Protein nanocondensates: the next frontier

Toledo, Pamela L.; Gianotti, Alejo R.; Vazquez, Diego S.; Ermácora, Mario R.

doi:10.1007/s12551-023-01105-1

Cited by 5 publications

(2 citation statements)

References 207 publications

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“…The study of nanocondensation is gaining interest due to its biological relevance, but also because it is not well described by the Flory-Huggins model which is often used to explain the liquid-liquid phase separation of protein 9 . This theoretical framework predicts that in sub-saturated solutions, clusters of > 3-5 proteins should not exist.…”

Section: Discussionmentioning

confidence: 99%

“…Pure in vitro reconstitutions as well as high resolution cell imaging have already provided a wealth of information on how weak multivalent interactions between intracellular components contribute to the process of LLPS, for example [5][6][7][8] . Current techniques tend to generate information on microscopic-sized condensates, however the existence of sub-micron assemblies, referred to as nanocondensates, is increasingly recognized 9 . These assemblies exist at subsaturated physiological concentrations making them biologically relevant.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of nanocondensates formation at the single molecule level

Houx,

Copie,

Gambin

et al. 2024

Preprint

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Understanding the molecular mechanisms of biomolecular condensate formation through liquid-liquid phase separation is crucial for deciphering cellular cues in normal and pathological contexts. Recent studies have highlighted the existence of sub-micron assemblies, known as nanocondensates or mesoscopic clusters, in the organization of a significant portion of the proteome. However, as smaller condensates are invisible to classical microscopy, new tools must be developed to quantify their numbers and properties. Here, we establish a simple analysis framework using single molecule fluorescence spectroscopy to quantify the formation of nanocondensates diffusing in solution. We used the low-complexity domain of TAR DNA-binding protein 43 (TDP-43) as a model system to show that we can recapitulate the phase separation diagram of the protein in various conditions. Single molecule spectroscopy reveals rapid formation of TDP-43 nanoclusters at ten-fold lower concentrations than described previously by microscopy. We demonstrate how straightforward fingerprinting of individual nanocondensates provides an exquisite quantification of their formation, size, density, and their temporal evolution. Overall, this study highlights the potential of single molecule spectroscopy to investigate the formation of biomolecular condensates and liquid-liquid phase separation mechanisms in protein systems.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quantification of nanocondensates formation at the single molecule level

Houx,

Copie,

Gambin

et al. 2024

Preprint

View full text Add to dashboard Cite

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Biophysical Reviews (ISSUE 4 2023): LAFeBS—highlighting biophysics in Latin America

Peluffo,

del V. Alonso,

Itri

et al. 2023

Biophys Rev

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TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways

Lin,

Wu,

Lin

et al. 2024

ACS Chem. Neurosci.

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Intrinsically disordered regions (IDRs) in proteins can undergo liquid–liquid phase separation (LLPS) for functional assembly, but this increases the chance of forming disease-associated amyloid fibrils. Not all amyloid fibrils form through LLPS however, and the importance of LLPS relative to other pathways in fibril formation remains unclear. We investigated this question in TDP-43, a motor neuron disease and dementia-causing protein that undergoes LLPS, using thioflavin T (ThT) fluorescence, NMR, transmission electron microscopy (TEM), and wide-angle X-ray scattering (WAXS) experiments. Using a fluorescence probe modified from ThT strategically designed for targeting protein assembly rather than β-sheets and supported by TEM images, we propose that the biphasic ThT signals observed under LLPS-favoring conditions are due to the presence of amorphous aggregates. These aggregates represent an intermediate state that diverges from the direct pathway to β-sheet-dominant fibrils. Under non-LLPS conditions in contrast (at low pH or at physiological conditions in a construct with key LLPS residues removed), the protein forms a hydrogel. Real-time WAXS data, ThT signals, and TEM images collectively demonstrate that the gelation process circumvents LLPS and yet still results in the formation of fibril-like structural networks. We suggest that the IDR of TDP-43 forms disease-causing amyloid fibrils regardless of the formation pathway. Our findings shed light on why both LLPS-promoting and LLPS-inhibiting mutants are found in TDP-43-related diseases.

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Protein nanocondensates: the next frontier

Cited by 5 publications

References 207 publications

Quantification of nanocondensates formation at the single molecule level

Quantification of nanocondensates formation at the single molecule level

Biophysical Reviews (ISSUE 4 2023): LAFeBS—highlighting biophysics in Latin America

TDP-43 Amyloid Fibril Formation via Phase Separation-Related and -Unrelated Pathways

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