CHEMICAL SCIENCE

Intercellular gelation induces spheroid formation


On May 22, 2023, the Brandeis Xu research team, Edward H. Egelman and Wang Feng Bin of the University of Virginia published a research result entitled “Cell spheroid creation by transcytotic intercellular gelation” in the journal Nature Nanotechnology.

The study utilized cryo-electron microscopy to determine the atomic structure of enzyme-responsive D-peptides and fluorescence imaging self-assembled spiral nanofibers. This study shows that transcytocytosis of D-peptides can induce the formation of nanofibers/gels between cells, which may interact with fibronectin to induce the formation of cellular spheroids. The corresponding authors are Bing Xu, Feng Bin Wang, and Edward H. Egelman; The first author is Guo Jiaqi.

Cell spheroids fill the gap between in vitro systems and in vivo animal models, and they better mimic in vivo microenvironment models of human organs than two-dimensional cell layers. However, the current methods used to generate spheroids have some problems, such as hanging drip and nonstick dishes, which are cumbersome, time-consuming, expensive, and poorly controlled. In addition, these methods cannot mimic the signature of extracellular matrix (ECM) production, i.e., regulated precursor secretion and subsequent self-assembly to form cell-to-cell junctions. In addition, ECM proteins have a variety of post-translational modifications that play an important role in cell adhesion and migration, among which phosphorylation is a modification that occurs at high frequency. However, until now, synthetic materials have been difficult to mimic the dynamic synthesis and transient phosphorylation of ECM proteins. Peptide assemblies formed by non-covalent interactions have dynamic and flexible assembly properties and have been shown to form hydrogel matrices to mimic ECM in cell culture and tissue engineering. However, most hydrogels are pre-prepared outside the cell and used to support or wrap the cells, which is different from the in-situ generation of ECMs around cells. The research team has previously demonstrated that enzymatically responsive D-phosphopeptide assemblies can induce the formation of HS-5 cell spheroids, but the detailed mechanism remains unclear. In addition, although peptide nanofibers have a variety of functions, much of their atomic structure has not yet been revealed.

In this study, the team reported that enzymatic D-phosphopeptides form intercellular D-peptide nanofibers through transcytosis (i.e., endocytosis and exocytosis) to promote the formation of cell spheroids as a hydrogel matrix. These D-phosphopeptides have antiprotease properties and undergo endocytosis and partial dephosphorylation during endoplasmic reticulum transport, forming nanofibers and becoming intercellular hydrogel matrix when exocrinous. Using cryo-electron microscopy (cryo-EM), the research team determined the atomic structure of the D-peptide nanofibers that lead to the formation of intercellular gels. In secretory vesicles, peptide nanofibers may interact with fibronectin to promote the formation of cellular spheroids. Minimizing endocytosis, blocking exocytosis, removing phosphoric acid triggers, or eliminating the transition of peptide assemblies from nanoparticles to nanofibers all prevent the formation of cell spheroids.

Figure 1. Self-assembled D-peptide nanofibers form intercellular hydrogels.

Figure 2: NBD–ffsy and BP–ffsy self-assemble into cross β-filaments in vitro.

Figure 3: Cell spheroids formed by adherent and suspended HS-5 cells consist of intercellular hydrogels colocalized with fibronectin.

Figure 4: Endocytosis and exocytosis are critical for the formation of cell spheroids.

Figure 5: Intracellular dephosphorylation is critical for the formation of cell spheroids.

Figure 6: D-peptide nanofibers promote fibronectin fiber production.

By using antiprotease properties and enzyme-responsive peptides to mimic the production of extracellular matrix (ECM) and ECM remodeling by forming spiral-shaped nanofibers as intercellular gels/matrices, this study demonstrates a versatile method combining transcytocytosis and enzymatic morphological transformation of peptide assemblies with potential applications in the fields of regenerative medicine and tissue engineering. (Source: Science Network)

Related paper information:https://doi.org/10.1038/s41565-023-01401-7



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