Assistant Professor, Department of Biology
Ph.D. Universita di Padova, Italy
B.Sc. Imperial College London, UK
Research Interests: Molecular mechanisms of synapse formation
Overview: Information is exchanged between neurons at synapses, which are essentially specialized sites of cell-cell adhesion . A mature synapse is defined as an accumulation of synaptic vesicles within the axon, in close apposition to a dendritic membrane studded with receptors (see figure)which are held in place by a submembranous scaffold (Sheng and Kim, 2002). The formation of such an intercellular structure requires spatially and temporally controlled changes in morphology and molecular content at sites of contacts. Recent advances in subcellular fluorescence microscopy have revealed that this process involves the rapid recruitment and stabilization of both pre- and postsynaptic elements. These studies have shown that major components of the synaptic vesicle and active zone machinery travel in clusters together with other presynaptic proteins, such as calcium channels, and are rapidly recruited to new sites of contact (Ahmari et al., 2000; Zhai et al., 2001; Washbourne et al., 2002) .
On the postsynaptic side, receptor subunits and components of the scaffold or post-synaptic density (PSD) are recruited separately and with distinct time courses within minutes to hours after initial contact (Friedman et al., 2000; Bresler et al., 2001; Washbourne et al., 2002; Bresler et al., 2004)
Despite these advances the basic mechanisms by which synapse formation is induced at discrete locations and by which the molecular machinery is recruited to sites of contact remain elusive. We are currently using both mammalian primary neuronal cultures and zebrafish embryos to investigate molecules that are involved in the mechanisms of synapse formation. Techniques currently employed are live confocal imaging of fluorescently-tagged synaptic components, electron microscopy, biochemistry and molecular biology.
Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.
PLoS One. 2017;12(4):e0176543
Authors: Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne P
[This corrects the article DOI: 10.1371/journal.pone.0159277.].
PMID: 28426753 [PubMed - in process]
Redundant Postsynaptic Functions of SynCAMs 1-3 during Synapse Formation.
Front Mol Neurosci. 2017;10:24
Authors: Fowler DK, Peters JH, Williams C, Washbourne P
Investigating the roles of synaptogenic adhesion molecules during synapse formation has proven challenging, often due to compensatory functions between additional family members. The synaptic cell adhesion molecules 1-3 (SynCAM1-3) are expressed both pre- and postsynaptically, share highly homologous domains and are synaptogenic when ectopically presented to neurons; yet their endogenous functions during synaptogenesis are unclear. Here we report that SynCAM1-3 are functionally redundant and collectively necessary for synapse formation in cultured hippocampal neurons. Only triple knockdown (KD) of SynCAM1-3 using highly efficient, chained artificial microRNAs (amiRNAs) reduced synapse density and increased synapse area. Electrophysiological recordings of quantal release events supported an increase in synapse size caused by SynCAM1-3 depletion. Furthermore, a combinatorial, mosaic lentiviral approach comparing wild type (WT) and SynCAM1-3 KD neurons in the same culture demonstrate that SynCAM1-3 set synapse number and size through postsynaptic mechanisms. The results demonstrate that the redundancy between SynCAM1-3 has concealed their synaptogenic function at the postsynaptic terminal.
PMID: 28197078 [PubMed - in process]
A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.
PLoS One. 2016;11(8):e0159277
Authors: Fowler DK, Stewart S, Seredick S, Eisen JS, Stankunas K, Washbourne P
Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.
PMID: 27500400 [PubMed - in process]
Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation.
BMC Genomics. 2015;16:1100
Authors: Tallafuss A, Kelly M, Gay L, Gibson D, Batzel P, Karfilis KV, Eisen J, Stankunas K, Postlethwait JH, Washbourne P
BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults.
RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages.
CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.
PMID: 26699284 [PubMed - in process]
Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.
Nucleic Acids Res. 2016 Mar 18;44(5):e48
Authors: Fowler DK, Williams C, Gerritsen AT, Washbourne P
Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.
PMID: 26582923 [PubMed - indexed for MEDLINE]
Temporally and spatially restricted gene expression profiling.
Curr Genomics. 2014 Aug;15(4):278-92
Authors: Tallafuss A, Washbourne P, Postlethwait J
Identifying gene function in specific cells is critical for understanding the processes that make cells unique. Several different methods are available to isolate actively transcribed RNA or actively translated RNA in specific cells at a chosen time point. Cell-specific mRNA isolation can be accomplished by the expression of transgenes in cells of interest, either directly from a specific promoter or using a modular system such as Gal4/UAS or Cre/lox. All of the methods described in this review, namely thiol-labeling of RNA (TU-tagging or RABT), TRAP (translating ribosome affinity purification) and INTACT (isolation of nuclei tagged in specific cell types), allow next generation sequencing, permitting the identification of enriched gene transcripts within the specific cell-type. We describe here the general concept of each method, include examples, evaluate possible problems related to each technique, and suggest the types of questions for which each method is best suited.
PMID: 25132798 [PubMed]
Synapse assembly and neurodevelopmental disorders.
Neuropsychopharmacology. 2015 Jan;40(1):4-15
Authors: Washbourne P
In this review we examine the current understanding of how genetic deficits associated with neurodevelopmental disorders may impact synapse assembly. We then go on to discuss how the critical periods for these genetic deficits will shape the nature of future clinical interventions.
PMID: 24990427 [PubMed - indexed for MEDLINE]
Complexes of Usher proteins preassemble at the endoplasmic reticulum and are required for trafficking and ER homeostasis.
Dis Model Mech. 2014 May;7(5):547-59
Authors: Blanco-Sánchez B, Clément A, Fierro J, Washbourne P, Westerfield M
Usher syndrome (USH), the leading cause of hereditary combined hearing and vision loss, is characterized by sensorineural deafness and progressive retinal degeneration. Mutations in several different genes produce USH, but the proximal cause of sensory cell death remains mysterious. We adapted a proximity ligation assay to analyze associations among three of the USH proteins, Cdh23, Harmonin and Myo7aa, and the microtubule-based transporter Ift88 in zebrafish inner ear mechanosensory hair cells. We found that the proteins are in close enough proximity to form complexes and that these complexes preassemble at the endoplasmic reticulum (ER). Defects in any one of the three USH proteins disrupt formation and trafficking of the complex and result in diminished levels of the other proteins, generalized trafficking defects and ER stress that triggers apoptosis. ER stress, thus, contributes to sensory hair cell loss and provides a new target to explore for protective therapies for USH.
PMID: 24626987 [PubMed - indexed for MEDLINE]