iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.

Heparan sulfate proteoglycan-mediated dynamin-dependent transport of neural stem cell exosomes in an in vitro blood-brain barrier model.

Drug delivery to the brain is greatly hampered by the presence of the blood-brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood-brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, that is, exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB.

Preparation of chaperone-loaded neural stem cell-derived extracellular vesicles to reduce protein aggregation in Huntington's disease cellular models.

Here, we present a protocol using genetic engineering techniques to prepare small extracellular vesicles (sEVs) enriched in the chaperone protein DNAJB6. We describe steps to prepare cell lines overexpressing DNAJB6, followed by the isolation and characterization of sEVs from cell conditioned media. Further, we describe assays to examine effects of DNAJB6-loaded sEVs on protein aggregation in Huntington's disease cellular models. The protocol can be readily repurposed to study protein aggregation in other neurodegenerative disorders or extended to other therapeutic proteins. For complete details on the use and execution of this protocol, please refer to Joshi et al. (2021).1.

DNAJB6-Containing Extracellular Vesicles as Chaperone Delivery Systems: A Proteomic Analysis.

Cell-derived extracellular vesicles (EVs) are effectors of cell-to-cell communication that are in the spotlight as promising candidates for in vivo drug delivery because of their ability to enter cells and deliver cargo. For example, proteins of interest can be loaded into EVs to mediate protein transfer into target cells. To determine causality between EV content and function, which is also important to assess the clinical safety of EVs, it is crucial to comprehensively characterize their complete molecular composition. Here, we investigated EVs loaded with the chaperone protein DNAJB6. Chaperone proteins assist in protein folding and have been suggested to alleviate protein aggregation diseases, such as Alzheimer's disease and Huntington's disease. We analyzed and compared the proteome of EVs isolated from wildtype HEK293T cells with that of EVs from HEK 293T cells overexpressing DNAJB6-WT or loss-of-function mutant DNAJB6-M3. Comprehensive analysis of proteomics data showed enhanced levels of DNAJB6 as well as protein-folding-related proteins in EVs derived from DNAJB6-overexpression cells. Interestingly, upregulation of a chaperone and its protein-folding-related proteins resulted in downregulation of another chaperone plus its related proteins, and vice versa. This implies the presence of compensatory mechanisms in the cellular expression of chaperones. Collectively, we provide the proteomic EV signatures underlying EV mediated DNAJB6 transmission by HEK293T cells, with the aim of establishing a causal relationship between EV protein content and EV function.

DNAJB6b-enriched small extracellular vesicles decrease polyglutamine aggregation in in vitro and in vivo models of Huntington disease.

Huntington disease (HD) is a devastating neurodegenerative disorder characterized by aggregation of huntingtin (HTT) protein containing expanded polyglutamine (polyQ) tracts. DNAJB6, a member of the DNAJ chaperone family, was reported to efficiently inhibit polyQ aggregation in vitro, in cell models, and in vivo in flies, xenopus, and mice. For the delivery of exogenous DNAJB6 to the brain, the DNAJB6 needs to be protected against (enzymatic) degradation and show good penetration into brain tissue. Here, we tested the potential of small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) for delivery of DNAJB6 as anti-amyloidogenic cargo. Administration of sEVs isolated from DNAJB6-overexpressing cells to cells expressing expanded polyQ tracts suppressed HTT aggregation. Furthermore, intrathecal injection of DNAJB6-enriched sEVs into R6/2 transgenic HD mice significantly reduced mutant HTT aggregation in the brain. Taken together, our data suggest that sEV-mediated molecular chaperone delivery may hold potential to delay disease onset in HD.

Endocytosis of Extracellular Vesicles and Release of Their Cargo from Endosomes.

Extracellular vesicles (EVs), such as exosomes, can mediate long-distance communication between cells by delivering biomolecular cargo. It is speculated that EVs undergo back-fusion at multivesicular bodies (MVBs) in recipient cells to release their functional cargo. However, direct evidence is lacking. Tracing the cellular uptake of EVs with high resolution as well as acquiring direct evidence for the release of EV cargo is challenging mainly because of technical limitations. Here, we developed an analytical methodology, combining state-of-the-art molecular tools and correlative light and electron microscopy, to identify the intracellular site for EV cargo release. GFP was loaded inside EVs through the expression of GFP-CD63, a fusion of GFP to the cytosolic tail of CD63, in EV producer cells. In addition, we genetically engineered a cell line which expresses anti-GFP fluobody that specifically recognizes the EV cargo (GFP). Incubation of anti-GFP fluobody-expressing cells with GFP-CD63 EVs resulted in the formation of fluobody punctae, designating cytosolic exposure of GFP. Endosomal damage was not observed in EV acceptor cells. Ultrastructural analysis of the underlying structures at GFP/fluobody double-positive punctae demonstrated that EV cargo release occurs from endosomes/lysosomes. Finally, we show that neutralization of endosomal pH and cholesterol accumulation in endosomes leads to blockage of EV cargo exposure. In conclusion, we report that a fraction of internalized EVs fuse with the limiting membrane of endosomes/lysosomes in an acidification-dependent manner, which results in EV cargo exposure to the cell cytosol.