The cells were settled on the surface of the carbon-coated coverslip and then slightly compressed using an agarose block (1.5%, dissolved in BSS, 1-mm thick) to observe the ventral cell surface33. strategies to escape risk of harm. Animals, including humans and snakes, possess the ability to avoid fires or electric shocks. Higher plants are not motile, but possess the ability to curl their leaves slant downwards1. In addition, chloroplasts inside plant cells can move away from the cell surface to the side when exposed to high-intensity light2. At the cellular level, mobile cells avoid harmful chemicals or repellents in a process referred to as negative chemotaxis. Bacteria exert negative chemotaxis to hydrogen peroxide and organic solvents such as alcohol. Upon exposure to repellants or intense light, ciliates and flagellates change the orientation of their swimming movement to avoid harm3,4. Cells of the cellular slime mold can alter their movement when exposed to repellents5. Repellents in mammalian cells such as leukocytes and neuronal cells have also been identified. These repellents are known to play roles DL-O-Phosphoserine in axonal guidance6, resolution of inflammation7, gastrulation8, and metastasis9. Mobilization of cytoplasmic Ca2+ (Cai2+) serves as an intracellular signal that is often observed when cells are exposed to repellents or dangers. In a recent study, we developed a novel laser-based cell poration method to introduce foreign molecules into single cells that precisely injure the cell membrane by regulating the wound size10. The wound pores in the cell membrane promptly close by employing a wound repair system, which involves the recruitment of several repair proteins, such as annexin and actin11. The exact molecular mechanisms underlying wounding remain to be elucidated, although Ca2+ entry is believed to be the first trigger. Here, the present study is the first to demonstrate that when cells are locally wounded in the cell membrane by laserporation, they move away from the site of wounding. Furthermore, we demonstrated that cell migration can be manipulated by repeated wounding. Results and Discussion Cells escape the site of wounding We used our novel laserporation method to create a local wound in the cell membrane of cells. Cells were placed on a coverslip coated with carbon by vapor deposition, after which a laser beam was focused on a small local spot beneath a single cell PDGFRA using total internal reflection fluorescence (TIRF) microscopy. The energy absorbed by the carbon created a small pore in the cell membrane in contact with the carbon coat. The wound pores are promptly closed by the wound repair system within a few seconds11. Using the powerful laserpolation method, we examined the behavior of cells locally wounded at different sites. A typical polarized migrating cell contains one or two DL-O-Phosphoserine pseudopods at its anterior side that project outward to propel the cell forward. When DL-O-Phosphoserine laserporation was applied at the anterior region of a migrating cell (wound size of 1C1.5?m in diameter), the cell stopped its movement and retracted the anterior pseudopod. Afterwards, a new pseudopod projected from the posterior region and the cell began to migrate towards the opposite direction (Fig.?1A, Anterior wound). On the other hand, when the laserporation was applied to the posterior region of a migrating cell, the cell did not change direction, although the velocity of cell migration was transiently increased (Fig.?1A, Posterior wound). When laserporation was locally applied in an immobile round-shaped cell, it began to migrate by extending a new pseudopod in the direction opposite DL-O-Phosphoserine to the wound site (Fig.?1A, Round cell). As a control, when the same strength of laser beam was applied to cells on coverslip without carbon coating, where no wound occurred (Fig.?1A, No coat), the cells did not show any response, suggesting that laser illumination does not induce the escape behavior. Figure?1B,C show the frequencies of cell migration in each direction after cells were wounded at the anterior or posterior sides on the coverslip, respectively, with or without carbon coating. Figure?1D,E show the changes in cell velocity over time after the cells were wounded at the anterior or the posterior regions, respectively. In both cases, the velocity of cell migration increased after a temporary decrease. Open in a separate window Figure 1 Cells escape the wounding site. (A) Cells were placed on a carbon-coated coverslip, and a laser beam was focused on a small local spot beneath a single.