Analytical Sciences

Membrane Dynamics Induced by a Phosphatidylinositol 3,4,5-Trisphosphate Optogenetic Tool

Yoshibumi UEDA,*1,*2 Tatsuhito II,*3 Yuki AONO,*1 Naotoshi SUGIMOTO,*4 Seiichi SHINJI,*5 Hiroshi YOSHIDA,*5 and Moritoshi SATO*1

*1 Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan
*2 AMED-PRIME, Japan Agency for Medical Research and Development, Tokyo, Japan
*3 Department of Veterinary Pathology, School of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino, Tokyo 180-8602, Japan
*4 Department of Physiology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa, Japan
*5 Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Bunkyo, Tokyo 113-8603, Japan

Membrane dynamic structures such as filopodia, lamellipodia, and ruffles have important cellular functions in phagocytosis and cell motility, and in pathological states, such as cancer metastasis. Phosphatidylinositol 3,4,5-trisphosphate (PIP₃) is a crucial lipid that regulates PIP₃ dynamics. Investigations of how PIP₃ is involved in these functions have mainly relied on pharmacological interventions, and therefore have not generated detailed spatiotemporal information concerning membrane dynamics upon PIP₃ production. In the present study, we applied an optogenetic approach using the CRY2–CIBN system. Using this system, we revealed that local PIP₃ generation induced directional cell motility and membrane ruffles in COS7 cells. Furthermore, combined with structured illumination microscopy (SIM), membrane dynamics were investigated with high spatial resolution. We observed PIP₃-induced apical ruffles and unique actin fiber behavior in that a single actin fiber protruded from the plasma membrane was taken up into the plasma membrane without depolymerization. This system has the potential to investigate other high-level cell motility and dynamic behaviors, such as cancer cell invasion and wound healing with high spatiotemporal resolution, and could provide new insights of biological sciences for membrane dynamics.

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