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Stroobants, A.

Publications and source records attributed to Stroobants, A..

3 recordsLinked to original sources

Disease-linked mutations in hnRNPA2B1 accelerate condensate maturation and promote amyloid aggregation

Heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) is a multifunctional RNA-binding protein that undergoes liquid-liquid phase separation (LLPS), contributing to the assembly of membraneless organelles across different cellular contexts. However, dysregulated phase separation can drive the transition from functional condensates to pathological protein aggregates. Two mutations in the low-complexity domain (LCD) of hnRNPA2B1 (D302V and P310L) have been linked to neurodegenerative diseases. How these mutations alter the interplay between LLPS and aggregation remains poorly understood. Here, we show that these disease-linked mutations accelerate condensate maturation and promote amyloid-like aggregation of hnRNPA2B1, with fibrils emerging from condensate-like cores. To dissect the mechanistic link between phase separation, aggregation, and disease, we focused on a conserved 25-amino-acid region within the LCD harboring both mutation sites. Deletion of this region markedly impairs LLPS and abolishes aggregation, revealing its contribution to both processes. Isolated peptides derived from this region have an intrinsic propensity for amyloid-like fibril formation, which is enhanced by the mutations. Although unable to phase separate, these peptides remodel condensate morphology and nucleate aggregation of the LCD. Together, our results identify a sequence-encoded mechanism linking disease-associated mutations to altered condensate behavior and amyloid aggregation, providing molecular insights into how aberrant phase transitions may contribute to hnRNPA2B1-associated disease.

biochemistry↗

MISO: Microfluidic protein isolation enables single particle cryo-EM structure determination from a single cell colony

Single particle cryo-EM enables reconstructing near-atomic or even atomic resolution 3D maps of proteins by visualizing thousands to a few million purified protein particles embedded in nanometer- thick vitreous ice. This corresponds to picograms of purified protein, which can potentially be isolated from a few thousand cells. Hence, cryo-EM holds the potential of one of the most sensitive analytical methods that deliver a high-resolution protein structure as a readout. In practice, more than a million times more starting biological material is required to prepare cryo-EM grids. To close the gap, we developed a micro isolation (MISO) method that combines microfluidics-based protein purification with cryo-EM grid preparation. We validated the method on soluble bacterial and eukaryotic membrane proteins. We showed that MISO enables protein structure determination starting from below one microgram of a target protein and going from cells to cryo-EM grids within a few hours. This scales down the purification by a factor of a few hundred to a few thousand and opens possibilities for the structural characterization of hitherto inaccessible proteins.

biophysics↗

Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting

Using single particle cryogenic electron microscopy (cryo-EM) high-resolution structures of proteins in different conformations can be reconstructed. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid initiation of reaction and rapid plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a spatial resolution of 2.1 [A]. We performed trEM experiments on GroEL:GroES chaperonin complex, these resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.

biophysics↗