Combined Image-Based Profiling and Biochemical Analysis of GCaMP Overexpression Effects on Mammalian Cells

Protein-based fluorescent sensors are a powerful addition to the biology toolbox for their ability to be stably expressed within living organisms, tissues, cells, and subcellular compartments, with the capacity to report on the presence of specific target molecules or other analytes. At the same time, sensor components will unavoidably present opportunities for unintended interaction with endogenous cellular machinery, potentially confounding both sensor function and cell health. Interactions with host components may not be readily predictable during the sensor design process, especially when simultaneously optimizing many other sensor parameters such as fluorescence response, dynamic range, and kinetics. Characterizing effects of sensor expression on cells is currently a laborious ad hoc process; new methods to characterize the cell expression effects of sensors and their variants could dramatically improve sensor design pipelines, laying the groundwork to recognize potentially problematic expression side effects earlier in the iterative design and testing workflow. Here, we take a dual high-content imaging-based and biochemical approach to examine sensor interactions with native cell biology, focusing on the widely used GCaMP calcium sensor. We identify a morphology-based signature of the cellular effects of high sensor expression in a neuroblastoma cell line. Subsequently, we identify biochemical interactions between GCaMP and a component of the mammalian cytoskeleton and track morphological features in sensor-expressing cells that lack these structural components. Our findings present an entry point for engineering new minimally cross-reactive sensor versions given a contextual biological understanding of sensor overexpression. We anticipate that as this and related workflows are incorporated into sensor engineering pipelines, bioorthogonality can be more systematically assessed and prioritized in diverse sensor scaffolds.