Publications by authors named "Joel Ryan"

20 Publications

  • Page 1 of 1

Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis.

Nat Microbiol 2021 Mar 18. Epub 2021 Mar 18.

Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.

Although many components of the cell division machinery in bacteria have been identified, the mechanisms by which they work together to divide the cell remain poorly understood. Key among these components is the tubulin FtsZ, which forms a Z ring at the midcell. FtsZ recruits the other cell division proteins, collectively called the divisome, and the Z ring constricts as the cell divides. We applied live-cell single-molecule imaging to describe the dynamics of the divisome in detail, and to evaluate the individual roles of FtsZ-binding proteins (ZBPs), specifically FtsA and the ZBPs EzrA, SepF and ZapA, in cytokinesis. We show that the divisome comprises two subcomplexes that move differently: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a moving complex that includes cell wall synthases. Our imaging analyses reveal that ZBPs bundle FtsZ filaments together and condense them into Z rings, and that this condensation is necessary for cytokinesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-021-00878-zDOI Listing
March 2021

HP1β carries an acidic linker domain and requires H3K9me3 for phase separation.

Nucleus 2021 Dec;12(1):44-57

Center for Molecular Biosystems (BioSysM), Faculty of Biology, Ludwig-Maximilians-Universität München, Munich, Germany.

Liquid-liquid phase separation (LLPS) mediated formation of membraneless organelles has been proposed to coordinate biological processes in space and time. Previously, the formation of phase-separated droplets was described as a unique property of HP1α. Here, we demonstrate that the positive net charge of the intrinsically disordered hinge region (IDR-H) of HP1 proteins is critical for phase separation and that the exchange of four acidic amino acids is sufficient to confer LLPS properties to HP1β. Surprisingly, the addition of mono-nucleosomes promoted H3K9me3-dependent LLPS of HP1β which could be specifically disrupted with methylated but not acetylated H3K9 peptides. HP1β mutants defective in H3K9me3 binding were less efficient in phase separationand failed to accumulate at heterochromatin . We propose that multivalent interactions of HP1β with H3K9me3-modified nucleosomes via its chromodomain and dimerization via its chromoshadow domain enable phase separation and contribute to the formation of heterochromatin compartments .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19491034.2021.1889858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939559PMC
December 2021

Recent evolution of a TET-controlled and DPPA3/STELLA-driven pathway of passive DNA demethylation in mammals.

Nat Commun 2020 11 24;11(1):5972. Epub 2020 Nov 24.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Human Biology and BioImaging, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. Here, we describe a recently evolved pathway in which global hypomethylation is achieved by the coupling of active and passive demethylation. TET activity is required, albeit indirectly, for global demethylation, which mostly occurs at sites devoid of TET binding. Instead, TET-mediated active demethylation is locus-specific and necessary for activating a subset of genes, including the naïve pluripotency and germline marker Dppa3 (Stella, Pgc7). DPPA3 in turn drives large-scale passive demethylation by directly binding and displacing UHRF1 from chromatin, thereby inhibiting maintenance DNA methylation. Although unique to mammals, we show that DPPA3 alone is capable of inducing global DNA demethylation in non-mammalian species (Xenopus and medaka) despite their evolutionary divergence from mammals more than 300 million years ago. Our findings suggest that the evolution of Dppa3 facilitated the emergence of global DNA demethylation in mammals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-19603-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686362PMC
November 2020

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency.

Sci Rep 2020 07 21;10(1):12066. Epub 2020 Jul 21.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-68600-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7374584PMC
July 2020

A unified multi-kingdom Golden Gate cloning platform.

Sci Rep 2019 07 12;9(1):10131. Epub 2019 Jul 12.

Faculty of Biology, Genetics, LMU Munich, D-82152, Martinsried, Germany.

Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-46171-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626145PMC
July 2019

Bacillus subtilis cell diameter is determined by the opposing actions of two distinct cell wall synthetic systems.

Nat Microbiol 2019 08 13;4(8):1294-1305. Epub 2019 May 13.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.

Rod-shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: the Rod complex moves around the cell circumference, whereas class A penicillin-binding proteins (aPBPs) do not. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each affects the growth and width of Bacillus subtilis as well as the mechanical anisotropy and orientation of material within their sacculi. Rod width is not determined by MreB, rather it depends on the balance between the systems: the Rod complex reduces diameter, whereas aPBPs increase it. Increased Rod-complex activity correlates with an increased density of directional MreB filaments and a greater fraction of directional PBP2a enzymes. This increased circumferential synthesis increases the relative quantity of oriented material within the sacculi, making them more resistant to stretching across their width, thereby reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems builds and maintains rods of different widths. Escherichia coli Rod mutants also show the same correlation between width and directional MreB filament density, suggesting this model may be generalizable to bacteria that elongate via the Rod complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-019-0439-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656618PMC
August 2019

Ubiquitome Analysis Reveals PCNA-Associated Factor 15 (PAF15) as a Specific Ubiquitination Target of UHRF1 in Embryonic Stem Cells.

J Mol Biol 2017 12 18;429(24):3814-3824. Epub 2017 Oct 18.

Department of Biology II and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Großhaderner Str. 2, 82152 Planegg-Martinsried, Germany. Electronic address:

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmb.2017.10.014DOI Listing
December 2017

DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation.

Hum Mol Genet 2017 04;26(8):1522-1534

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddx057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393148PMC
April 2017

STED Imaging in Drosophila Brain Slices.

Methods Mol Biol 2017 ;1563:143-150

Max Planck Institute of Neurobiology, Am Klopferspitz 18, 82152, Martinsried, Munich, Germany.

Super-resolution microscopy is a very powerful tool to investigate fine cellular structures and molecular arrangements in biological systems. For instance, stimulated emission depletion (STED) microscopy has been successfully used in recent years to investigate the arrangement and colocalization of different protein species in cells in culture and on the surface of specimens. However, because of its extreme sensitivity to light scattering, super-resolution imaging deep inside tissues remains a challenge. Here, we describe the preparation of thin slices from the fruit fly (Drosophila melanogaster) brain, subsequent immunolabeling and imaging with STED microscopy. This protocol allowed us to image small dendritic branches from neurons located deep in the fly brain with improved resolution compared with conventional light microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-6810-7_10DOI Listing
February 2018

Introduction to Modern Methods in Light Microscopy.

Methods Mol Biol 2017 ;1563:1-15

Department of Biology, University of North Carolina at Chapel Hill, 4358 Genome Sciences Building, Chapel Hill, NC, 27599, USA.

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-6810-7_1DOI Listing
February 2018

High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells.

J Clin Invest 2016 12 31;126(12):4569-4584. Epub 2016 Oct 31.

Current chemotherapies for T cell acute lymphoblastic leukemia (T-ALL) efficiently reduce tumor mass. Nonetheless, disease relapse attributed to survival of preleukemic stem cells (pre-LSCs) is associated with poor prognosis. Herein, we provide direct evidence that pre-LSCs are much less chemosensitive to existing chemotherapy drugs than leukemic blasts because of a distinctive lower proliferative state. Improving therapies for T-ALL requires the development of strategies to target pre-LSCs that are absolutely dependent on their microenvironment. Therefore, we designed a robust protocol for high-throughput screening of compounds that target primary pre-LSCs maintained in a niche-like environment, on stromal cells that were engineered for optimal NOTCH1 activation. The multiparametric readout takes into account the intrinsic complexity of primary cells in order to specifically monitor pre-LSCs, which were induced here by the SCL/TAL1 and LMO1 oncogenes. We screened a targeted library of compounds and determined that the estrogen derivative 2-methoxyestradiol (2-ME2) disrupted both cell-autonomous and non-cell-autonomous pathways. Specifically, 2-ME2 abrogated pre-LSC viability and self-renewal activity in vivo by inhibiting translation of MYC, a downstream effector of NOTCH1, and preventing SCL/TAL1 activity. In contrast, normal hematopoietic stem/progenitor cells remained functional. These results illustrate how recapitulating tissue-like properties of primary cells in high-throughput screening is a promising avenue for innovation in cancer chemotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI86489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127686PMC
December 2016

Identification of microtubule growth deceleration and its regulation by conserved and novel proteins.

Mol Biol Cell 2016 05 16;27(9):1479-87. Epub 2016 Mar 16.

Institut Jacques Monod, CNRS, UMR 7592, University Paris Diderot, Sorbonne Paris Cité, F-75205 Paris, France

Microtubules (MTs) are cytoskeletal polymers that participate in diverse cellular functions, including cell division, intracellular trafficking, and templating of cilia and flagella. MTs undergo dynamic instability, alternating between growth and shortening via catastrophe and rescue events. The rates and frequencies of MT dynamic parameters appear to be characteristic for a given cell type. We recently reported that all MT dynamic parameters vary throughout differentiation of a smooth muscle cell type in intact Caenorhabditis elegans. Here we describe local differences in MT dynamics and a novel MT behavior: an abrupt change in growth rate (deceleration) of single MTs occurring in the cell periphery of these cells. MT deceleration occurs where there is a decrease in local soluble tubulin concentration at the cell periphery. This local regulation of tubulin concentration and MT deceleration are dependent on two novel homologues of human cylicin. These novel ORFs, which we name cylc-1 and -2, share sequence homology with stathmins and encode small, very basic proteins containing several KKD/E repeats. The TOG domain-containing protein ZYG-9(TOGp) is responsible for the faster polymerization rate within the cell body. Thus we have defined two contributors to the molecular regulation for this novel MT behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E16-01-0056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850035PMC
May 2016

Investigating the regulation of stem and progenitor cell mitotic progression by in situ imaging.

Curr Biol 2015 May 26;25(9):1123-34. Epub 2015 Mar 26.

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Genome stability relies upon efficacious chromosome congression and regulation by the spindle assembly checkpoint (SAC). The study of these fundamental mitotic processes in adult stem and progenitor cells has been limited by the technical challenge of imaging mitosis in these cells in situ. Notably, how broader physiological changes, such as dietary intake or age, affect mitotic progression in stem and/or progenitor cells is largely unknown. Using in situ imaging of C. elegans adult germlines, we describe the mitotic parameters of an adult stem and progenitor cell population in an intact animal. We find that SAC regulation in germline stem and progenitor cells is distinct from that found in early embryonic divisions and is more similar to that of classical tissue culture models. We further show that changes in organismal physiology affect mitotic progression in germline stem and progenitor cells. Reducing dietary intake produces a checkpoint-dependent delay in anaphase onset, and inducing dietary restriction when the checkpoint is impaired increases the incidence of segregation errors in mitotic and meiotic cells. Similarly, developmental aging of the germline stem and progenitor cell population correlates with a decline in the rate of several mitotic processes. These results provide the first in vivo validation of models for SAC regulation developed in tissue culture systems and demonstrate that several fundamental features of mitotic progression in adult stem and progenitor cells are highly sensitive to organismal physiological changes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2015.02.054DOI Listing
May 2015

A hybrid electrical/chemical circuit in the spinal cord generates a transient embryonic motor behavior.

J Neurosci 2014 Jul;34(29):9644-55

Departments of Pathology and Cell Biology and Neuroscience, Research Centre of the University of Montréal Hospital Centre and Groupe de Recherche sur le Systeme Nerveux Central, Université de Montréal, Montréal, Québec H3T 1J4, Canada

Spontaneous network activity is a highly stereotyped early feature of developing circuits throughout the nervous system, including in the spinal cord. Spinal locomotor circuits produce a series of behaviors during development before locomotion that reflect the continual integration of spinal neurons into a functional network, but how the circuitry is reconfigured is not understood. The first behavior of the zebrafish embryo (spontaneous coiling) is mediated by an electrical circuit that subsequently generates mature locomotion (swimming) as chemical neurotransmission develops. We describe here a new spontaneous behavior, double coiling, that consists of two alternating contractions of the tail in rapid succession. Double coiling was glutamate-dependent and required descending hindbrain excitation, similar to but preceding swimming, making it a discrete intermediary developmental behavior. At the cellular level, motoneurons had a distinctive glutamate-dependent activity pattern that correlated with double coiling. Two glutamatergic interneurons, CoPAs and CiDs, had different activity profiles during this novel behavior. CoPA neurons failed to show changes in activity patterns during the period in which double coiling appears, whereas CiD neurons developed a glutamate-dependent activity pattern that correlated with double coiling and they innervated motoneurons at that time. Additionally, double coils were modified after pharmacological reduction of glycinergic neurotransmission such that embryos produced three or more rapidly alternating coils. We propose that double coiling behavior represents an important transition of the motor network from an electrically coupled spinal cord circuit that produces simple periodic coils to a spinal network driven by descending chemical neurotransmission, which generates more complex behaviors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.1225-14.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608330PMC
July 2014

Octameric CENP-A nucleosomes are present at human centromeres throughout the cell cycle.

Curr Biol 2013 May 25;23(9):764-9. Epub 2013 Apr 25.

Institute for Research in Immunology and Cancer (IRIC), Faculty of Medicine, University of Montreal, Montreal, QC H3T 1J4, Canada.

The presence of a single centromere on each chromosome that signals formation of a mitotic kinetochore is central to accurate chromosome segregation. The histone H3 variant centromere protein-A (CENP-A) is critical for centromere identity and function; CENP-A chromatin acts as an epigenetic mark to direct both centromere and kinetochore assembly. Interpreting the centromere epigenetic mark ensures propagation of a single centromere per chromosome to maintain ploidy. Thus, understanding the nature of CENP-A chromatin is crucial for all cell divisions. However, there are ongoing debates over the fundamental composition of centromeric chromatin. Here we show that natively assembled human CENP-A nucleosomes are octameric throughout the cell cycle. Using total internal reflection fluorescence (TIRF)-coupled photobleaching-assisted copy-number counting of single nucleosomes obtained from cultured cells, we find that the majority of CENP-A nucleosomes contain CENP-A dimers. In addition, we detect the presence of H2B and H4 in these nucleosomes. Surprisingly, CENP-A associated with the chaperone HJURP can exist as either monomer or dimer, indicating possible assembly intermediates. Thus, our findings indicate that octameric CENP-A nucleosomes mark the centromeric region to ensure proper epigenetic inheritance and kinetochore assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2013.03.037DOI Listing
May 2013

Touch responsiveness in zebrafish requires voltage-gated calcium channel 2.1b.

J Neurophysiol 2012 Jul 4;108(1):148-59. Epub 2012 Apr 4.

Départment de Pathologie et Biologie Cellulaire, Groupe de Recherche sur le Système Nerveux Central et Centre d'Excellence en Neuromique de l'Université de Montéral, Université de Montréal, Montreal, Quebec, Canada.

The molecular and physiological basis of the touch-unresponsive zebrafish mutant fakir has remained elusive. Here we report that the fakir phenotype is caused by a missense mutation in the gene encoding voltage-gated calcium channel 2.1b (CACNA1Ab). Injection of RNA encoding wild-type CaV2.1 restores touch responsiveness in fakir mutants, whereas knockdown of CACNA1Ab via morpholino oligonucleotides recapitulates the fakir mutant phenotype. Fakir mutants display normal current-evoked synaptic communication at the neuromuscular junction but have attenuated touch-evoked activation of motor neurons. NMDA-evoked fictive swimming is not affected by the loss of CaV2.1b, suggesting that this channel is not required for motor pattern generation. These results, coupled with the expression of CACNA1Ab by sensory neurons, suggest that CaV2.1b channel activity is necessary for touch-evoked activation of the locomotor network in zebrafish.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/jn.00839.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434603PMC
July 2012

touché Is required for touch-evoked generator potentials within vertebrate sensory neurons.

J Neurosci 2010 Jul;30(28):9359-67

Département de Pathologie et Biologie Cellulaire, Groupe de Recherche sur le Système Nerveux Central, Centre d'Excellence en Neuromique de l'Université de Montréal, Université de Montréal, Montréal, Québec, Canada.

The process by which light touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing channelrhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP-positive peripheral neurites. In wild-type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild-type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that cutaneous mechanoreceptors with generator potentials are necessary for responsiveness to light touch in zebrafish.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.1639-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2921932PMC
July 2010

Impaired dopaminergic neuron development and locomotor function in zebrafish with loss of pink1 function.

Eur J Neurosci 2010 Feb 5;31(4):623-33. Epub 2010 Feb 5.

Center for Advanced Research in Environmental Genomics, Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario, Canada K1N6N5.

Mutations in the human PTEN-induced kinase 1 (PINK1) gene are linked to recessive familial Parkinson's disease. Animal models of altered PINK1 function vary greatly in their phenotypic characteristics. Drosophila pink1 mutants exhibit mild dopaminergic neuron degeneration and locomotion defects. Such defects are not observed in mice with targeted null mutations in pink1, although these mice exhibit impaired dopamine release and synaptic plasticity. Here, we report that in zebrafish, morpholino-mediated knockdown of pink1 function did not cause large alterations in the number of dopaminergic neurons in the ventral diencephalon. However, the patterning of these neurons and their projections are perturbed. This is accompanied by locomotor dysfunction, notably impaired response to tactile stimuli and reduced swimming behaviour. All these defects can be rescued by expression of an exogenous pink1 that is not a target of the morpholinos used. These results indicate that normal PINK1 function during development is necessary for the proper positioning of populations of dopaminergic neurons and for the establishment of neuronal circuits in which they are implicated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1460-9568.2010.07091.xDOI Listing
February 2010

Glutamate drives the touch response through a rostral loop in the spinal cord of zebrafish embryos.

Dev Neurobiol 2009 Oct;69(12):780-95

Institute of Neuroscience, University of Oregon, Eugene, Oregon 97403, USA.

Characterizing connectivity in the spinal cord of zebrafish embryos is not only prerequisite to understanding the development of locomotion, but is also necessary for maximizing the potential of genetic studies of circuit formation in this model system. During their first day of development, zebrafish embryos show two simple motor behaviors. First, they coil their trunks spontaneously, and a few hours later they start responding to touch with contralateral coils. These behaviors are contemporaneous until spontaneous coils become infrequent by 30 h. Glutamatergic neurons are distributed throughout the embryonic spinal cord, but their contribution to these early motor behaviors in immature zebrafish is still unclear. We demonstrate that the kinetics of spontaneous coiling and touch-evoked responses show distinct developmental time courses and that the touch response is dependent on AMPA-type glutamate receptor activation. Transection experiments suggest that the circuits required for touch-evoked responses are confined to the spinal cord and that only the most rostral part of the spinal cord is sufficient for triggering the full response. This rostral sensory connection is presumably established via CoPA interneurons, as they project to the rostral spinal cord. Electrophysiological analysis demonstrates that these neurons receive short latency AMPA-type glutamatergic inputs in response to ipsilateral tactile stimuli. We conclude that touch responses in early embryonic zebrafish arise only after glutamatergic synapses connect sensory neurons and interneurons to the contralateral motor network via a rostral loop. This helps define an elementary circuit that is modified by the addition of sensory inputs, resulting in behavioral transformation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dneu.20741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771646PMC
October 2009