Fig. 2
From: Modular assembly of synthetic proteins that span the plasma membrane in mammalian cells
An additional transmembrane domain anchors proteins on the PM with the fluorescent protein, Venus, facing extracellularly. Pictorial representation of receptor anchoring and orientation (a). COS-7 cells transfected with the plasma membrane labelled Lyn-Ceru showed a matte-like appearance (b and c) while those transfected with the ER labelled STIM1-mRFP showed a web-like fluorescence distribution (e and f). Merged images illustrate resultant co-localization (d and g). TLP-V-TM skewed more towards a matte-like plasma membrane appearance (b and c). Fluorescence quenching with [30 μM]f HCl showed no effect on membrane-labelled Lyn-Ceru at time = 0 and time = 1 min (h and i) but did sequester Venus (j and k). Likewise, fluorescence quenching with [60 μM]f NaCl showed no effect on membrane-labelled Lyn-Ceru at time = 0 and time = 1 min (l and m) but again sequestered Venus (n and o). ER: endoplasmic reticulum, PM: plasma membrane, hv: emitted fluorescent light, HCl: hydrogen chloride, NaCl: sodium chloride, TM: transmembrane domain TLR4, TLP: fusion of TM with signal peptidase cleavage site from human immunoglobulin K, V: Venus fluorescent protein, LC: Lyn-Ceru, STIM1: stromal interaction molecule 1. Scale bars are 10 μm. Images are false colored: CFP, cyan; YFP, green; mRFP, red. All insets show zoomed regions (4x) of structures in dotted rectangles. All experiments were repeated at least 3 times