Small Plasma Membrane-Targeted Fluorescent Dye for Long-Time Imaging and Protein Degradation Analyses

Abstract

Plasma membrane (PM)-targeted fluorescent dyes have become an important tool to visualize morphological and dynamic changes in the cell membrane. However, most of these PM dyes are either too large and thus might potentially perturb the membrane and affect its functions or exhibit a short retention time on the cell membrane. The rapid internalization problem is particularly severe for PM dyes based on cationic and neutral hydrophobic fluorescent dyes, which can be easily transported into the cells by transmembrane potential and passive diffusion mechanisms. In this paper, we report a small but highly specific PM fluorescent dye, PM-1, which exhibits a very long retention time on the plasma membrane with a half-life of approximately 15 h. For biological applications, we demonstrated that PM-1 can be used in combination with protein labeling probes to study ectodomain shedding and endocytosis processes of cell surface proteins and successfully demonstrated that native transmembrane human carbonic anhydrase IX (hCAIX) is degraded via the ectodomain shedding mechanism. In contrast, hCAIX undergoes endocytic degradation in the presence of sheddase inhibitors. We believe that PM-1 can be a versatile tool to provide detailed insights into the dynamic processes of the cell surface proteins.

Fluorescent Solvatochromic Probes for Long‐Term Imaging of Lipid Order in Living Cells

“…[ 30 , 31 ] Therefore, we utilized a technique in which a linker consisting of a zwitterionic group and a long hydrophobic chain is coupled to selectively localize to the plasma membrane. [ 17 , 32 ] Compared to ketone‐based probes, modification methods for ester‐based FπCM are well described, and a high yield of the product FπCM‐SO 3 was obtained ( Figure 5 b ; see also Section S2 , Supporting Information). As shown in Figure 5b , the sensitivity to membrane order in GUVs was not disturbed by the amphiphilic anchor group.…”

“…[ 15 ] Nevertheless, even Nile Red probes present significant phototoxicity to specific cell membranes. [ 13 ] Recent studies have explored exchangeable dyes based on Nile Red [ 15 ] and other dyes [ 17 ] to achieve long‐term measurements using strong excitation power of stimulated emission depletion (STED) microscopy. [ 18 ] However, this approach requires a low affinity of the dye for bio‐membranes, which decreases the signal intensity in conventional microscopy.…”

Lipid-directed covalent fluorescent labeling of plasma membranes for long-term imaging, barcoding and manipulation of cells

“…However, this approach is not so convenient and efficient, because this labeling reaction requires high concentrations of the labeling agent (~5 μM). 44 Therefore, we hypothesized that the labeling could be accelerated by designing a reactive probe capable of first binding to the cell surface noncovalently. Then, the high local concentration of the probe would favor the fast labeling of neighboring proteins.…”