Förster Resonance Energy Transfer — An approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling

Background

Signaling messengers and effector proteins provide an orchestrated molecular machinery to relay extracellular signals to the inside of cells and thereby facilitate distinct cellular behaviors. Formations of intracellular macromolecular complexes and segregation of signaling cascades dynamically regulate the flow of a biological process.

Scope of review

In this review, we provide an overview of the development and application of FRET technology in monitoring cyclic nucleotide-dependent signalings and protein complexes associated with these signalings in real time and space with brief mention of other important signaling messengers and effector proteins involved in compartmentalized signaling.

Major conclusions

The preciseness, rapidity and specificity of cellular responses indicate restricted alterations of signaling messengers, particularly in subcellular compartments rather than globally. Not only the physical confinement and selective depletion, but also the intra- and inter-molecular interactions of signaling effectors modulate the direction of signal transduction in a compartmentalized fashion. To understand the finer details of various intracellular signaling cascades and crosstalk between proteins and other effectors, it is important to visualize these processes in live cells. Förster Resonance Energy Transfer (FRET) has been established as a useful tool to do this, even with its inherent limitations.

General significance

FRET technology remains as an effective tool for unraveling the complex organization and distribution of various endogenous signaling proteins, as well as the spatiotemporal dynamics of second messengers inside a single cell to distinguish the heterogeneity of cell signaling under normal physiological conditions and during pathological events.     

Asymmetrical Macromolecular Complex Formation of Lysophosphatidic Acid Receptor 2 (LPA2) Mediates Gradient Sensing in Fibroblasts

Chemotactic migration of fibroblasts toward growth factors relies on their capacity to sense minute extracellular gradients and respond to spatially confined receptor-mediated signals. Currently, mechanisms underlying the gradient sensing of fibroblasts remain poorly understood. Using single-particle tracking methodology, we determined that a lysophosphatidic acid (LPA) gradient induces a spatiotemporally restricted decrease in the mobility of LPA receptor 2 (LPA₂) on chemotactic fibroblasts. The onset of decreased LPA₂ mobility correlates to the spatial recruitment and coupling to LPA₂-interacting proteins that anchor the complex to the cytoskeleton. These localized PDZ motif-mediated macromolecular complexes of LPA₂ trigger a Ca²⁺ puff gradient that governs gradient sensing and directional migration in response to LPA. Disruption of the PDZ motif-mediated assembly of the macromolecular complex of LPA₂ disorganizes the gradient of Ca²⁺ puffs, disrupts gradient sensing, and reduces the directional migration of fibroblasts toward LPA. Our findings illustrate that the asymmetric macromolecular complex formation of chemoattractant receptors mediates gradient sensing and provides a new mechanistic basis for models to describe gradient sensing of fibroblasts.     

Building the Future of Bioinformatics through Student-Facilitated Conferencing

Sharing results, techniques, and challenges is paramount to advance our understanding of any field of science. In the scientific community this exchange of ideas is mainly made possible through national and international conferences. Scientists have the opportunity to showcase their work, receive feedback, and improve their presentation skills. However, conferences can be large and intimidating for young researchers. In addition, for many of the more prestigious conferences, the very high number of submissions and low selection rate are major limitations to aspiring young researchers aiming to present their work to the scientific community. To improve student participation and proliferation of information, regional student groups have successfully organized conferences and symposia specifically aimed at students. This gives more students the opportunity to present their work and receive valuable experience and insight from peers and leaders in the field. At the same time, it is an ideal way for students to gain familiarity with the conference experience. In this paper, we highlight some of the benefits of participating in such student conferences, and we review the challenges we have encountered when organizing them. Both topics are illustrated in detail with examples from different ISCB Student Council Regional Student Groups.     

Compartmentalized Accumulation of cAMP near Complexes of Multidrug Resistance Protein 4 (MRP4) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Contributes to Drug-induced Diarrhea

Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3′-azido-3′-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.