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Live Imaging of ESCRT Proteins in Microfluidically Isolated Hippocampal Axons

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Book cover The ESCRT Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1998))

Abstract

Live imaging of microfluidically isolated axons permits study of the dynamic behavior of fluorescently tagged proteins and vesicles in these neuronal processes. We use this technique to study the motility and transport of ESCRT proteins in axons of primary hippocampal neurons. This chapter details the preparation of microfluidic chambers, as well as the seeding, fluidic isolation, and lentiviral transduction of hippocampal neurons in these chambers, optimized for the study of ESCRT protein dynamics.

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References

  1. Sweeney NT, Brenman JE, Jan YN, Gao F-B (2006) The coiled-coil protein shrub controls neuronal morphogenesis in Drosophila. Curr Biol 16(10):1006–1011. https://doi.org/10.1016/j.cub.2006.03.067

    Article  CAS  PubMed  Google Scholar 

  2. Rodal AA, Blunk AD, Akbergenova Y, Jorquera RA, Buhl LK, Littleton JT (2011) A presynaptic endosomal trafficking pathway controls synaptic growth signaling. J Cell Biol 193(1):201

    Article  CAS  Google Scholar 

  3. Talbot K, Ansorge O (2006) Recent advances in the genetics of amyotrophic lateral sclerosis and frontotemporal dementia: common pathways in neurodegenerative disease. Hum Mol Genet 15(suppl_2):R182–R187. https://doi.org/10.1093/hmg/ddl202

    Article  CAS  PubMed  Google Scholar 

  4. Lee J-A, Beigneux A, Ahmad ST, Young SG, Gao F-B (2007) ESCRT-III dysfunction causes autophagosome accumulation and neurodegeneration. Curr Biol 17(18):1561–1567. https://doi.org/10.1016/j.cub.2007.07.029

    Article  CAS  PubMed  Google Scholar 

  5. Cox LE, Ferraiuolo L, Goodall EF, Heath PR, Higginbottom A, Mortiboys H, Hollinger HC, Hartley JA, Brockington A, Burness CE, Morrison KE, Wharton SB, Grierson AJ, Ince PG, Kirby J, Shaw PJ (2010) Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS). PLoS One 5(3):e9872. https://doi.org/10.1371/journal.pone.0009872

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Vernay A, Therreau L, Blot B, Risson V, Dirrig-Grosch S, Waegaert R, Lequeu T, Sellal F, Schaeffer L, Sadoul R, Loeffler J-P, René F (2016) A transgenic mouse expressing CHMP2Bintron5 mutant in neurons develops histological and behavioural features of amyotrophic lateral sclerosis and frontotemporal dementia. Hum Mol Genet 25(15):3341–3360. https://doi.org/10.1093/hmg/ddw182

    Article  CAS  PubMed  Google Scholar 

  7. Walker WP, Oehler A, Edinger AL, Wagner K-U, Gunn TM (2016) Oligodendroglial deletion of ESCRT-I component TSG101 causes spongiform encephalopathy. Biol Cell 108(11):324–337. https://doi.org/10.1111/boc.201600014

    Article  CAS  PubMed  Google Scholar 

  8. Tamai K, Toyoshima M, Tanaka N, Yamamoto N, Owada Y, Kiyonari H, Murata K, Ueno Y, Ono M, Shimosegawa T, Yaegashi N, Watanabe M, Sugamura K (2008) Loss of Hrs in the central nervous system causes accumulation of ubiquitinated proteins and neurodegeneration. Am J Pathol 173(6):1806–1817. https://doi.org/10.2353/ajpath.2008.080684

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Park JW, Vahidi B, Taylor AM, Rhee SW, Jeon NL (2006) Microfluidic culture platform for neuroscience research. Nat Protoc 1:2128. https://doi.org/10.1038/nprot.2006.316

    Article  CAS  PubMed  Google Scholar 

  10. Batista AFR, Martínez JC, Hengst U (2017) Intra-axonal synthesis of SNAP25 is required for the formation of presynaptic terminals. Cell Rep 20(13):3085–3098. https://doi.org/10.1016/j.celrep.2017.08.097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kaech S, Banker G (2007) Culturing hippocampal neurons. Nat Protoc 1:2406. https://doi.org/10.1038/nprot.2006.356

    Article  CAS  Google Scholar 

  12. Waites CL, Specht CG, Härtel K, Leal-Ortiz S, Genoux D, Li D, Drisdel RC, Jeyifous O, Cheyne JE, Green WN, Montgomery JM, Garner CC (2009) Synaptic SAP97 isoforms regulate AMPA receptor dynamics and access to presynaptic glutamate. J Neurosci 29(14):4332

    Article  CAS  Google Scholar 

  13. Pacifici M, Peruzzi F (2012) Isolation and culture of rat embryonic neural cells: a quick protocol. J Vis Exp (63):e3965. https://doi.org/10.3791/3965

  14. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D (2002) Germline transmission and tissue-specific expression of transgenes delivered by Lentiviral vectors. Science 295(5556):868

    Article  CAS  Google Scholar 

  15. Leal-Ortiz S, Waites CL, Terry-Lorenzo R, Zamorano P, Gundelfinger ED, Garner CC (2008) Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis. J Cell Biol 181(5):831

    Article  CAS  Google Scholar 

  16. Sheehan P, Zhu M, Beskow A, Vollmer C, Waites CL (2016) Activity-dependent degradation of synaptic vesicle proteins requires Rab35 and the ESCRT pathway. J Neurosci 36(33):8668

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by NIH grants 5T32NS064928 to V.B.; F31GM11661701 to J.C.M.; 5T32HD007430-20 to L.R., R01NS080967 to C.L.W., and Hirschl Research Scientist Award to U.H. and C.L.W.

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Authors and Affiliations

  1. Neurobiology and Behavior PhD Program, Columbia University, New York, NY, USA

    Veronica Birdsall & Lisa Randolph

  2. Medical Scientist Training Program, Columbia University, New York, NY, USA

    Jose C. Martinez

  3. The Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University, New York, NY, USA

    Ulrich Hengst

  4. Department of Pathology and Cell Biology, Columbia University, New York, NY, USA

    Ulrich Hengst & Clarissa L. Waites

  5. Department of Neuroscience, Columbia University, New York, NY, USA

    Clarissa L. Waites

Authors
  1. Veronica Birdsall
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  2. Jose C. Martinez
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  3. Lisa Randolph
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  4. Ulrich Hengst
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  5. Clarissa L. Waites
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Corresponding author

Correspondence to Clarissa L. Waites .

Editor information

Editors and Affiliations

  1. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

    Emmanuel Culetto

  2. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ.Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette, France

    Renaud Legouis

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Birdsall, V., Martinez, J.C., Randolph, L., Hengst, U., Waites, C.L. (2019). Live Imaging of ESCRT Proteins in Microfluidically Isolated Hippocampal Axons. In: Culetto, E., Legouis, R. (eds) The ESCRT Complexes. Methods in Molecular Biology, vol 1998. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9492-2_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9492-2_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9491-5

  • Online ISBN: 978-1-4939-9492-2

  • eBook Packages: Springer Protocols

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