - Abstract
Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements. more...
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- 2019
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21. On-Site Ribosome Remodeling by Locally Synthesized Ribosomal Proteins in Axons
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Max Koppers, Christine E. Holt, Julie Qiaojin Lin, Hosung Jung, Hovy Ho-Wai Wong, Florian Ströhl, Jean-Michel Cioni, Roberta Cagnetta, Toshiaki Shigeoka, Janaina de Freitas Nascimento, William A. Harris, Mark Carrington, Clemens F. Kaminski, Julia Schaeffer, Asha Dwivedy, Francesca W. van Tartwijk, Lin, Qiaojin [0000-0002-2669-6478], van Tartwijk, Francesca [0000-0002-9795-2571], Carrington, Mark [0000-0002-6435-7266], Kaminski, Clemens [0000-0002-5194-0962], Holt, Christine [0000-0003-2829-121X], and Apollo - University of Cambridge Repository more...
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Ribosomal Proteins ,0301 basic medicine ,mRNA ,Neurogenesis ,neural wiring ,VDP::Mathematics and natural science: 400::Basic biosciences: 470::Cell biology: 471 ,Library science ,Regulatory Sequences, Ribonucleic Acid ,Article ,General Biochemistry, Genetics and Molecular Biology ,ribosome remodeling ,Xenopus laevis ,03 medical and health sciences ,local translation ,0302 clinical medicine ,Political science ,Rps4x ,Animals ,RNA, Messenger ,lcsh:QH301-705.5 ,health care economics and organizations ,Cells, Cultured ,axon ,European research ,Brain ,Axons ,Engineering and Physical Sciences ,axon branching ,030104 developmental biology ,ribosome ,Axonal branching ,lcsh:Biology (General) ,nervous system ,Research council ,Ribosomes ,030217 neurology & neurosurgery ,axonal protein synthesis ,VDP::Matematikk og Naturvitenskap: 400::Basale biofag: 470::Cellebiologi: 471 - Abstract
Summary Ribosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons., Graphical Abstract, Highlights • Axonal ribosomal protein (RP) synthesis is cue regulated via a 5′ UTR loop-forming motif • Axonal RP synthesis generates a free cytosolic pool of ribosomal proteins in axons • Axonally synthesized RPs join axonal ribosomes in a nucleolus-independent manner • Local RP synthesis is required for ribosome function and branch architecture, Local protein synthesis in axons supplies new ribosomal proteins far from the nucleolus, the known site of ribosome biogenesis. Shigeoka et al. provide evidence that axonally synthesized ribosomal proteins join pre-existing ribosomes and maintain translation activity in axons, which is required for axon terminal branching. more...
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- 2019
22. Local translation in neurons: visualization and function
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Kelsey C. Martin, Erin M. Schuman, Christine E. Holt, Schuman, Erin M [0000-0001-7505-1855], and Apollo - University of Cambridge Repository
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Neurons ,0303 health sciences ,Cell type ,Microscopy ,Computer science ,Effector ,Translation (biology) ,Visualization ,03 medical and health sciences ,0302 clinical medicine ,Post translational ,Structural Biology ,Protein Biosynthesis ,Proteome ,Protein biosynthesis ,Animals ,Humans ,RNA, Messenger ,Molecular Biology ,Neuroscience ,Protein Processing, Post-Translational ,Ribosomes ,030217 neurology & neurosurgery ,Function (biology) ,030304 developmental biology - Abstract
Neurons are among the most compartmentalized and interactive of all cell types. Like all cells, neurons use proteins as the main sensors and effectors. The modification of the proteome in axons and dendrites is used to guide the formation of synaptic connections and to store information. In this Review, we discuss the data indicating that an important source of protein for dendrites, axons and their associated elements is provided by the local synthesis of proteins. We review the data indicating the presence of the machinery required for protein synthesis, the direct visualization and demonstration of protein synthesis, and the established functional roles for local translation for many different neuronal functions. Finally, we consider the open questions and future directions in this field. more...
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- 2019
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23. Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain
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Amelia J Thompson, Eva K Pillai, Ivan B Dimov, Sarah K Foster, Christine E Holt, Kristian Franze
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- 2019
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24. On-site ribosome remodeling by locally synthesized ribosomal proteins in axons
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Christine E. Holt, Asha Dwivedy, Florian Ströhl, Roberta Cagnetta, Max Koppers, Francesca W. van Tartwijk, Mark Carrington, Hovy Ho-Wai Wong, Jean-Michel Cioni, Clemens F. Kaminski, Toshiaki Shigeoka, Hosung Jung, Janaina de Freitas Nascimento, William A. Harris, and Julie Qiaojin Lin more...
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0303 health sciences ,Nucleolus ,Chemistry ,Translation (biology) ,Ribosomal RNA ,Ribosome ,Cell biology ,03 medical and health sciences ,0302 clinical medicine ,Start codon ,nervous system ,Cytoplasm ,Ribosomal protein ,Sequence motif ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
SUMMARYRibosomes are known to be assembled in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Using subcellular proteomics and live-imaging, we show that locally synthesized RPs incorporate into axonal ribosomes in a nucleolus-independent fashion. We revealed that axonal RP translation is regulated through a novel sequence motif, CUIC, that forms a RNA-loop structure in the region immediately upstream of the initiation codon. Inhibition of axonal CUIC-regulated RP translation leads to defects in local translation activity and axon branching, demonstrating the physiological relevance of the axonal ribosome remodeling. These results indicate that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus. more...
- Published
- 2018
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25. Targeted Electroporation in the CNS in Xenopus Embryos
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Hovy Ho-Wai, Wong and Christine E, Holt
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Central Nervous System ,Electroporation ,Embryo, Nonmammalian ,Xenopus ,Animals ,Time-Lapse Imaging ,Retina - Abstract
Electroporation allows targeting of genetic materials (e.g., DNA, RNA, antisense morpholinos) to the tissue of interest with high spatial and temporal specificity. Here, we describe a highly efficient and reproducible electroporation strategy for targeting the central nervous system in Xenopus. This versatile approach can be combined with live imaging or other existing experimental procedures to aid the investigation of different research questions. more...
- Published
- 2018
26. Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain
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Amelia J, Thompson, Eva K, Pillai, Ivan B, Dimov, Sarah K, Foster, Christine E, Holt, and Kristian, Franze
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musculoskeletal diseases ,Retinal Ganglion Cells ,animal structures ,Embryo, Nonmammalian ,Xenopus ,Short Report ,durotaxis ,Mitosis ,Cell Count ,macromolecular substances ,Physics of Living Systems ,Xenopus laevis ,stiffness ,Animals ,Optic Tract ,mechanotransduction ,atomic force microscopy ,axon guidance ,technology, industry, and agriculture ,Brain ,Axons ,Biomechanical Phenomena ,body regions ,Cell Body ,mechanics ,Developmental Biology - Abstract
Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements., eLife digest Neurons in the brain form an intricate network that follows a precise template. For example, in a young frog embryo, the neurons from the eyes send out thin structures, called axons, which navigate along a well-defined path and eventually connect with the visual centres of the brain. This journey requires the axons to take a sharp turn so they can wire with the right brain structures. Axons find their paths not only by following chemical signals but also by reacting to the stiffness of their environment. In an older frog embryo for instance, the brain is stiffer at the front, and softer at the back. As neurons from the eyes make their way through the brain, they turn to follow this gradient, moving away from stiffer areas towards the softer regions. Here, Thompson, Pillai et al. investigate when and how this stiffness gradient is established in frogs. To do so, a new technique was developed. Called time-lapse in vivo atomic force microscopy, the method measures how brain stiffness changes over time in a live embryo, while also taking images of the growing axons. The experiments show that the stiffness gradient arose within tens of minutes, just as the first ‘pioneering’ axons from the eyes began to grow across the brain. These axons then responded to the gradient, turning towards the softer tissue. Changes in the number of cells in the underlying brain tissue governed the formation of the gradient, with rapidly stiffening areas containing more cells than those that remained soft. In fact, using drugs that stop cells from dividing reduced both the mechanical gradient and the turning response of the axons. The technique developed by Thompson, Pillai et al. is a useful tool that can help elucidate how variations in stiffness control the brain wiring process. It could also be used to look into how other developmental or regenerative processes, such as the way neurons reconnect after injuries to the brain or spinal cord, may be regulated by mechanical tissue properties. more...
- Published
- 2018
27. Simultaneous in vivo time-lapse stiffness mapping and fluorescence imaging of developing tissue
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Kristian Franze, Eva K Pillai, Ivan B Dimov, Amelia J Thompson, and Christine E. Holt
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0303 health sciences ,Fluorescence-lifetime imaging microscopy ,biology ,Chemistry ,Dynamics (mechanics) ,Xenopus ,Stiffness ,Embryonic Tissue ,biology.organism_classification ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,medicine ,Biophysics ,Tissue mechanics ,Tissue stiffness ,medicine.symptom ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that in the developing Xenopus brain, a stiffness gradient evolves over time because of differential cell proliferation. Subsequently, axons turn to follow this gradient, underpinning the importance of time-resolved mechanics measurements. more...
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- 2018
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28. Tumor protein Tctp regulates axon development in the embryonic visual system
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Christine E. Holt, Ho Wai Wong, Cláudio Gouveia Roque, Julie Qiaojin Lin, Lin, Qiaojin [0000-0002-2669-6478], Holt, Christine [0000-0003-2829-121X], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Retinal Ganglion Cells ,Blastomeres ,Embryo, Nonmammalian ,Xenopus ,tpt1 ,Mitochondrial Dynamics ,Morpholinos ,Xenopus laevis ,Retinal ganglion cell ,Axon ,Cells, Cultured ,Membrane Potential, Mitochondrial ,biology ,Retinotectal projection ,Axon extension ,Axon guidance ,Tumor Protein, Translationally-Controlled 1 ,Anatomy ,Tctp ,3. Good health ,Cell biology ,Mitochondria ,medicine.anatomical_structure ,Research Article ,Neurogenesis ,RNA localisation ,Retina ,03 medical and health sciences ,Translationally-controlled tumor protein ,medicine ,Biological neural network ,Biomarkers, Tumor ,In Situ Nick-End Labeling ,Animals ,Visual Pathways ,Molecular Biology ,Optic Lobe, Nonmammalian ,biology.organism_classification ,Axons ,Rats, Inbred F344 ,Rats ,Myeloid Cell Leukemia Sequence 1 Protein ,030104 developmental biology ,nervous system ,Neural circuitry assembly ,Developmental Biology - Abstract
The transcript encoding translationally controlled tumor protein (Tctp), a molecule associated with aggressive breast cancers, was identified among the most abundant in genome-wide screens of axons, suggesting that Tctp is important in neurons. Here, we tested the role of Tctp in retinal axon development in Xenopus laevis. We report that Tctp deficiency results in stunted and splayed retinotectal projections that fail to innervate the optic tectum at the normal developmental time owing to impaired axon extension. Tctp-deficient axons exhibit defects associated with mitochondrial dysfunction and we show that Tctp interacts in the axonal compartment with myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl2 family. Mcl1 knockdown gives rise to similar axon misprojection phenotypes, and we provide evidence that the anti-apoptotic activity of Tctp is necessary for the normal development of the retinotectal projection. These findings suggest that Tctp supports the development of the retinotectal projection via its regulation of pro-survival signalling and axonal mitochondrial homeostasis, and establish a novel and fundamental role for Tctp in vertebrate neural circuitry assembly., Highlighted article: The cancer-associated protein Tctp controls neural circuitry in Xenopus via its regulation of pro-survival signalling and axonal mitochondrial homeostasis. more...
- Published
- 2016
29. Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo
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Toshiaki Shigeoka, Jane Jung, Hosung Jung, and Christine E. Holt
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0301 basic medicine ,Immunoprecipitation ,Chemistry ,Translation (biology) ,Ribosome ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,nervous system ,Retinal ganglion cell ,Ribosomal protein ,In vivo ,medicine ,Neuron ,Axon ,030217 neurology & neurosurgery - Abstract
Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated. more...
- Published
- 2018
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30. Targeted Electroporation in the CNS in Xenopus Embryos
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Christine E. Holt and Hovy Ho-Wai Wong
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0301 basic medicine ,Morpholino ,Electroporation ,Central nervous system ,Xenopus ,RNA ,Biology ,biology.organism_classification ,Cell biology ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Live cell imaging ,medicine ,Preclinical imaging ,DNA - Abstract
Electroporation allows targeting of genetic materials (e.g., DNA, RNA, antisense morpholinos) to the tissue of interest with high spatial and temporal specificity. Here, we describe a highly efficient and reproducible electroporation strategy for targeting the central nervous system in Xenopus. This versatile approach can be combined with live imaging or other existing experimental procedures to aid the investigation of different research questions. more...
- Published
- 2018
- Full Text
- View/download PDF
31. Tctp in Neuronal Circuitry Assembly
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Cláudio Gouveia, Roque and Christine E, Holt
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Neural Pathways ,Biomarkers, Tumor ,Humans ,Tumor Protein, Translationally-Controlled 1 ,RNA, Messenger ,Axons ,Retina ,Signal Transduction - Abstract
Although tctp expression in many areas of the human brain was reported more than 15 years ago, little was known about how it functions in neurons. The early notion that Tctp is primarily expressed in mitotic cells, together with reports suggesting a relative low abundance in the brain, has perhaps potentiated this almost complete disregard for the study of Tctp in the context of neuron biology. However, recent evidence has challenged this view, as a number of independent genome-wide profiling studies identified tctp mRNA among the most enriched in the axonal compartment across diverse neuronal populations, including embryonic retinal ganglion cells. Considering the emerging parallels between axon guidance and cancer cell invasion, the axonal expression of cancer-associated tctp was suggestive of it holding an unexplored role in the wiring of neuronal circuits. Our study revealed that Tctp is necessary for the accurate and timely development of axon projections during the formation of vertebrate retinal circuits via its association with the survival machinery of the axon. Globally, the findings indicate that compromised pro-survival signaling in Tctp-deficient axons results in mitochondrial dysfunction and a subsequent decrease in axonal mitochondrial density. These effects likely translate into a metabolic state inadequate to support the normal guidance and extension processes of a developing axon. more...
- Published
- 2017
32. Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo
- Author
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Toshiaki, Shigeoka, Jane, Jung, Christine E, Holt, and Hosung, Jung
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Mice ,Protein Biosynthesis ,Animals ,Immunoprecipitation ,RNA ,Breeding ,Ribosomes ,Axons ,Chromatography, Affinity - Abstract
Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated. more...
- Published
- 2017
33. Axon-Axon Interactions Regulate Topographic Optic Tract Sorting via CYFIP2-Dependent WAVE Complex Function
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Jean-Michel, Cioni, Hovy Ho-Wai, Wong, Dario, Bressan, Lay, Kodama, William A, Harris, and Christine E, Holt
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Male ,Retinal Ganglion Cells ,axon ,Superior Colliculi ,optic tract ,CYFIP ,Cell Communication ,Axons ,Article ,topographic sorting ,Animals, Genetically Modified ,growth cone ,Xenopus laevis ,filopodia ,nervous system ,Animals ,WAVE ,Female ,Visual Pathways ,sense organs ,retinal ganglion cell ,Zebrafish ,Adaptor Proteins, Signal Transducing - Abstract
Summary The axons of retinal ganglion cells (RGCs) are topographically sorted before they arrive at the optic tectum. This pre-target sorting, typical of axon tracts throughout the brain, is poorly understood. Here, we show that cytoplasmic FMR1-interacting proteins (CYFIPs) fulfill non-redundant functions in RGCs, with CYFIP1 mediating axon growth and CYFIP2 specifically involved in axon sorting. We find that CYFIP2 mediates homotypic and heterotypic contact-triggered fasciculation and repulsion responses between dorsal and ventral axons. CYFIP2 associates with transporting ribonucleoprotein particles in axons and regulates translation. Axon-axon contact stimulates CYFIP2 to move into growth cones where it joins the actin nucleating WAVE regulatory complex (WRC) in the periphery and regulates actin remodeling and filopodial dynamics. CYFIP2’s function in axon sorting is mediated by its binding to the WRC but not its translational regulation. Together, these findings uncover CYFIP2 as a key regulatory link between axon-axon interactions, filopodial dynamics, and optic tract sorting., Highlights • CYFIP1 and CYFIP2 serve non-redundant functions in retinal axon growth and guidance • CYFIP2 regulates growth cone filopodial dynamics and axon-axon responses • CYFIP2 interacts with RNPs and the WRC in distinct cellular compartments • Axon sorting is mediated by CYFIP2’s interaction with the WRC, Axon sorting within nerve tracts is important for the establishment of neural connectivity in the vertebrate nervous system. In this paper, Cioni et al. reveal new insights on how retinal axons segregate in the optic tract through CYFIP2-mediated axon-axon signaling. more...
- Published
- 2017
34. RNA-based mechanisms underlying axon guidance
- Author
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Bo Lu, Christine E. Holt, and Toshiaki Shigeoka
- Subjects
Pioneer axon ,Proteome ,RNA ,Axon guidance ,Translation (biology) ,Cell Biology ,Anatomy ,Biology ,Growth cone ,Neuroscience ,Neuronal circuitry ,Embryonic stem cell - Abstract
Axon guidance plays a key role in establishing neuronal circuitry. The motile tips of growing axons, the growth cones, navigate by responding directionally to guidance cues that pattern the embryonic neural pathways via receptor-mediated signaling. Evidence in vitro in the last decade supports the notion that RNA-based mechanisms contribute to cue-directed steering during axon guidance. Different cues trigger translation of distinct subsets of mRNAs and localized translation provides precise spatiotemporal control over the growth cone proteome in response to localized receptor activation. Recent evidence has now demonstrated a role for localized translational control in axon guidance decisions in vivo. more...
- Published
- 2013
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35. RNA-binding protein Vg1RBP regulates terminal arbor formation but not long-range axon navigation in the developing visual system
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Christine E. Holt, Adrianna Kalous, James I. Stake, and Joel K. Yisraeli
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Biology ,Axon hillock ,Retinal ganglion ,Cell biology ,Cellular and Molecular Neuroscience ,medicine.anatomical_structure ,nervous system ,Developmental Neuroscience ,Pioneer axon ,medicine ,Axon guidance ,Telodendron ,Axon ,Growth cone ,Tectum ,Neuroscience - Abstract
Local synthesis of β-actin is required for attractive turning responses to guidance cues of growth cones in vitro but its functional role in axon guidance in vivo is poorly understood. The transport and translation of β-actin mRNA is regulated by the RNA-binding protein, Vg1RBP (zipcode-binding protein-1). To examine whether Vg1RBP plays a role in axon navigation in vivo, we disrupted Vg1RBP function in embryonic Xenopus laevis retinal ganglion cells by expressing a dominant-negative Vg1RBP and by antisense morpholino knockdown. We found that attractive turning to a netrin-1 gradient in vitro was abolished in Vg1RBP-deficient axons but, surprisingly, the long-range navigation from the retina to the optic tectum was unaffected. Within the tectum, however, the branching and complexity of axon terminals were significantly reduced. High-resolution time-lapse imaging of axon terminals in vivo revealed that Vg1RBP-GFP-positive granules accumulate locally in the axon shaft immediately preceding the emergence a filopodial-like protrusion. Comparative analysis of branch dynamics showed that Vg1RBP-deficient axons extend far fewer filopodial-like protrusions than control axons and indicate that Vg1RBP promotes filopodial formation, an essential step in branch initiation. Our findings show that Vg1RBP is required for terminal arborization but not long-range axon navigation and suggest that Vg1RBP-regulated mRNA translation promotes synaptic complexity. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 303–318, 2014 more...
- Published
- 2013
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36. Role of microRNAs in Semaphorin function and neural circuit formation
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Anaïs Bellon, Christine E. Holt, and Marie-Laure Baudet
- Subjects
Nervous system ,Neuronal Plasticity ,animal structures ,Semaphorins ,Cell Biology ,Anatomy ,Biology ,Nervous System ,MicroRNAs ,Signalling ,medicine.anatomical_structure ,nervous system ,Semaphorin ,Cell Movement ,Mirna expression ,microRNA ,medicine ,Animals ,Humans ,Axon guidance ,Signal transduction ,Neuroscience ,Function (biology) ,Signal Transduction ,Developmental Biology - Abstract
Since the discovery of the first microRNA (miRNA) almost 20 years ago, insight into their functional role has gradually been accumulating. This class of non-coding RNAs has recently been implicated as key molecular regulators in the biology of most eukaryotic cells, contributing to the physiology of various systems including immune, cardiovascular, nervous systems and also to the pathophysiology of cancers. Interestingly, Semaphorins, a class of evolutionarily conserved signalling molecules, are acknowledged to play major roles in these systems also. This, combined with the fact that Semaphorin signalling requires tight spatiotemporal regulation, a hallmark of miRNA expression, suggests that miRNAs could be crucial regulators of Semaphorin function. Here, we review evidence suggesting that Semaphorin signalling is regulated by miRNAs in various systems in health and disease. In particular, we focus on neural circuit formation, including axon guidance, where Semaphorin function was first discovered. more...
- Published
- 2013
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37. RNA Docking and Local Translation Regulate Site-Specific Axon Remodeling In Vivo
- Author
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Hovy Ho-Wai, Wong, Julie Qiaojin, Lin, Florian, Ströhl, Cláudio Gouveia, Roque, Jean-Michel, Cioni, Roberta, Cagnetta, Benita, Turner-Bridger, Romain F, Laine, William A, Harris, Clemens F, Kaminski, and Christine E, Holt more...
- Subjects
Blastomeres ,Embryo, Nonmammalian ,neural wiring ,Biotin ,In Vitro Techniques ,Retina ,Article ,Morpholinos ,Xenopus laevis ,Organ Culture Techniques ,retinotectal projection ,Animals ,local protein synthesis ,Cycloheximide ,β-actin ,RNA trafficking ,Protein Synthesis Inhibitors ,RNA labeling ,axon guidance ,fungi ,Gene Expression Regulation, Developmental ,RNA localization ,Carbocyanines ,Oligonucleotides, Antisense ,Actins ,Axons ,Mitochondria ,Luminescent Proteins ,axon branching ,nervous system ,FRAP ,RNA ,Deoxyuracil Nucleotides ,Anisomycin - Abstract
Summary Nascent proteins can be positioned rapidly at precise subcellular locations by local protein synthesis (LPS) to facilitate localized growth responses. Axon arbor architecture, a major determinant of synaptic connectivity, is shaped by localized growth responses, but it is unknown whether LPS influences these responses in vivo. Using high-resolution live imaging, we examined the spatiotemporal dynamics of RNA and LPS in retinal axons during arborization in vivo. Endogenous RNA tracking reveals that RNA granules dock at sites of branch emergence and invade stabilized branches. Live translation reporter analysis reveals that de novo β-actin hotspots colocalize with docked RNA granules at the bases and tips of new branches. Inhibition of axonal β-actin mRNA translation disrupts arbor dynamics primarily by reducing new branch emergence and leads to impoverished terminal arbors. The results demonstrate a requirement for LPS in building arbor complexity and suggest a key role for pre-synaptic LPS in assembling neural circuits., Highlights • Tracking endogenous RNA shows that RNA docking predicts axon branch emergence in vivo • Axon arbor complexity in vivo depends on local protein synthesis • Axonal β-actin synthesis regulates branching by increased branch initiation • Live imaging reveals de novo synthesis of β-actin hotspots during branch formation, Wong et al. track endogenous RNA granules in single axons in vivo and show that they predict positions of axonal branch formation in the tectum. They also show that perturbation of local protein synthesis in these axons autonomously regulates branch formation. more...
- Published
- 2017
38. Coupling of NF-protocadherin signaling to axon guidance by cue-induced translation
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William A. Harris, Vasja Urbančič, Christine E. Holt, Asha Dwivedy, Timothy G. Bayley, Aih Cheun Lee, Marie-Laure Baudet, and Louis C. Leung
- Subjects
Retinal Ganglion Cells ,Embryo, Nonmammalian ,Time Factors ,genetic structures ,Optic tract ,Green Fluorescent Proteins ,Neuroepithelial Cells ,Protocadherin ,Xenopus Proteins ,Biology ,Transfection ,Retina ,Article ,Xenopus laevis ,chemistry.chemical_compound ,Organ Culture Techniques ,Chlorocebus aethiops ,Animals ,Visual Pathways ,Protein Synthesis Inhibitors ,Analysis of Variance ,Microscopy, Confocal ,General Neuroscience ,Semaphorin-3A ,Retinal ,Translation (biology) ,SEMA3A ,Adhesion ,Cadherins ,Axons ,Protocadherins ,Cell biology ,Coupling (electronics) ,Electroporation ,nervous system ,chemistry ,Protein Biosynthesis ,COS Cells ,Axon guidance ,sense organs ,Cues ,Neuroscience ,Anisomycin ,Signal Transduction ,Transcription Factors - Abstract
Cell adhesion molecules and diffusible cues both regulate axon pathfinding, yet how these two modes of signaling interact is poorly understood. The homophilic cell adhesion molecule NF-protocadherin (NFPC) is expressed in the mid-dorsal optic tract neuroepithelium and in the axons of developing retinal ganglion cells (RGC) in Xenopus laevis. Here we report that targeted disruption of NFPC function in RGC axons or the optic tract neuroepithelium results in unexpectedly localized pathfinding defects at the caudal turn in the mid-optic tract. Semaphorin 3A (Sema3A), which lies adjacent to this turn, stimulates rapid, protein synthesis-dependent increases in growth cone NFPC and its cofactor, TAF1, in vitro. In vivo, growth cones exhibit marked increases in NFPC translation reporter activity in this mid-optic tract region that are attenuated by blocking neuropilin-1 function. Our results suggest that translation-linked coupling between regionally localized diffusible cues and cell adhesion can help axons navigate discrete segments of the pathway. more...
- Published
- 2013
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39. Single Molecule Translation Imaging Visualizes the Dynamics of Local β-Actin Synthesis in Retinal Axons
- Author
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Florian Ströhl, Julie Qiaojin Lin, Romain F. Laine, Hovy Ho-Wai Wong, Vasja Urbančič, Roberta Cagnetta, Christine E. Holt, Clemens F. Kaminski, Stroehl, Florian [0000-0002-2603-0780], Lin, Qiaojin [0000-0002-2669-6478], Holt, Christine [0000-0003-2829-121X], Kaminski, Clemens [0000-0002-5194-0962], and Apollo - University of Cambridge Repository more...
- Subjects
axon and dendritic guidance ,Science ,Xenopus ,Gene Expression ,Actins ,Axons ,Retina ,Article ,Cell Line ,Molecular Imaging ,Genes, Reporter ,Protein Biosynthesis ,microscopy ,Medicine ,Animals ,Humans ,RNA, Messenger ,Cells, Cultured - Abstract
Local mRNA translation occurs in growing axons enabling precise control of the proteome in response to signals. To measure quantitatively the spatiotemporal dynamics of protein synthesis in growth cones, we further developed a technique for single molecule translation imaging (SMTI). We report that Netrin-1 triggers a burst of β-actin synthesis at multiple non-repetitive sites, particularly in the periphery. The response is remarkably rapid starting within 20 seconds of cue application. more...
- Published
- 2016
40. Hermes Regulates Axon Sorting in the Optic Tract by Post-Trancriptional Regulation of Neuropilin 1
- Author
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Hanna, Hörnberg, Jean-Michel, Cioni, William A, Harris, and Christine E, Holt
- Subjects
Retinal Ganglion Cells ,Superior Colliculi ,Embryo, Nonmammalian ,RNA-binding protein ,Growth Cones ,Development/Plasticity/Repair ,RNA-Binding Proteins ,Semaphorin-3A ,Xenopus Proteins ,axon sorting ,Axons ,Neuropilin-1 ,Xenopus laevis ,topography ,Gene Knockdown Techniques ,Animals ,visual system ,Visual Pathways ,sense organs ,Protein Processing, Post-Translational ,Zebrafish ,Research Articles - Abstract
The establishment of precise topographic maps during neural development is facilitated by the presorting of axons in the pathway before they reach their targets. In the vertebrate visual system, such topography is seen clearly in the optic tract (OT) and in the optic radiations. However, the molecular mechanisms involved in pretarget axon sorting are poorly understood. Here, we show in zebrafish that the RNA-binding protein Hermes, which is expressed exclusively in retinal ganglion cells (RGCs), is involved in this process. Using a RiboTag approach, we show that Hermes acts as a negative translational regulator of specific mRNAs in RGCs. One of these targets is the guidance cue receptor Neuropilin 1 (Nrp1), which is sensitive to the repellent cue Semaphorin 3A (Sema3A). Hermes knock-down leads to topographic missorting in the OT through the upregulation of Nrp1. Restoring Nrp1 to appropriate levels in Hermes-depleted embryos rescues this effect and corrects the axon-sorting defect in the OT. Our data indicate that axon sorting relies on Hermes-regulated translation of Nrp1. SIGNIFICANCE STATEMENT An important mechanism governing the formation of the mature neural map is pretarget axon sorting within the sensory tract; however, the molecular mechanisms involved in this process remain largely unknown. The work presented here reveals a novel function for the RNA-binding protein Hermes in regulating the topographic sorting of retinal ganglion cell (RGC) axons in the optic tract and tectum. We find that Hermes negatively controls the translation of the guidance cue receptor Neuropilin-1 in RGCs, with Hermes knock-down resulting in aberrant growth cone cue sensitivity and axonal topographic misprojections. We characterize a novel RNA-based mechanism by which axons restrict their translatome developmentally to achieve proper targeting. more...
- Published
- 2016
41. Differing Semaphorin 3A Concentrations Trigger Distinct Signaling Mechanisms in Growth Cone Collapse
- Author
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Christine E. Holt, Richard P. C. Manns, Geoffrey M.W. Cook, and Roger J. Keynes
- Subjects
Growth Cones ,Chick Embryo ,Biology ,Article ,Growth cone collapse ,Glycogen Synthase Kinase 3 ,Semaphorin ,Dorsal root ganglion ,Cell Movement ,Ganglia, Spinal ,Nerve Growth Factor ,medicine ,Protein biosynthesis ,Animals ,Cycloheximide ,Growth cone ,Cells, Cultured ,Collapse (medical) ,Protein Synthesis Inhibitors ,Glycogen Synthase Kinase 3 beta ,Dose-Response Relationship, Drug ,TOR Serine-Threonine Kinases ,General Neuroscience ,Semaphorin-3A ,SEMA3A ,Axons ,Neuropilin-1 ,medicine.anatomical_structure ,Protein Biosynthesis ,Biophysics ,sense organs ,medicine.symptom ,Signal transduction ,Neuroscience ,Signal Transduction - Abstract
Semaphorin-3A (Sema3A) is a major guidance cue in the developing nervous system. Previous studies have revealed a dependence of responses to Sema3A on local protein synthesis (PS) in axonal growth cones, but a recent study has called this dependence into question. To understand the basis of this discrepancy we used the growth cone collapse assay on chick dorsal root ganglion (DRG) neurons. We show that the dependence of growth cone collapse on protein synthesis varies according to Sema3A concentration, from near-total at low concentration (625ng/ml). Further, we show that neuropilin-1 (NP-1) mediates both PS-dependent and PS–independent collapse. Our findings are consistent with the operation of at least two distinct Sema3A signaling pathways: one that is PS-dependent, involving mammalian target of rapamycin (mTOR), and one that is PS-independent, involving GSK-3β activation and operative at all concentrations of Sema3A examined. The results provide a plausible explanation for the discrepancy in PS-dependence reported in the literature, and indicate that different signaling pathways activated within growth cones can be modulated by changing the concentration of the same guidance cue. more...
- Published
- 2012
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42. Translational regulation in growth cones
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Catherine M. O'Hare, Christine E. Holt, and Hosung Jung
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Genetics ,Regulation of gene expression ,Messenger RNA ,Three prime untranslated region ,Growth Cones ,Gene Expression Regulation, Developmental ,Translation (biology) ,Biology ,Article ,Cell biology ,Protein Biosynthesis ,Translational regulation ,Protein biosynthesis ,Animals ,RNA, Messenger ,Signal transduction ,Growth cone ,3' Untranslated Regions ,Signal Transduction ,Developmental Biology - Abstract
Axonal growth cones (GCs) steer in response to extrinsic cues using mechanisms that include local protein synthesis. This adaptive form of gene regulation occurs with spatial precision and depends on subcellular mRNA localisation. Recent genome-wide studies have shown unexpectedly complex and dynamically changing mRNA repertoires in growing axons and GCs. Axonal targeting of some transcripts seems to be highly selective and involves sequence diversity in non-coding regions generated by transcriptional and/or post-transcriptional mechanisms. New evidence reports direct coupling of a guidance receptor to the protein synthesis machinery and other findings demonstrate that some guidance cues can repress translation. The recent findings shed further light on the exquisitely regulated process that enables distant cellular compartments to respond to local stimuli. more...
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- 2011
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43. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization
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Yi-Chun Loraine Tung, Brian Y.H. Lam, James W. Fawcett, Christine E. Holt, Giovanni Coppola, Giles S.H. Yeo, Krishna H. Zivraj, Laura F. Gumy, Dianna E. Willis, and Jeffery L. Twiss
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Sensory Receptor Cells ,Blotting, Western ,Nerve Tissue Proteins ,Sensory system ,Biology ,Article ,Immunoenzyme Techniques ,Rats, Sprague-Dawley ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Tubulin ,medicine ,Animals ,RNA, Messenger ,Axon ,Molecular Biology ,Cells, Cultured ,Oligonucleotide Array Sequence Analysis ,030304 developmental biology ,0303 health sciences ,Messenger RNA ,Reverse Transcriptase Polymerase Chain Reaction ,Microarray analysis techniques ,Gene Expression Profiling ,Embryo, Mammalian ,Embryonic stem cell ,Molecular biology ,Axons ,Nerve Regeneration ,Rats ,Cell biology ,Gene expression profiling ,Nociception ,medicine.anatomical_structure ,nervous system ,030217 neurology & neurosurgery - Abstract
mRNAs are transported, localized, and translated in axons of sensory neurons. However, little is known about the full repertoire of transcripts present in embryonic and adult sensory axons and how this pool of mRNAs dynamically changes during development. Here, we used a compartmentalized chamber to isolate mRNA from pure embryonic and adult sensory axons devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localized in sensory axons and that this repertoire changes during development toward adulthood. Embryonic axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are enriched in mRNAs encoding immune molecules with a role in nociception. Additionally, we show Tubulin-beta3 (Tubb3) mRNA is present only in embryonic axons, with Tubb3 locally synthesized in axons of embryonic, but not adult neurons where it is transported, thus validating our experimental approach. In summary, we provide the first complete catalog of embryonic and adult sensory axonal mRNAs. In addition we show that this pool of axonal mRNAs dynamically changes during development. These data provide an important resource for studies on the role of local protein synthesis in axon regeneration and nociception during neuronal development. more...
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- 2010
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44. Local translation of mRNAs in neural development
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Christine E. Holt and Hosung Jung
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nervous system ,Cell bodies ,Protein biosynthesis ,Biological neural network ,Translation (biology) ,Anatomy ,Biology ,Growth cone ,Molecular Biology ,Biochemistry ,Neural development ,Neuroscience - Abstract
Growing axons encounter numerous developmental signals to which they must promptly respond in order to properly form complex neural circuitry. In the axons, these signals are often transduced into a local increase or decrease in protein levels. Contrary to the traditional view that the cell bodies are the exclusive source of axonal proteins, it is becoming increasingly clear not only that de novo protein synthesis takes place in axons, but also that it is required for the axons to respond to certain signals. Here we review the current knowledge of local mRNA translation in developing neurons with a special focus on protein synthesis occurring in axons and growth cones. more...
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- 2010
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45. Subcellular Profiling Reveals Distinct and Developmentally Regulated Repertoire of Growth Cone mRNAs
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Giles S.H. Yeo, Laura F. Gumy, Yi Chun Loraine Tung, Krishna H. Zivraj, James W. Fawcett, Michael Piper, and Christine E. Holt
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Retinal Ganglion Cells ,genetic structures ,Growth Cones ,Xenopus ,Article ,03 medical and health sciences ,Mice ,Xenopus laevis ,0302 clinical medicine ,Species Specificity ,Gene expression ,medicine ,Animals ,RNA, Messenger ,Axon ,Growth cone ,Cells, Cultured ,030304 developmental biology ,Laser capture microdissection ,0303 health sciences ,biology ,General Neuroscience ,Gene Expression Profiling ,Gene Expression Regulation, Developmental ,biology.organism_classification ,Molecular biology ,Cell biology ,Gene expression profiling ,medicine.anatomical_structure ,Axon guidance ,Soma ,sense organs ,030217 neurology & neurosurgery ,Subcellular Fractions - Abstract
Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons, yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Here laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse andXenopus, coupled with unbiased genomewide microarray profiling. An unexpectedly large pool of mRNAs defined predominant pathways in protein synthesis, oxidative phosphorylation, cancer, neurological disease, and signaling. Comparative profiling of “young” (pathfinding) versus “old” (target-arriving)Xenopusgrowth cones revealed that the number and complexity of transcripts increases dramatically with age. Many presynaptic protein mRNAs are present exclusively in old growth cones, suggesting that functionally related sets of mRNAs are targeted to growth cones in a developmentally regulated way. Remarkably, a subset of mRNAs was significantly enriched in the growth cone compared with the axon compartment, indicating that mechanisms exist to localize mRNAs selectively to the growth cone. Furthermore, some receptor transcripts (e.g., EphB4), present exclusively in old growth cones, were equally abundant in young and old cell bodies, indicating that RNA trafficking from the soma is developmentally regulated. Our findings show that the mRNA repertoire in growth cones is regulated dynamically with age and suggest that mRNA localization is tailored to match the functional demands of the growing axon tip as it transforms into the presynaptic terminal. more...
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- 2010
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46. Outsourcing CREB translation to axons to survive
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Christine E. Holt and Andrew C. Lin
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biology ,Translation (biology) ,Cell Biology ,CREB ,Sensory neuron ,Cell biology ,Nerve growth factor ,medicine.anatomical_structure ,nervous system ,biology.protein ,Retrograde signaling ,medicine ,Axon ,Growth cone ,Transcription factor - Abstract
Nerve growth factor induces sensory neuron survival via retrograde signalling from the axon to the cell body. Local translation of the transcription factor CREB in the axon, followed by its transport to the nucleus, is involved in this process. more...
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- 2008
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47. Mechanosensing is critical for axon growth in the developing brain
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Luciano da Fontoura Costa, Graham K. Sheridan, Christine E. Holt, Eva K Pillai, Sarah Foster, David E. Koser, Hanno Svoboda, Kristian Franze, Amelia J Thompson, Jochen Guck, Matheus P. Viana, and Asha Dwivedy more...
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Retinal Ganglion Cells ,0301 basic medicine ,Nervous system ,Neurogenesis ,durotaxis ,Biology ,Mechanotransduction, Cellular ,Retina ,Article ,biomechanics ,Xenopus laevis ,03 medical and health sciences ,Mechanosensitive ion channel ,brain stiffness ,medicine ,Animals ,Visual Pathways ,Mechanotransduction ,Zebrafish ,axon guidance ,General Neuroscience ,PIEZO1 ,stiffness gradient ,Brain ,CÉREBRO ,mechanosensitivity ,Axons ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,Retinal ganglion cell ,Mechanosensitive channels ,Axon guidance ,stretch-activated ion channels ,AFM ,Neuroscience - Abstract
During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo. more...
- Published
- 2016
48. Ena/VASP function in retinal axons is required for terminal arborization but not pathway navigation
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Jeffrey S. Miller, Cécile Lebrand, Asha Dwivedy, Christine E. Holt, and Frank B. Gertler
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Retinal Ganglion Cells ,genetic structures ,Protein family ,Xenopus ,Green Fluorescent Proteins ,Growth Cones ,macromolecular substances ,Biology ,Retinal ganglion ,Article ,EVH1 domain ,Animals ,Pseudopodia ,Growth cone ,Molecular Biology ,Cells, Cultured ,Microscopy, Video ,Microfilament Proteins ,Ena/Vasp homology proteins ,Phosphoproteins ,Actin cytoskeleton ,Mitochondria ,Cell biology ,nervous system ,sense organs ,Cell Adhesion Molecules ,Filopodia ,Developmental Biology - Abstract
The Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family of proteins is required for filopodia formation in growth cones and plays a crucial role in guidance cue-induced remodeling of the actin cytoskeleton. In vivo studies with pharmacological inhibitors of actin polymerization have previously provided evidence for the view that filopodia are needed for growth cone navigation in the developing visual pathway. Here we have re-examined this issue using an alternative strategy to generate growth cones without filopodia in vivo by artificially targeting Xena/XVASP (Xenopushomologs of Ena/VASP) proteins to mitochondria in retinal ganglion cells(RGCs). We used the specific binding of the EVH1 domain of the Ena/VASP family of proteins with the ligand motif FP4 to sequester the protein at the mitochondria surface. RGCs with reduced function of Xena/XVASP proteins extended fewer axons out of the eye and possessed dynamic lamellipodial growth cones missing filopodia that advanced slowly in the optic tract. Surprisingly,despite lacking filopodia, the axons navigated along the optic pathway without obvious guidance errors, indicating that the Xena/XVASP family of proteins and filopodial protrusions are non-essential for pathfinding in retinal axons. However, depletion of Xena/XVASP proteins severely impaired the ability of growth cones to form branches within the optic tectum, suggesting that this protein family, and probably filopodia, plays a key role in establishing terminal arborizations. more...
- Published
- 2007
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49. Dynamic Axonal Translation in Developing and Mature Visual Circuits
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Toshiaki, Shigeoka, Hosung, Jung, Jane, Jung, Benita, Turner-Bridger, Jiyeon, Ohk, Julie Qiaojin, Lin, Paul S, Amieux, and Christine E, Holt
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Retinal Ganglion Cells ,Superior Colliculi ,Proteome ,Gene Expression Regulation, Developmental ,Synaptic Transmission ,Axons ,Article ,Alternative Splicing ,Mice ,nervous system ,Protein Biosynthesis ,Animals ,RNA, Messenger ,Ribosomes - Abstract
Summary Local mRNA translation mediates the adaptive responses of axons to extrinsic signals, but direct evidence that it occurs in mammalian CNS axons in vivo is scant. We developed an axon-TRAP-RiboTag approach in mouse that allows deep-sequencing analysis of ribosome-bound mRNAs in the retinal ganglion cell axons of the developing and adult retinotectal projection in vivo. The embryonic-to-postnatal axonal translatome comprises an evolving subset of enriched genes with axon-specific roles, suggesting distinct steps in axon wiring, such as elongation, pruning, and synaptogenesis. Adult axons, remarkably, have a complex translatome with strong links to axon survival, neurotransmission, and neurodegenerative disease. Translationally co-regulated mRNA subsets share common upstream regulators, and sequence elements generated by alternative splicing promote axonal mRNA translation. Our results indicate that intricate regulation of compartment-specific mRNA translation in mammalian CNS axons supports the formation and maintenance of neural circuits in vivo., Graphical Abstract, Highlights • Dynamic translatome of retinal axons in vivo matches changing subcellular function • Adult CNS axons translate mRNAs for synaptic transmission and axon survival in vivo • Target mRNAs of key translation regulators show developmental co-regulation • Axon-specific sequence motifs link alternative splicing to axonal translation, Local mRNA translation in axons of developing and adult CNS neurons in vivo shows dynamic regulation, suggesting functional relevance for neural circuit formation and maintenance. more...
- Published
- 2015
50. Erratum to: Differential requirement of F-actin and microtubule cytoskeleton in cue-induced local protein synthesis in axonal growth cones
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Michael Piper, Trina Bo Lu, Christine E. Holt, Aih Cheun Lee, William A. Harris, Francisca P. G. van Horck, and Heather McNeilly
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Developmental Neuroscience ,Microtubule cytoskeleton ,Protein biosynthesis ,Biology ,Growth cone ,Differential (mathematics) ,Actin ,Cell biology - Published
- 2015
- Full Text
- View/download PDF
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