Intracellular signaling dynamics and their role in coordinating tissue repair

Abstract

Tissue repair is a complex process that requires effective communication and coordination between cells across multiple tissues and organ systems. Two of the initial intracellular signals that encode injury signals and initiate tissue repair responses are calcium and extracellular signal-regulated kinase (ERK). However, calcium and ERK signaling control a variety of cellular behaviors important for injury repair including cellular motility, contractility, and proliferation, as well as the activity of several different transcription factors, making it challenging to relate specific injury signals to their respective repair programs. This knowledge gap ultimately hinders the development of new wound healing therapies that could take advantage of native cellular signaling programs to more effectively repair tissue damage. The objective of this review is to highlight the roles of calcium and ERK signaling dynamics as mechanisms that link specific injury signals to specific cellular repair programs during epithelial and stromal injury repair. We detail how the signaling networks controlling calcium and ERK can now also be dissected using classical signal processing techniques with the advent of new biosensors and optogenetic signal controllers. Finally, we advocate the importance of recognizing calcium and ERK dynamics as key links between injury detection and injury repair programs that both organize and execute a coordinated tissue repair response between cells across different tissues and organs. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Biological Mechanisms > Cell Signaling Laboratory Methods and Technologies > Imaging Models of Systems Properties and Processes > Organ, Tissue, and Physiological Models.

Keywords: ERK signaling; calcium signaling; molecular mechanisms; signaling dynamics; tissue repair.

Figure 1

Overview of the role intracellular signaling dynamics play in tissue gap repair. (Left) Tissue injury induces both biochemical stimuli released from damaged cells and a mechanical stimulus through the release of tension. Nearby cells then sense these stimuli, which trigger the activation of intracellular signaling pathways such as calcium and ERK. These pathways can encode information by varying the concentration of a molecule or the level of activation of a protein in time and space. (Center) Molecular mechanisms inside the cell transduce dynamic calcium and ERK signals into cellular‐ and tissue‐level responses. (Right) Intracellular responses, such as protein activation and gene transcription, and tissue‐wide responses, such as cell migration and matrix production, coordinate tissue repair. ERK, extracellular signal‐regulated kinase

Figure 2

Mechanisms of epithelial and stromal injury repair. (Top) Epithelial injury repair. After injury, actin and myosin are moved to the injury edge of the epithelial cells, forming long networks of actomyosin bundles, termed the “Actomyosin Purse String” (red line). The actomyosin purse string is then contracted via the activation of myosin, which pulls the cells together and physically closes the gap. See Martin and Lewis (1992) for first image of purse string and (Tetley et al., 2019) for a recent time‐lapse video of the process. (Bottom) Stromal injury repair. After injury, fibroblasts migrate to the wound edge and secrete a provisional fibronectin matrix to fill in the gap (red lines). The fibroblasts then migrate onto the provisional matrix and continue to secrete fibronectin until the gap is closed. During the remodeling phase, the fibronectin is replaced with collagen to fully restore mechanical integrity to the tissue. See Sakar et al. (2016) for time‐lapse images of the stromal repair process

Figure 3

Calcium and ERK signaling networks for injury sensing and repair. Calcium and ERK can be activated by several different tissue injury signals, and can in turn activate several different tissue repair pathways. Cytosolic calcium can either come from extracellular influx via stretch activated channels, or from endoplasmic reticulum efflux. Calcium can then control several different cellular behaviors, including motility, contraction, and gene transcription. ERK can be activated by various receptor tyrosine kinases and can in turn control cellular contraction, motility, and gene transcription. One of the challenges in studying calcium and ERK signaling during injury is understanding which pathways are being activated at a given time by a given molecule, given the diverse and overlapping pathways containing calcium and ERK. Each pathway is referenced throughout the text. ERK, extracellular signal‐regulated kinase

Figure 4

Genetically encoded biosensors for quantifying cellular signaling dynamics. (Left) Intensity‐based sensors. These sensors convert the binding of an analyte into a change in fluorescence. See Grynkiewicz, Poenie, and Tsien (1985) and Nakai, Ohkura, and Imoto (2001)) for examples of dye‐ and fluorescent protein‐based intensity sensors and (Shannon et al., 2017) for a study of calcium dynamics after epithelial injury using the GCaMP genetically encoded calcium sensor as illustrated. (Center) FRET‐based sensors. These ratiometric sensors take advantage of FRET to convert a change in distance between two fluorophores due to conformational changes in the protein into a difference in the intensity ratio between two fluorophores. As an example, in the unbound state, the donor fluorophore could be spatially separated from the acceptor fluorophore, and is not able to transfer energy, therefore there is high donor fluorescence and low acceptor fluorescence. In the bound state, the analyte binding would cause the fluorophores to move closer together, resulting in energy transfer between the two, therefore there is decreased donor fluorescence and increased acceptor fluorescence. See Heim and Tsien (1996) and Legant, Chen, and Vogel (2012) for examples of fluorescent protein and dye‐based FRET sensors in use, and Handly et al. (2015) for an application of an EKAR‐based ERK FRET sensor to quantifying epithelial wounding dynamics as illustrated. (Right) Translocase sensors. These sensors use a combination of nuclear import and export tags together with a kinase binding domain to control the nuclear translocation of a fluorescent protein. In the dephosphorylated state, the sensor resides in the nucleus. Upon kinase activation and subsequent sensor phosphorylation, the sensor is transported out of the nucleus into the cytosol. See Regot, Hughey, Bajar, Carrasco, and Covert (2014) for multiple examples of kinase translocation reporters and Mayr, Sturtzel, Stadler, Grissenberger, and Distel (2018) for an example application of the ERKKTR to understanding post‐wounding ERK dynamics in zebrafish muscle. ERK, extracellular signal‐regulated kinase

Figure 5

Illustration of how optogenetic controllers enable the application of classical signal processing techniques to biological systems. (a) Optogenetic controllers provide unprecedented spatiotemporal control over intracellular signals, including calcium and ERK, as illustrated by the dials tuning signal frequency. This control allows signal processing approaches such as black box techniques to be applied to the molecular processes that transduce intracellular signals into repair responses. (b) Optogenetic controllers also allow for precise spatiotemporal control over intracellular signal in a multicellular context, permitting us to determine if intracellular signaling observed across a tissue induces coordinated cellular behaviors that are merely additive of many single cells together or emergent and only exist in a group context. (c, Left) Optogenetics can be used to activate repair programs in uninjured tissues, testing the causal relationship between intracellular signaling and tissue‐wide repair programs. See Wilson et al. (2017) for an example of this technique applied to ERK activation of immediate early genes. (c, Center) Optogenetics can also test sufficiency of signaling motifs by attempting to recover tissue repair programs after blocking injury stimulation using a drug. If optogenetically induced‐intracellular signaling recovers tissue repair then the observed intracellular signaling is sufficient to control the observed tissue repair program. (c, Right) Optogenetics can also tune intracellular responses during injury repair by controlling the dynamics of intracellular signaling. This could potentially link different signaling frequencies to different levels of behavior, such as increased or decreased matrix production. ERK, extracellular signal‐regulated kinase