TY  - CHAP
AU  - Hochmair, Janine
AU  - Exner, Christian
AU  - Betzel, Christian
AU  - Mandelkow, Eckhard
AU  - Wegmann, Susanne
TI  - Light Microscopy and Dynamic Light Scattering to Study Liquid-Liquid Phase Separation of Tau Proteins In Vitro.
VL  - 2551
CY  - New York, NY
PB  - Springer US
M1  - DZNE-2022-01748
SN  - 978-1-0716-2596-5 (print)
T2  - Methods in Molecular Biology
SP  - 225 - 243
PY  - 2023
AB  - Tau is an intrinsically disordered protein that binds and stabilizes axonal microtubules (MTs) in neurons of the central nervous system. The binding of Tau to MTs is mediated by its repeat domain and flanking proline-rich domains. The positively charged (basic) C-terminal half of Tau also mediates the assembly Tau into fibrillar aggregates in Alzheimer's disease (AD) and tauopathy brains. In recent years, another assembly form of Tau has been identified: Tau can undergo liquid-liquid phase separation (LLPS), which leads to its condensation into liquid-dense phases, either by complex coacervation with polyanions like heparin or RNA or through 'self-coacervation' at high Tau concentrations. Condensation of Tau in the absence of polyanions can be enhanced by the presence of molecular crowding agents in a dilute Tau solution. In vitro experiments using recombinant purified Tau are helpful to study the physicochemical determinants of Tau LLPS, which can then be extrapolated into the cellular context. Tau condensation is a new aspect of Tau biology that may play a role for the initiation of Tau aggregation, but also for its physiological function(s), for example, the binding to microtubules. Here we describe how to study the condensation of Tau in vitro using light microscopy, including fluorescence recovery after photobleaching (FRAP), to assess the shape and molecular diffusion in the condensates, a proxy for the degree of condensate percolation. We also describe turbidity measurements of condensate-containing solutions to assess the overall amount of LLPS and time-resolved dynamic light scattering (trDLS) to study the formation and size of Tau condensates.
KW  - Humans
KW  - tau Proteins: metabolism
KW  - Microscopy
KW  - Dynamic Light Scattering
KW  - Alzheimer Disease: metabolism
KW  - Coacervation (Other)
KW  - Condensation (Other)
KW  - Crowding agents (Other)
KW  - FRAP (Other)
KW  - LLPS (Other)
KW  - MAPT (Other)
KW  - Polyethylene glycol (Other)
KW  - RNA (Other)
KW  - tau Proteins (NLM Chemicals)
KW  - polyanions (NLM Chemicals)
LB  - PUB:(DE-HGF)7
C6  - pmid:36310206
DO  - DOI:10.1007/978-1-0716-2597-2_15
UR  - https://pub.dzne.de/record/165615
ER  -