Using Fluorescence On/Off to Trace Tandem Nanofiber Assembly/Disassembly in Living Cells

To track an intact biological process inside cells, continuous showing of the assembly/disassembly process is needed and fluorescence is advantageous in characterizing these processes. However, using fluorescence "on/off" to observe a sequential assembly/disassembly process in living cells has not been reported. Herein, we rationally designed a probe PEA-NBD-Yp and employed its fluorescence "on/off" to trace tandem assembly/disassembly of nanofibers in living HeLa cells. In vitro experiments validated that PEA-NBD-Yp could be efficiently dephosphorylated by ALP to yield PEA-NBD-Y, which self-assembled into nanofibers with the NBD fluorescence "on". Also, the PEA-NBD-Y nanofiber was disassembled by GSH, accompanied by fluorescence "off". Living cell imaging (together with ALP-inhibition or GSH-blocking) experiments sequentially showed the self-assembling nanofibers on the cell outer membrane with fluorescence "on" (On₁), translocated inside cells (On₂), and disassembled by GSH with fluorescence "off" (Off₂). We anticipate that our strategy of one probe conferring temporal "on/off" fluorescence signals might provide people with a new tool to deeply understand a biological event in living cells in the near future.