Why study cell signalling

Delbridge laboratory: Cardiac phenomics. Head of Laboratory Professor Lea Delbridge. Designing new optogenetic tools for biomedical research. Determining the molecular basis of disease-causing mutations in synaptic proteins. Developing a novel class of therapeutics for muscle wasting and frailty. Developing innovative animal and cell culture models to study and treat muscular dystrophies.

Development of an electromechanical flow-cytometry based cell stimulation system. Development of Novel Antimalarial Drugs. Diabetic cardiomyopathy — an epidemic disease. Discovering and characterizing new genes involved in innate immunity, tissue injury and fibrosis. Project Leader Professor Christine Wells. Towards understanding new therapies for obesity and diabetes: discovering new regulators of lipid metabolism. Engineering G protein-coupled receptors for structural biology and drug discovery with directed evolution.

Exercise-induced liver-muscle cross-talk and mitochondrial adaptations. Exploiting nanoparticles as vaccines. Functional neuroanatomy of genitourinary vasculature. Genetics of blood brain barrier formation. Project Leader Professor Ben Hogan. Gregorevic laboratory: Muscle Research and Therapeutics. Gunnersen laboratory: Neuron development and plasticity.

Hogan laboratory: Vascular Cell and Developmental Biology. Head of Laboratory Professor Ben Hogan. Immune responses to pathogenic bacteria. Similar strategies have also been successfully used in other signaling systems 63 — Single-cell dynamic imaging can also reveal new phenomena that can be further studied using other techniques.

For example, the HIV virus is known to integrate into the host genome and lay dormant in some T lymphocytes, which presents a major obstacle for treatment with antiretroviral therapy Although these latent reservoirs of virus exhibit stochastic, low level activation, the molecular regulators controlling viral activation are still incompletely understood Instead, using smFISH and chromatin immunoprecipitation ChIP , the authors found that the chromatin environment regulates transcriptional bursting and can explain clone-to-clone variability.

FISH-based strategies have also been used to elucidate the role dynamics can play in vivo during development. For example, it had been shown that myogenesis requires transient, not sustained, activation of Notch, but the mechanism of transient Notch activation was not clear Recently, it was revealed that the Notch pathway also uses dynamics to encode and decode information about the identity of the activating stimulus These experiments revealed that Dll1 stimulation leads to pulsatile Notch activation, while Dll4 creates sustained Notch activation, with differences in gene expression as a result.

The results were reproduced in an in vivo model by electroporating either Dll1 or Dll4 into one side of the neural crest of a chick embryo and then using hybridization chain reaction HCR FISH to stain for MyoD1, a muscle regulatory factor. Their results revealed that MyoD1 is upregulated by Dll1, which creates pulsatile dynamics, while Dll4, which creates sustained dynamics, downregulated MyoD1 In systems where endpoint measurements of gene expression are insufficient, multiple fluorescent reporters can be used to measure signaling and transcriptional output simultaneously.

Finally, it is now also possible to measure signaling dynamics and genome-wide transcriptional responses in the same single cell. RNA-seq provided a method to measure the entire transcriptome of a single cell, but it remained unsolved how to connect those data with measurements of transcription factor activation dynamics.

Lane et al. The ability to measure global gene expression resultant from heterogeneous dynamics is exceedingly useful, because it allows for phenotypic characterization of single cell dynamics without a need for a priori knowledge of the target genes. Frequently, a cellular response to signals that it receives is to differentially regulate the expression or secretion of proteins. For example, a large part of the immune response is coordination of cytokine and chemokine secretion by immune cells at the site of infection.

Therefore, another promising avenue for research in cellular dynamics is to study changes in protein expression and secretion in conjunction with measurements of dynamics. Immunofluorescence can be used to measure intracellular protein expression, similar to measurements of gene expression using smFISH. Alternatively, microfluidic devices or microwell-based assays can be used to measure protein secretion from single cells. For example, protein quantification can be used to understand how signaling pathways in cells control differentiation.

Hormones such as glucocorticoids strongly induce adipogenesis in vivo and in vitro , but basal rates of preadipocyte differentiation are low in living animals, despite large daily spikes in glucocorticoid hormone production This raises the question of how the differentiation pathway in preadipocytes is able to filter daily, pulsatile signals.

Live cell imaging of endogenous adipogenic transcription factors CEBPB and PPARG, and staining for markers of fat cell differentiation, revealed that the transcriptional circuit in preadipocytes effectively filters out pulsatile signals, but responds to continuous signals of the same total magnitude Figure 2C.

A model predicted that such a response could be achieved if the system had both fast and slow feedback loops, and further protein and mRNA staining revealed FABP4 as a potential slow-feedback partner in the pathway Most often, protein expression is measured at the experiment's endpoint, but sometimes more frequent measurements of downstream protein expression changes are required.

Simultaneously measuring ERK activity and induction of Fra-1, a target of ERK, using live-cell reporters instead revealed that linear integration of ERK activity was the primary determinant of downstream responses Single-cell protein secretion is more challenging to measure, because of the low amounts of protein secreted and the need for isolating individual cells.

One strategy for studying single cell protein secretion is to use total internal reflection microscopy to measure secreted proteins in a microwell by a sandwich immunoassay Alternatively, multiple microfluidic strategies to combine live-cell imaging and antibody-based detection of secreted proteins have been developed 30 , Cells are initially captured in single cell wells, where they can be exposed to precise doses and durations of stimuli and imaged using a fluorescent microscope.

The media from each cell's well can be sampled and measured with antibodies for secreted proteins at various time points during the experiment.

Depending on the device, it is also possible to stain cells using immunofluorescence, allowing for both secreted and intracellular proteins to be measured Finally, changes in protein expression can also be controlled by chromatin regulators, which impart histone and DNA modifications 80 , It is now possible to study how single cells use different chromatin regulators to produce varying dynamics of gene expression.

Bintu et al. They showed that epigenetic silencing and reactivation are digital processes in single cells and that different chromatin regulators modulate the fraction of cells silenced. Further, using a stochastic model, they describe the different dynamics for both silencing and reactivation, created by each chromatin regulator How cells use chromatin modifications to process signal information is still poorly understood 83 , but studies of single cell chromatin regulation dynamics provide a promising avenue for future research.

Studying cellular decoding is more challenging than studying cellular encoding, for technical and biological reasons. The primary biological challenge is that cells have complicated signaling pathways that interact with each other and control heterogeneous outputs. Thus, it is technically difficult to prove that the dynamics are the causative factor in the phenotypic measurement.

It has also been difficult to perturb signaling dynamics in ways that would help to establish causality of phenotype, especially in single cells. Here we summarize strategies that have been successfully used to modulate signaling dynamics, as well as significant technical advances that have enabled novel ways of controlling signaling dynamics in single cells.

Recent advancements in synthetic biology have opened exciting new ways of precisely and selectively controlling dynamic signaling. One such advancement is the field of optogenetics, which exploits light to control protein function and cell activities with high spatio-temporal resolution Figure 3A. Optogenetic tools are generally faster and more selective than pharmacological stimulation, and their ability to generate flexible temporal patterns brought us a concept of engineering system identification to study the characteristics of cellular signaling pathway in a more direct manner.

Here we introduce a few examples of optogenetic strategies applicable to signaling dynamics, but more detailed information about limitations and other applications have also been recently reviewed 84 — Figure 3. Engineering approaches for manipulating dynamic signaling patterns. A Optogenetic tools can dynamically and selectively activate a pathway in isolation from endogenous receptor signaling contexts.

Ras activates cRaf at the membrane, and thus it activates the downstream ERK pathway. C Microfluidic devices were used to control the flux of small-molecule inhibitors of the Notch and Wnt signaling pathway. In-phase oscillations of these two pathways led to proper mesoderm segmentation, whereas out-of-phase oscillation impaired segmentation.

One commonly used system is Phy-PIF, an optogenetic system with a fast deactivation rate. Both binding and dissociation are induced by light stimulation; red light induces binding and infrared light induces dissociation This system was recently used to show that a mutation in B-Raf in a human cancer line led to slower decay kinetics of the pathway, meaning that a larger space of input frequencies and strengths are interpreted as growth signals in this cell line Cry2 is another widely used optogenetic system.

It has a slower activation and deactivation rate compared to other systems, but it has the unique property that it exhibits both hetero- and homo-dimerization upon blue light stimulation.

Cry2 binds to the N-terminal domain of CIB1 in a blue-light dependent manner. Using this system, it was shown that pulsatile ERK activation led to higher cell proliferation than sustained activation. Several genes that are induced better by pulsatile ERK activation were also identified. In addition to this heterodimerization between Cry2 and CIB1, Cry2 is known to have a propensity for oligomerization. This property of oligomerization was later improved with a small change in sequences 93 , As many signaling events are initiated by homo-oligomerization, particularly receptor signaling, they have been widely applied to many signaling pathways 95 — A light-oxygen-voltage-sensing LOV domain is a photosensory motif found in many proteins across diverse species.

Blue light stimulation induces covalent bond formation between the LOV domain and its flavin cofactor, leading to a partial unfolding between the LOV domain and C-terminal A -helix. For example, a light-switchable gene promoter system was developed by fusing a fungal LOV domain and the Gal4 transcription factor lacking the dimerization domain This system was also applied to control temporal patterns of proneural gene Ascl1 expression in neural progenitor cells 24 , and the oscillatory and sustained expression of Ascl1 were shown to induce proliferation and differentiation, respectively.

Contrary to the examples above exploiting translocation or recruitment, there are many optogenetic tools that can directly control allostery and fragment complementation.

This includes Dronpa-based strategies 25 , , LOV domain-based proteins utilizing photo-uncaging — , and a number of light-sensitive channels and receptors.

For example, Hannanta-Anan and Chow used melanopsin to generate a wave of calcium release By systematically controlling the calcium oscillation amplitude, frequency, and duty cycles, they found downstream NFAT integrates total elevated calcium concentrations due to its slow export rate. Dimerization can also be induced chemically; the dimerization of FKBP and FRB with rapamycin is a classic example that has been used in a wide variety of applications for many years — Though many tools are essentially irreversible due to high affinity binding, acute induction of dimerization or translocation has been an effective strategy to investigate causation of signaling events.

For instance, Santos et al. The chemical dimerization of these complexes in nuclei triggered cyclin B1 nuclear translocation, which was confirmed by translocation of a fluorescent protein fused to cyclin B1, but not fused to FRB.

Another potential approach to control dynamics is to engineer a fully synthetic version of the pathway in an orthogonal cellular environment. As an example, the ERK pathway was reconstructed in yeast, and this minimal cascade itself was shown to generate ultrasensitivity Synthetic systems provide an easy way to manipulate pathway parameters and circuit structures with small-molecule inputs.

Fluidic control was a standard approach to dynamically manipulate a stimulation pattern before genetic or synthetic approaches became popular.

A simple fluidic setup with a pump has been used for several decades to control input flux, including early studies of glucagon signaling and calcium signaling Microfluidic devices now represent a dramatic improvement, providing us with more precise control to generate virtually any kind of temporal pattern, to study both dynamic encoding and decoding. In terms of dynamic decoding, the filter characteristics of the yeast stress pathway has been extensively studied using microfluidic devices 16 , , By modulating the amplitude, frequency, and duration of periodic Msn2 nuclear translocation, it was shown that each promoter transcribed by Msn2 has a distinct sensitivity to amplitude and pulse frequency.

These differences can differentially regulate at least four classes of genes downstream of Msn2 Microfluidics have also been used to manipulate cellular phenotypes by controlling signaling dynamics. With a microfluidic device, Ryu et al.

As another example, Sonnen et al. They used microfluidics to generate either in-phase or out-of-phase oscillations of these two pathways and showed the out-of-phase oscillations impair segmentation Figure 3C. Many of the examples above show the versatility and capability of microscopy to interrogate relationships between signaling dynamics and downstream phenotypes in single cells.

It is becoming clear that signaling dynamics can be decoded to distinct gene expression programs leading to diverse cellular phenotypes; yet most studies were only able to measure a few genes due to technical challenges. Here we will go over some of the recent technical advancements that potentially expand the throughput and accessibility of this measurement modality.

As we saw in the examples above, FISH and immunofluorescence are commonly used techniques for capturing downstream responses. FISH can be implemented in a high-throughput manner, as one can strip or bleach probes and thus iterate detection — The downside of these techniques is their cost and sensitivity, since many probes are required to bind one species of mRNA and thereby amplify a specific signal. Two recently developed methods utilize different signal amplification schemes, enabling higher sensitivity and gain.

PLISH amplifies a target region by rolling circle amplification after generating closed circle probe oligonucleotides by RNA-templated proximity ligation.

The second technique, called click-amplifying FISH clampFISH , uses non-enzymatic click chemistry to generate closed circle oligonucleotides , Kinases When a ligand binds a receptor, it triggers changes within the cell that influence other signalling molecules. Transcription factors Many signalling pathways influence which genes are switched on in a cell.

Cell death signalling Many signalling pathways in humans involve specialised proteins. Switching off signals Cells in our body are constantly barraged with different signals from their environment.

Cell signalling and disease When cells do not respond appropriately to their environment, or do not work cooperatively with other cells, disease can result. Some examples of disrupted cell signalling in disease include: Cancer cells have constant activation of signalling pathways instructing the cells to grow and divide. This often occurs because of changes mutations in receptors, protein kinases or transcription factors that keep the proteins an active state.

Some immunodeficiencies occur because immune cells lack the receptors for ligands that instruct immune cells to divide and develop, or lack the specific kinases that transmit these signals.

Targeting cell signalling Cell signalling influences how cells behave in health and disease. Associate Professor Jeff Babon. Associate Professor. Laboratory head. Read more about Associate Professor Jeff Babon. Associate Professor Grant Dewson. Laboratory Head. Read more about Associate Professor Grant Dewson. Associate Professor Gemma Kelly. Read more about Associate Professor Gemma Kelly. Professor David Komander. Division Head.

Read more about Professor David Komander. Dr Alisa Glukhova. Read more about Dr Alisa Glukhova. Dr Bernhard Lechtenberg. Read more about Dr Bernhard Lechtenberg. Professor Geoff Lindeman. Joint Division Head. Read more about Professor Geoff Lindeman. Associate Professor Isabelle Lucet. Read more about Associate Professor Isabelle Lucet. Associate Professor James Murphy.

Read more about Associate Professor James Murphy. Associate Professor Sandra Nicholson. Research Highlights 10 November How lymphatic vessels arise from veins is still poorly understood.

A study reports the discovery of a ribosome biogenesis regulator Ddx21 as a previously unappreciated specific factor that is important for the first steps of lymphatic but not blood vessel development. The finding may lead to better strategies to selectively target lymphangiogenesis.

Research Highlights 08 October This requires assembly of a multiprotein—lipid complex on the plasma membrane. In a tour de force of modeling, Mysore et al.

Research Highlights 30 September Advanced search. Skip to main content Thank you for visiting nature.