Ghani, K. | Search bioRxiv

bioRxiv2024

EPHA1 and EPHB4 tyrosine kinase receptors regulate epithelial morphogenesis

Organ formation and homeostasis require the coordination of cell-cell adhesion, epithelial cell polarity and orientation of cell division to organize epithelial tissue architecture. We have previously identified proximity protein networks acting downstream of members of the EPH family of tyrosine kinase receptors and found within these networks an enrichment of components associated with cell morphogenesis and cell-cell junctions. Here, we show that two EPH receptors, EPHA1 and EPHB4, are localized to the basolateral domain of Caco-2 cells in spheroidal cultures. Depletion of either EPHA1 or EPHB4 disrupts spheroid morphogenesis, without affecting cell polarity, but via randomizing mitotic spindle orientation during cell division. Strikingly, EPHA1 and EPHB4 exert this function independently of their catalytic activity but still requiring EFN ligand binding. Consistent with this, the most abundantly expressed EPHB4 ligand in Caco-2 cells, EFNB2, is also compartmentalized at the basolateral domain in spheroids, and is required for epithelial morphogenesis. Taken together, our data reveal a new role for EPHRs in epithelial morphogenesis.

cell biology↗

bioRxiv2020

Efficient production of Moloney murine leukemia virus-like particles pseudotyped with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak that started in China at the end of 2019 has rapidly spread to become pandemic. Several investigational vaccines that have already been tested in animals and humans were able to induce neutralizing antibodies against the SARS-CoV-2 spike (S) protein, however protection and long-term efficacy in humans remain to be demonstrated. We have investigated if a virus-like particle (VLP) derived from Moloney murine leukemia virus (MLV) could be engineered to become a candidate SARS-CoV-2 vaccine amenable to mass production. First, we showed that a codon optimized version of the S protein could migrate efficiently to the cell membrane. However, efficient production of infectious viral particles was only achieved with stable expression of a shorter version of S in its C-terminal domain ({Delta}S) in 293 cells that express MLV Gag-Pol (293GP). The incorporation of {Delta}S was 15-times more efficient into VLPs as compared to the full-length version, and that was not due to steric interference between the S cytoplasmic tail and the MLV capsid. Indeed, a similar result was also observed with extracellular vesicles released from parental 293 and 293GP cells. The amount of {Delta}S incorporated into VLPs released from producer cells was robust, with an estimated 1.25 g/ml S2 equivalent (S is comprised of S1 and S2). Thus, a scalable platform that has the potential for production of pan-coronavirus VLP vaccines has been established. The resulting nanoparticles could potentially be used alone or as a boost for other immunization strategies for COVID-19. IMPORTANCESeveral candidate COVID-19 vaccines have already been tested in humans, but their protective effect and long-term efficacy are uncertain. Therefore, it is necessary to continue developing new vaccine strategies that could be more potent and/or that would be easier to manufacture in large-scale. Virus-like particle (VLP) vaccines are considered highly immunogenic and have been successfully developed for human papilloma virus as well as hepatitis and influenza viruses. In this study, we report the generation of a robust Moloney murine leukemia virus platform that produces VLPs containing the spike of SARS-CoV-2. This vaccine platform that is compatible with lyophilization could simplify storage and distribution logistics immensely.

microbiology↗