Applications Of Cell Surface Engineering On Cell Plasma Membrane Imaging

Cell plasma membrane is a vital organelle that serves as the boundary of the cell and ensures the cell integrity.Besides,cell plasma membrane is also functionally related to various biological events such as cell adhesion,cell migration,cell proliferation,endocytosis,exocytosis,apoptosis,and signal transduction.Important information on cell behavior and state can be obtained by studying the structure and function of plasma membrane.The simplest and most powerful method for plasma membrane study is fluorescence labeling,which can not only observe the membrane structures but also dynamically trace their time-dependent changes.However,commonly used commercial dyes(e.g.,dyes from DiD and FM families)have drawbacks such as facile cellular internalization,low photostability,and incompatibility with immunofluorescence staining,etc.On the other hand,cell surface engineering as a newly developed field has received increasing interest due to its capability to introduce new properties and functions to the cells by cell surface modification.In this work,through hydrophobic anchoring-based cell surface modification strategy,we have developed several novel plasma membrane imaging reagents,which significantly improved the efficiency of membrane labeling.Since the lipophilic cholesterol has excellent membrane anchoring ability,we first developed a cholesterol-based,biotin-containing,single-site membrane anchoring reagent,cholesterol-polyethylene glycol-biotin(chol-PEG-biotin).After surface biotinylation using chol-PEG-biotin,the cells could efficiently recruit avidin-conjugated quantum dots to the plasma membrane and realize quantum dot-based anti-photobleaching plasma membrane imaging,making long-time observation of the plasma membrane possible.To inhibit cellular internalization of membrane imaging dyes,a multi-site membrane imaging reagent was synthesized by using glycol chitosan(GC)as the backbone,and PEG-cholesterols(anchoring units)and FITC molecules(fluorescence units)as the side chains.Since this membrane imaging reagent,abbreviated as GC-PEG chol-FITC,contains several membrane anchoring sites and has a large molecular weight,the cellular internalization of GC-PEG chol-FITC was significantly decreased.Next,we updated the multi-site membrane anchoring strategy with the biotin-avidin recognition mediated two-step synergistic cell surface modification method,which could effectively inhibit the signal decline caused by the detachment of the dyes from the plasma membranes.This method not only achieved stable plasma membrane labeling for up to 8 h,but also realized "wash-free"staining.Additionally,we further discovered that the multi-site membrane anchoring reagent has the conformation-adjusting ability,enabling it to stain not only the plasma membranes of mammalian cells through hydrophobic interaction,but also the cell walls of fungal and bacterial cells though electrostatic interaction.Thus,GC-PEG chol-FITC is universally applicable for labeling all kinds of cell surfaces.Moreover,since our multi-site membrane staining reagent contains a large number of amine groups,cell fixation using paraformaldehyde could induce crosslinking between the staining reagents and the membrane proteins,thus making GC-PEG chol-FITC have the ability to tolerate permeabilization treatment by detergents.Such a permeabilization-tolerant ability makes it compatible with immunofluorescence staining of intracellular proteins,enabling simultaneous labeling of plasma membrane and intracellular proteins.In summary,the plasma membrane labeling strategies developed here not only functionally improve the imaging efficiency,but also provide new insight into the interaction mechanisms between biomaterials and cell surfaces,which will promote the development of cell surface engineering.