InteractiveFly: GeneBrief

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The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. This study shows that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps; see Gbp1), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, this study showed that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. Experimental and computational evidence is presented that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium (O'Connor, 2021).

When a tissue is wounded, the cells surrounding the wound rapidly respond to repair the damage. Despite the non-specific nature of cellular damage, there is remarkable specificity to the earliest cellular response: cells around the wound increase cytosolic calcium, and this damage response is conserved across the animal kingdom. The calcium response is not limited to cells at the wound margin but extends even to distal cells. Multiple molecular mechanisms have been identified that regulate wound-induced gene expression or cell behavior downstream of calcium, but the upstream signals remain unclear. How exactly do cells detect wounds? Thia study investigate the molecular mechanisms by which a wound initiates cytosolic calcium signals (O'Connor, 2021).

The immediate increase in cellular calcium in turn initiates repair or defense responses. Calcium has been well established as a versatile and universal intracellular signal that plays a role in the modulation of numerous intracellular processes. Several calcium-regulated processes are required for proper wound repair, including actomyosin dynamics, JNK pathway activation and plasma membrane repair. Unsurprisingly, an increase in cytosolic calcium is necessary for wound repair. Nonetheless, there is less clarity on the mechanisms that trigger increased cytosolic calcium in cells near to and distant from the wound. In some cases, wound-induced cytoplasmic calcium enters from the extracellular environment, either directly through plasma membrane damage. In others, calcium is released from the endoplasmic reticulum (ER) through the IP3 Receptor and initiated by an unknown G-protein-coupled receptor (GPCR) or receptor tyrosine kinase (RTK). Further, calcium responses can be initiated by mechanical stimuli alone. Elucidating the mechanisms by which calcium signaling is triggered in vivo is critical to understanding how wound information is transmitted through a tissue in order to change cellular behavior and properly repair the wound (O'Connor, 2021).

By live imaging laser wounds in Drosophila pupae, previously work showed that damaged cells around wounds become flooded within milliseconds by extracellular calcium entering through microtears in the plasma membrane. Although this calcium influx expands one or two cell diameters through gap junctions, it does not extend to more distal cells. Strikingly, after a delay of 45-75 s, a second independent calcium response expands outward to reach a larger number of distal cells. This study identified the relevant signal transduction pathway and receptor, the GPCR Mthl10. Downstream, signals are relayed through Gαq and PLCβ to release calcium from the ER. Upstream, Mthl10 is activated around wounds by the cytokine ligands Growth-blocking peptides (Gbps). Further, experimental and computational evidence is provided that the initiating event for the distal calcium response in vivo is a wound-induced release of proteases that activate the latent Gbp cytokines, cleaving them from inactive/pro-forms into active signaling molecules (O'Connor, 2021).

It was already known that Gbps are synthesized in an inactive pro-form, requiring proteolytic cleavage for activation, and that they are secreted by the fat body. Although Gbps are present in unwounded tissues, they activate Mthl10 only in the presence of a wound. Interestingly, Gbps have cleavage consensus sequences for multiple protease families. Further, the addition of cell lysate or the addition of the unrelated proteases trypsin or clostripain to unwounded tissue is sufficient to generate a calcium signal in wing discs through Mthl10/Gbp signaling. These results lead to a model in which the lysis of cells inherent in wounding releases non-specific cellular proteases into the extracellular environment. These proteases cleave and activate extracellular Gbps, which in turn activate the Mthl10 GPCR on cells around the wound, initiating wound-induced calcium signaling. Such cell lysis and protease release should be a general feature of cell destruction, whether caused by trauma, pathogen-induced lysis, or a lytic form of cell death such as pyroptosis or necroptosis (immunologically silent apoptosis may well be an exception). A variety of epithelial damage mechanisms may thus converge through the Gbps to signal via the GPCR Mthl10 and alert surrounding cells to the presence of a nearby wound. This molecular mechanism is supported by a computational model that accurately describes the pattern and timing of wound-induced calcium, predicted its dependence on wound size and initial levels of Gbps, and led to the observation that cell lysis is not immediate but rather takes place over tens of seconds. Thus, this study offers a model for how surrounding cells detect the damage of cell lysis, utilizing a Gbp-based protease-detector system (O'Connor, 2021).

Two superimposed mechanisms increase cytoplasmic calcium levels around wounds Laser wounds generate complex yet reproducible patterns of increased cytoplasmic calcium, and the complexity of this pattern has undoubtedly made it difficult to unravel its underlying mechanisms. Within the first ~90 s after wounding, two mechanisms drive the increase of calcium, and the complexity is generated by the temporal and spatial superimposition of these two mechanisms. Previously, it was reported that a different type of cellular damage initiates a different mechanism for increasing cytoplasmic calcium. In that report, wound-induced microtears were identified in the plasma membranes of surviving cells, and these microtears provided an entry for extracellular calcium to flood into the cytoplasm and then flow out to neighboring undamaged cells via gap junctions. This direct entry of calcium through damaged plasma membranes is evident within milliseconds after wounding. In this report we describe a second mechanism that extends to more distal cells, initiated by cell lysis at wounding. The dynamics of protease release from lysed cells, along with the gradual accumulation of active Gbp and its rapid diffusion, all contribute to the appearance of this distal calcium response 45-75 s after wounding. The earliest and closest cells to be activated by Mthl10/Gbp signaling cannot be identified visually because the initial flood of calcium through microtears takes time to subside (O'Connor, 2021).

Three tools allowed deciphering od these superimposed mechanisms. The first tool was the laser itself, which generates a highly stereotyped pattern of damage within a circular wound bed. Although cell lysis and plasma membrane damage are features of nearly every wound, their reproducible pattern in a laser wound allowed distinguishing the signaling mechanisms each type of damage potentiated. The second tool was a spatiotemporal analytical framework to measure radius over time, which clearly identified two peaks, the first induced by microtears and the second induced by cell lysis. The third tool was experimental, using RNAi-knockdown of genes in a limited region and comparing it with an internal control. The ability to identify asymmetry between the control and experimental sides of wounds allowed bypassing of concerns about variable wound sizes, which otherwise would have made it difficult to recognize patterns and interpret data. Complex overlapping patterns may have obscured the mechanisms upstream of wound-induced calcium in other systems as well as the current one (O'Connor, 2021).

Previous studies identified other molecules and phenomena upstream of wound-induced calcium. Studies in cell culture found that wound-generated cell lysis releases ATP, which diffuses extracellularly to bind to purinergic receptors and activate calcium release from intracellular stores. Although reproducible in many types of cultured cells, there has been little evidence to support ATP signaling from lytic cells in vivo, likely because extracellular ATP is rapidly hydrolyzed by nucleotidases in vivo. Interestingly, ATP does appear to signal damage and promote motility in response to injuries associated with cell swelling in zebrafish, animals that inhabit a hypotonic aqueous environment; however, even in this wounding paradigm, ATP does not signal from lytic cells at an appreciable level. No evidence was found for ATP signaling upstream of calcium in the wounding experiments, as knockdown of the only fly adenosine receptor did not alter the calcium pattern around wounds (O'Connor, 2021).

Some in vivo studies have implicated a TrpM ion channel upstream of calcium release. This role of TrpM was first identified in laser-wounding studies of the C. elegans hypodermis, a giant syncytial cell where great overlap in the spatial extent of microtear-initiated calcium, which would diffuse quickly throughout a syncytium, and receptor-mediated calcium released from the ER would be expected. In the hypodermis, loss of TrpM reduced by half the intensity of wound-induced calcium signaling, but without spatial and temporal analysis, the exact contribution of TrpM is not known. In the Drosophila notum, a previous study identified TrpM as a regulator of wound-induced actin remodeling, and a slight reduction in wound-induced calcium intensity over time was noted in TrpM knockdowns. In contrast, this study did not observe any change in the spatial or temporal aspects of the calcium response in TrpM knockdown cells compared with the internal control, and given wound-to-wound variability, it would have been hard to identify a small effect without an internal control. A study in the fly embryo determined that wound-induced calcium originates from both the external environment and internal stores, suggesting to that two superimposed calcium response mechanisms may have been at play in these experiments. That study found when TrpM was knocked down, calcium intensity was reduced by half, but again without spatial and temporal analysis or an internal control, it is difficult to know what pathway TrpM regulates. Tissue mechanics are upstream of increases in cytoplasmic calcium in a non-wounding context. Several labs have reported calcium flashes and waves in unwounded wing discs, dissected from larvae and cultured ex vivo. Cell and organ culture requires serum to support metabolism outside the organism, and in fly culture, this 'serum' is generated by grinding whole adult flies and collecting the supernatant. Because such serum would undoubtedly contain secreted signals from wounded cells, calcium signaling in wing discs ex vivo is probably a wound response; indeed, it was founs to be transduced by the same mechanism as wound signals, requiring protease, Gbps, and Mthl10. One aspect of calcium signaling in wing discs that we have not tested in our wounding model, however, is the role of mechanical tension. In carefully controlled mechanical experiments, fly serum was found to induce calcium flashes in wing discs specifically on the release of mechanical compression, indicating that tension is a requirement for calcium signaling in these wing discs. It is interesting to consider the TrpM results in light of these mechanical studies, as some TrpM channels can be mechanosensitive. Together, these data suggest that there may be a role for mechanical tension in wound-induced calcium responses (O'Connor, 2021).

Two independent mechanisms were founs that increase cytoplasmic calcium, and in the cells at the wound margin these mechanisms would appear to act redundantly. Such redundancy indicates that the role of calcium in these cells is very important for wound healing. One biological pathway that may be downstream of calcium in these cells is recruitment of actin and myosin to the wound margin to form an actomyosin purse string that cinches the wound closed. What about calcium in the distal cells, regulated by Gbp/Mthl10? There are many possible functions, but currently, all of them are speculative. One possibility is that the cytosolic calcium response initiates distal epithelial cells to modify their cellular behavior from a stationary/non-proliferative state to a migratory and/or proliferative state necessary to repair the wound. Alternatively, an increase in cytosolic calcium may act to modulate an inflammatory response through DUOX leading to the formation of hydrogen peroxide to recruit inflammatory cells to the wound or through the calcium-dependent activation of cytoplasmic Phospholipase A2 leading to the rapid recruitment of immune cells to tissue damage. This possibility is intriguing because Gbp is known to activate an immune response leading to the upregulation of antimicrobial peptides and to increased activity of phagocytic plasmatocytes in a calcium-dependent manner. Interestingly, loss of Methuselah-like (Mthl) GPCRs results in increased lifespans, and Gbps are nutrition-sensitive peptides whose expression is reduced under starvation conditions. Increased lifespan, caloric restriction and decreased inflammation have all been linked, and Gbp/Mthl10 activation at wounds may be part of this link (O'Connor, 2021).

Although the cytokine and GPCR families are widely conserved, Gbp and Mthl10 do not have direct orthologs in chordates. Nonetheless, similarities exist between the Gbp/Mthl10 mechanism and wound responses in other organisms. Damage- or pathogen-induced activation of proteins by proteolytic cleavage has been well documented in the cases of Spatzle in the Toll pathway, thrombin and fibrin in the blood coagulation pathway, and IL-1β and IL-18 in the pyroptosis pathway. Additionally, wound-defense signaling in plants relies on an immunomodulatory plant elicitor peptide that is cleaved into its active form by cysteine proteases upon damage-induced cytosolic calcium, and the plant defense hormone systemin is cleaved into its active form by phytaspases in response to damage or predation. Because the basic circuitry is similar across kingdoms, the current study suggests an ancient strategy for wound detection based on proprotein cleavage, activated by proteases released via cell lysis. As these examples make clear, proteases are already known to play critical roles in blood clotting and immune signaling, and this study finds that proteases are also instructive signals in epithelial wound detection (O'Connor, 2021).

As noted above, the Gbp ligands and Mthl10 receptor are not present in mammals, so the extent of mechanistic conservation is unclear. Further, this study did not experimentally tested this wound-detection mechanism in other developmental stages of Drosophila. For the computational model, several simplifications were made: the use of one variable for all proteases and one variable for all Gbps, rather than having separate Gbp1 and Gbp2; the use of simplified receptor/ligand dynamics that do not include uptake or recycling; and the use of a ligand-receptor-binding threshold rather than inclusion of the signal transduction cascade between receptor-binding and calcium release. Finally, this study does not describe or address the mechanism behind the calcium flares that continue for at least one houe after wounding (O'Connor, 2021).

Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress

This study used Drosophila to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca(2+) signaling pathway mediates innate immune responses to GBP, this study conducted a dsRNA library screen for genes that modulate Ca(2+) mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. The metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This was established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. It was further reported that organismal fitness pays a price for the utilization of Mthl10 to integrate all of these homeostatic attributes of GBP: Elevated GBP expression reduces lifespan. Conversely, Mthl10 knockdown extended lifespan (Sung, 2017).

The most important development to emerge from this study is the deorphanization of Mthl10, through the placement of this GPCR at the epicenter of a molecular pathway that pits stress responses against lifespan. Various immunological and metabolic properties of a single cytokine, GBP, are integrated through its interactions with Mthl10. In particular, it was shown how the operation of the GBP/Mthl10 axis usefully matches nutrient supply to the degree of a metabolically expensive inflammatory response; this is an important topic in immunology. The model for GBP/Mthl10 functionality also shows how it has the potential to exacerbate metabolic inflammation; this may be one of the reasons that nutrient excess in Drosophila can model human metabolic syndrome. Furthermore, these homeostatic functions for Mthl10 were linked to its strong influence upon longevity. This provides a molecular foundation for a theory of aging, namely, that a shortened lifespan can be the ultimate price that a young organism pays to successfully combat short-term environmental stresses (Sung, 2017).

These findings were considered in relation to previous work that provides a detailed analysis of the expression pattern of Mthl10 in Drosophila embryos and larvae. For example, due to extensive expression of Mthl10 in imaginal discs, it has been proposed this gene may influence organogenesis. It is therefore relevant that cytokines-including the Mthl10 ligand, GBP-are well-known to regulate tissue remodeling and development. Additionally, the determination that Mthl10 regulates GBP-mediated innate immune responses seems pertinent to earlier observations that Mthl10 is expressed in hematopoietic tissue (which has immunological functions) and also crystal cells, which encapsulate foreign material. Nevertheless, the possibility cannot be excluded that other ligands for Mthl10 remain to be identified, perhaps as a consequence of the expression of alternate Mthl10 transcripts (Sung, 2017).

The significance of Mthl10 to longevity and metabolism is shared by Mth. In fact, it was the first gene duplication within the Mth superclade that is believed to have given rise to Mthl10, which did not then undergo any further expansion in Drosophila. In contrast, five further rounds of gene duplication apparently occurred before Mth emerged. Thus, it is concluded that the connection between lifespan and metabolic homeostasis that was observed for Mthl10 is an ancestral trait rather than adaptive specifically to Mth (Sung, 2017).

It is not unusual for gene regulatory networks to be widely conserved, even when certain components might undergo evolutionary turnover. Indeed, recent work has shown that although selection pressure has caused GPCR ectodomains and their ligands to codiversify, there has nevertheless been considerable conservation of the receptor's intracellular interactions with G proteins; as a result, flies and mammals share many of the same downstream signaling cascades. Indeed, GBP exhibits some sequence similarity with the human defensin BD2; both are small, cationic cytokines produced by protease action upon larger, precursor proteins. Furthermore, human BD2 acts through an uncharacterized GPCR to stimulate PLC/Ca2+ signaling to initiate inflammatory responses; the current study demonstrates that GBP is also a GPCR ligand that initiates PLC/Ca2+ signaling. Thus, it is proposed that there is general applicability to the concepts that emerge from our integration of immunological, metabolic, and lifespan functions for the GBP/Mthl10 axis (Sung, 2017).

Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution

Functional studies of the methuselah/methuselah-like (mth/mthl) gene family have focused on the founding member mth, but little is known regarding the developmental functions of this receptor or any of its paralogs. A comprehensive analysis of developmental expression and sequence divergence in the mth/mthl gene family members was undertaken. Using in situ hybridization techniques, expression was detected of six genes (mthl1, 5, 9, 11, 13, and 14) in the embryo during gastrulation and development of the gut, heart, and lymph glands. Four receptors (mthl3, 4, 6, and 8) are expressed in the larval central nervous system, imaginal discs, or both, and two receptors (mthl10 and mth) are expressed in both embryos and larvae. Phylogenetic analysis of all mth/mthl genes in five Drosophila species, mosquito and flour beetle structured the mth/mthl family into several subclades. mthl1, 5, and 14 are present in most species, each forming a separate clade. A newly identified Drosophila mthl gene (CG31720; herein mthl15) formed another ancient clade. The remaining Drosophila receptors, including mth, are members of a large “superclade” that diversified relatively recently during dipteran evolution, in many cases within the melanogaster subgroup. Comparing the expression patterns of the mth/mthl “superclade” paralogs to the embryonic expression of the singleton ortholog in Tribolium suggests both subfunctionalization and acquisition of novel functionalities. Taken together, these findings shed novel light on mth as a young member of an adaptively evolving developmental gene family (Patel, 2012).

REFERENCES

Search PubMed for articles about Drosophila Mthl10

O'Connor, J. T., Stevens, A. C., Shannon, E. K., Akbar, F. B., LaFever, K. S., Narayanan, N. P., Gailey, C. D., Hutson, M. S. and Page-McCaw, A. (2021). Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection. Dev Cell. PubMed ID: 34273275

Patel M. V., Hallal, D. A., Jones, J. W., Bronner, D. N., Zein, R., Caravas, J., Husain, Z., Friedrich, M., Vanberkum, M. F. (2012). Dramatic expansion and developmental expression diversification of the methuselah gene family during recent Drosophila evolution. J Exp Zool B Mol Dev Evol318(5):368-387. PubMed ID: 22711569

Sung, E. J., et al. (2017). Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc Natl Acad Sci U S A 114(52): 13786-13791. PubMed ID: 29229844

Thomsen A. R. B., Jensen, D. D., Hicks, G. A., Bunnett, N. W. (2018). Therapeutic Targeting of Endosomal G-Protein-Coupled Receptors. Trends Pharmacol Sci39(10):879-891. PubMed ID: 30180973