Colocalization of Known Focal Adhesion Proteins and Novel Focal Adhesion Protein GPR98 in Human Neurites during Neuronal Development in 2 and 3 Dimensions

Abstract

Focal adhesions are critical to cell motility, morphology, and neuronal circuit formation. Proper neuronal circuit formation relies on neurons reaching the right location, assuming a specific morphology, and connecting with signaling partners. While much about focal adhesions has been studied in non-human neurites, or in non-neuronal human tissues, there is little known about focal adhesions in human neurons or during the process of human neuronal differentiation and neural circuit formation. We use a human induced pluripotent stem cell line that endogenously expresses the focal adhesion protein paxillin to model human neuronal differentiation. We measure the size, shape, and colocalization of focal adhesion proteins including vinculin, α-actinin, and zyxin. In this study we were able to track focal adhesion composition in human-derived samples in a traditional 2-dimensional cell culture model and in a 3-dimensional model, spheroids. While 3-dimensional models of neurons are on the rise with spheroids, organoids, and assembloids, many studies don’t measure the stiffness of their substrates or only measure stiffness after fixation. We determined the stiffness of our 3-dimensional model to have an average Youngs Modulus of 494 Pa for a culture of at least 98 days of maturity. Most notably, we found that the range of stiffness present within a spheroid increased with average stiffness. With these 2-dimensional and 3-dimensional human model systems we were able to investigate a novel adhesion associated protein GPR98 as little work has been done to explore its role in neurodevelopment outside of Usher’s Syndrome. In both our 2-dimensional and 3-dimensional models we were able to locate, distinguish, and analyze the colocalization of GPR98. GRP98 showed colocalization with paxillin, vinculin, and α-actinin. Most notably, GPR98 presented a change in localization of expression at 24 hours of neuronal differentiation in the 2-dimensional model that necessitates further investigation; to that end we created CRISPR activation and interference lines to further investigate these proteins. There is still much to understand about focal adhesion proteins and their colocalizations during human neurodevelopment and how these interactions change in a soft environment of the developing brain.