Figueiredo, R. | bioRxiv Science

bioRxiv2023

JAGGER localization and function is dependent on GPI anchor addition

In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiples pollen tubes to one ovule - polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein (AGP) which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted {omega}-site regions and GAS (GPI attachment signal) domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

plant biology↗

bioRxiv2023

A small molecule cocktail for robust induction of blood-brain barrier properties

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/{beta}-catenin signaling, and inhibition of the TGF-{beta} pathway in endothelial cells robustly induce BBB properties in vitro. To target this novel interaction, we present a small molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/{beta}-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells towards the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories. Significance StatementThe blood-brain barrier (BBB) hinders drug delivery to the brain and is implicated in neurological diseases. To better understand these processes in humans, there is a need for culture models that mimic the complexity of the BBB. However, state-of-the-art human BBB models either suffer from a non-physiological, mixed epithelial-endothelial identity or have weak barrier tightness, which greatly limits their usability. We identified a molecule combination that synergistically enhances barrier tightness in several in vitro models and induces complex BBB properties in human stem cell-derived endothelial cells by targeting a novel link between three signaling pathways. The molecule combination has the potential to improve BBB culture models across laboratories to advance both basic research and drug development for the human brain.

bioengineering↗