Trends in Cell Biology
Volume 19, Issue 11, November 2009, Pages 575-586
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Review
Special Issue – Imaging Cell Biology
Green light to illuminate signal transduction events

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When cells are exposed to hormones that act on cell surface receptors, information is processed through the plasma membrane into the cell interior via second messengers generated in the inner leaflet of the plasma membrane. Individual biochemical steps along this cascade have been characterized from ligand binding to receptors through to activation of guanine nucleotide binding proteins and their downstream effectors such as adenylate cyclase or phospholipase C. However, the complexity of temporal and spatial integration of these molecular events requires that they are studied in intact cells. The great expansion of fluorescent techniques and improved imaging technologies such as confocal and TIRF microscopy combined with genetically-engineered protein modules has provided a completely new approach to signal transduction research. Spatial definition of biochemical events followed with real-time temporal resolution has become a standard goal, and several new techniques are now breaking the resolution barrier of light microscopy.

Introduction

The concept of second messengers arose from the realization that many hormones and neurotransmitters do not penetrate the cell membrane but bind to receptors at the surface of the cell. To communicate external cues to the cell interior, these receptors must generate intracellular messengers to initiate a cellular response. The information flow from cell-surface receptors all the way to the response machinery of the cells, irrespective of whether the response is contraction, secretion or gene activation, has been the subject of intense studies over the past 50 years. Anomalies in such pathways are the underlying cause of most human diseases 1, 2, 3, highlighting the importance of signal transduction research.

Classical studies targeting what are now known as G protein-coupled receptors (GPCRs) found that these receptors communicate with GTP-binding proteins and activate enzymes that generate second messengers including cyclic adenosine- or guanosine monophosphates (cAMP and cGMP) [4], the Ca2+-mobilizing inositol 1,4,5-trisphosphate (InsP3) [5], or Ca2+ itself. These second messengers then activate a variety of proteins including ion channels, transcription factors and other regulatory proteins either directly or via the modulation of protein- and lipid kinases or phosphatases [6]. Each of these processes has been delineated in vitro with biochemical methods employing extracted cell components, and these have rapidly advanced the field. However, several observations suggest that this linear chain of information transfer is an oversimplification and the overall increase in second messenger concentration does not necessarily correlate with the biological response [7]. Apparently similar increases in cytoplasmic cAMP can reflect very different local cAMP elevations, and can therefore have completely different biological outcomes depending on the receptors or the Ca2+ channels involved 8, 9. As more molecular details are revealed as to how second messengers interact with their downstream targets it is becoming obvious that these intracellular signals are highly restricted in space and time; it is therefore necessary to obtain information both on their intracellular locations and temporal patterns. At the same time, significant progress is being made in our understanding of the changes of structure and conformation taking place in a large number of proteins following their activation. The genomic era has revealed the modular nature of most signal transduction proteins and has identified many basic protein folds [10]; these now can be identified by simple sequence analysis tools. Moreover, fluorescence methods including the appearance of genetically encoded fluorescent proteins and parallel advances in microscopy techniques have all contributed to a change in how signal transduction research is conducted. Current standards demand that each step in the second-messenger activation cascade is followed in single living cells, and at a spatial resolution that can break the limits of light microscopy (see Lidke and Wilson, and Larson et al. in this issue, and Box 1). Here we summarize the current state of fluorescence techniques as they are applied to the individual elements of the information chain from the receptors all the way to protein kinases.

Section snippets

Basic principles of fluorescence techniques used to follow signaling events

Many studies use proteins tagged with the green fluorescent protein (GFP) or its variants [11] to determine the localization of proteins within the cell. This method has contributed tremendously to our knowledge of cell biology. Fluorescence tagging gives excellent information on the steady-state distribution of proteins and, combined with photobleaching or photoactivation, can also address the dynamics of protein trafficking [12]. However, the GFP tag itself can affect protein localization,

Measuring the activation state of GPCRs

GPCRs undergo a conformation change upon binding of agonist-ligands (Figure 2A). This conformation change is the result of the repositioning of the transmembrane helices that eventually affect regions in the inner side of the protein. One such change is the movement of intracellular (IC) loops (usually but not exclusively the 3rd IC loop) that affects the interaction with heterotrimeric G proteins and perhaps with other proteins such as G protein receptor kinases (GRKs) [26]. Another

Heterotrimeric G proteins

G-protein coupling and activation is almost inseparable from GPCR function. Heterotrimeric G proteins are membrane-associated transducing modules that consist of a larger α-subunit that is palmitoylated and smaller β and γ subunits that form a stable dimer that is also anchored to membranes by lipid modification [37]. It has long been postulated that activated GPCRs cause a dissociation of Gα and Gβγ subunits and allow the exchange of GDP to GTP within the Gα subunits to generate the active

Probes to measure Ca2+, cAMP, or InsP3 dynamics

The use of the fluorescent Ca2+ indicators was first introduced by Tsien and colleagues 14, 56; this has revolutionized research on Ca2+ signaling and set the standard for the development of new probes for monitoring kinetic changes in other second messengers at the single cell level. In fact, it was the design of genetically encoded single-molecule FRET Ca2+-sensors from the same group that inspired the design of most of the FRET sensors used today. These probes, named cameleons, used the Ca2+

Probes to measure protein kinase or phosphatase activities

Protein phosphorylation cascades are the ultimate means of information flow along diverse signaling pathways [6]. The principle underlying the analysis of kinase activities in single cells is again based on FRET changes elicited when phosphorylation of a peptide induces binding to a downstream effector protein [82]. This phosphorylation-dependent binding can be translated to changes in FRET using the usual CFP/YFP pairs of fluorophores. In the case of PKA the first such reporter, AKAR (A-kinase

Making lipid messengers visible

Although the importance of lipid messengers in cell regulation has long been established, the study of lipids has always been more difficult than of proteins. Lipids require special extraction and separation procedures, they are mostly detected by isotope incorporation with complex labeling kinetics, and are not as easy as proteins to detect with antibodies. However, the importance of phosphoinositides, serving not only as precursors of important messengers such as DAG and Ins(1,4,5)P3, but

Concluding remarks

Progress in signal transduction research over the last 40 years has been breathtaking. It is noteworthy, however, that the diversity of cell surface receptors and the complexity of the kinase cascades contrasts with the small number of second messenger mechanisms. In a majority of signaling cascades cAMP or Ca2+ serve as the second messenger, raising the question of why cells need the variety of receptors if most of these couple to one or other of these second messengers. The answer must lie in

Acknowledgements

The research of TB is supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health.

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