Constitutive Expression of Squid Reflectin Protein Impairs Tight Junction and Polarity in MDCK Cells
HU Biru#, SONG Junyi#*
Cephalopods assemble periodic membrane architectures within iridocytes using reflectin proteins to generate tunable structural colors. However, the mechanisms underlying their intracellular trafficking, spatial organization and functional execution remain poorly understood. Previous studies have demonstrated that RefA1 (reflectin A1) obtained via heterologous expression in Escherichia coli displays high affinity for phospholipid membranes, while RefA1 expressed in HEK-293T cells by transient transfection undergoes directional trafficking along the microtubule cytoskeleton. Therefore, the MDCK cell line, which possesses vesicular transport capacity and cytoskeletal plasticity, was selected as the research platform in this study. A cell model stably expressing squid-derived reflectin RefA1 was established to investigate its intracellular trafficking behavior and impacts on cellular structures. Results showed that RefA1 failed to yield the anticipated microtubule guided subplasmalemmal enrichment, yet drastically disrupted cellular morphology and polarity. Cells expressing RefA1 presented reduced cell area, disassembled tight junctions, and became trapped in a “proliferation-detachment” cycle, preventing the formation of an intact cell monolayer. Transcriptomic analysis revealed that RefA1 severely perturbed the molecular networks governing cell junction assembly and polarity establishment. Specifically, it repressed the expression of polarity-determining factors including CLDN4, CDH1 and PARD6B, while upregulating contractility-regulatory genes such as MYLK2 and ROCK1. Collectively, these findings reveal for the first time that reflectin proteins not only interact with cell membranes and the cytoskeleton, but also actively modulate the establishment of cell polarity. This work offers novel insights into the cellular basis of photonic structures in cephalopods, and provides new clues for protein-mediated cellular morphogenesis engineering and biomimetic material design.
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