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Spectrally Resolved FRET Microscopy of α-Synuclein Phase-Separated Liquid Droplets

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Protein Aggregation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2551))

Abstract

Liquid-liquid phase separation (LLPS) has emerged as an important phenomenon associated with formation of membraneless organelles. Recently, LLPS has been shown to act as nucleation centers for disease-associated protein aggregation and amyloid fibril formation. Phase-separated α-synuclein droplets gradually rigidify during the course of protein aggregation, and it is very challenging to understand the biomolecular interactions that lead to liquid-like to solid-like transition using conventional ensemble measurements. Here, we describe a spectrally-resolved fluorescence microscopy based Förster resonance energy transfer (FRET) imaging to probe interactions of α-synuclein in individual droplets during LLPS-mediated aggregation. By acquiring entire emission spectral profiles of individual droplets upon sequential excitation of acceptors and donors therein, this technique allows for the quantification of sensitized emission proportional to the extent of FRET, which enables interrogation of the evolution of local interactions of donor-/acceptor-labeled α-synuclein molecules within each droplet. The present study on single droplets is not only an important development for studying LLPS but can also be used to investigate self-assembly or aggregation in biomolecular systems and soft materials.

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Author notes

  1. Jaladhar Mahato and Soumik Ray have been equally contributed to this chapter.

Authors and Affiliations

  1. Department of Chemistry, IIT Bombay, Mumbai, India

    Jaladhar Mahato & Arindam Chowdhury

  2. Department of Biosciences and Bioengineering, IIT Bombay, Mumbai, India

    Soumik Ray & Samir K. Maji

Authors
  1. Jaladhar Mahato
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  2. Soumik Ray
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  3. Samir K. Maji
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  4. Arindam Chowdhury
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Corresponding authors

Correspondence to Samir K. Maji or Arindam Chowdhury .

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Editors and Affiliations

  1. Cambridge, MA, USA

    Andrzej Stanisław Cieplak

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Mahato, J., Ray, S., Maji, S.K., Chowdhury, A. (2023). Spectrally Resolved FRET Microscopy of α-Synuclein Phase-Separated Liquid Droplets. In: Cieplak, A.S. (eds) Protein Aggregation. Methods in Molecular Biology, vol 2551. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2597-2_27

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  • DOI: https://doi.org/10.1007/978-1-0716-2597-2_27

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2596-5

  • Online ISBN: 978-1-0716-2597-2

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