Fluorescent proteins: a cell biologist's user guide

Abstract

Fluorescent Proteins (FPs) have revolutionized cell biology. The value of labeling and visualizing proteins in living cells is evident from the thousands of publications since the cloning of Green Fluorescent Protein (GFP). Biologists have been flooded with a cornucopia of FPs; however, the FP toolbox has not necessarily been optimized for cell biologists. Common FP plasmids are suboptimal for the construction of proteins fused to FP. More problematic are commercial and investigator-constructed FP-fusion proteins that disrupt important cellular targeting information. Even when cell biologists correctly construct FP-fusion proteins, it is rarely self-evident which FP should be used. Important FP information, such as oligomer formation or photostability, is often obscure or anecdotal. This brief guide is offered to assist the biologist to exploit FPs in the analysis of cellular processes.

Figure 1. GFP from Jellyfish to expression in mammalian cells

A. The jellyfish Aequorea victoria. Image provided by Sierra Blakely. B. GFP targeted to the endoplasmic reticulum of a mammalian fibroblast. C. Ribbon diagram of the β-barrel structure typical of FPs. Image produced by Richard Wheeler from PDB:1EMA rendered in PyMOL. D. Co-expression of an endoplasmic reticulum targeted red fluorescent protein and a Golgi complex targeted GFP in a mammalian fibroblast. Scale bars = 10μm.

Figure I. FP fusion protein design

A. Illustration of a typical resident ER luminal protein primary sequence. The Signal Sequence (SS) occurs at the extreme NH₂ terminus and the KDEL ER retrieval motif occurs at the absolute COOH terminus. Placement of an FP at either the NH₂ terminus or COOH terminus will disrupt essential position-dependent targeting information and are strongly discouraged (red Xs). In contrast, placement of the FP after the SS or before the KDEL will generate a protein correctly targeted to and retained in the ER lumen. B. The resulting FP-fusion protein, when the FP is placed immediately before the KDEL sequence. Note that the NH2 and COOH termini of GFP occur at the same end of the β barrel structure.