Epithelial mechanics are maintained by inhibiting cell fusion with age in Drosophila.

A characteristic of normal aging and age-related diseases is the remodeling of the cellular organization of a tissue through polyploid cell growth. Polyploidy arises from an increase in nuclear ploidy or the number of nuclei per cell. However, it is not known whether age-induced polyploidy is an adaption to stressors or a precursor to degeneration. Here, we find that abdominal epithelium of the adult fruit fly becomes polyploid with age through generation of multinucleated cells by cell fusion. Inhibition of fusion does not improve the lifespan of the fly, but does enhance its biomechanical fitness, a measure of the healthspan of the animal. Remarkably, Drosophila can maintain their epithelial tension and abdominal movements with age when cell fusion is inhibited. Epithelial cell fusion also appears to be dependent on a mechanical cue, as knockdown of Rho kinase, E-cadherin or α-catenin is sufficient to induce multinucleation in young animals. Interestingly, mutations in α-catenin in mice result in retina pigment epithelial multinucleation associated with macular disease. Therefore, we have discovered that polyploid cells arise by cell fusion and contribute to the decline in the biomechanical fitness of the animal with age.

Binucleation ramps up gene expression meeting the physiological demands of an organism.

In this issue of PLOS Biology, van Rijnberk and colleagues show how polyploidy, via binucleation, enables Caenorhabditis elegans intestinal cells to ramp up gene expression supplying the oocytes with the necessary lipids for optimal organismal growth and reproductive fitness.

The endocycle restores tissue tension in the Drosophila abdomen post wound repair.

Polyploidy frequently arises in response to injury, aging, and disease. Despite its prevalence, major gaps exist in our understanding of how polyploid cells alter tissue function. In the adult Drosophila epithelium, wound healing is dependent on the generation of multinucleated polyploid cells resulting in a permanent change in the epithelial architecture. Here, we study how the wound-induced polyploid cells affect tissue function by altering epithelial mechanics. The mechanosensor nonmuscle myosin II is activated and upregulated in wound-induced polyploid cells and persists after healing completes. Polyploidy enhances relative epithelial tension, which is dependent on the endocycle and not cell fusion post injury. Remarkably, the enhanced epithelial tension mimics the relative tension of the lateral muscle fibers, which are permanently severed by the injury. As a result, we found that the wound-induced polyploid cells remodel the epithelium to maintain fly abdominal movements, which may help compensate for lost tissue tension.

Polyploidy in Tissue Repair and Regeneration.

Polyploidy is defined as a cell with three or more whole genome sets and enables cell growth across the kingdoms of life. Studies in model organisms have revealed that polyploid cell growth can be required for optimal tissue repair and regeneration. In mammals, polyploid cell growth contributes to repair of many tissues, including the liver, heart, kidney, bladder, and eye, and similar strategies have been identified in Drosophila and zebrafish tissues. This review discusses the heterogeneity and versatility of polyploidy in tissue repair and regeneration. Polyploidy has been shown to restore tissue mass and maintain organ size as well as protect against oncogenic insults and genotoxic stress. Polyploid cells can also serve as a reservoir for new diploid cells in regeneration. The numerous mechanisms to generate polyploid cells provide an unlimited resource for tissues to exploit to undergo repair or regeneration.

Wound-induced polyploidization is dependent on Integrin-Yki signaling.

A key step in tissue repair is to replace lost or damaged cells. This occurs via two strategies: restoring cell number through proliferation or increasing cell size through polyploidization. Studies in Drosophila and vertebrates have demonstrated that polyploid cells arise in adult tissues, at least in part, to promote tissue repair and restore tissue mass. However, the signals that cause polyploid cells to form in response to injury remain poorly understood. In the adult Drosophila epithelium, wound-induced polyploid cells are generated by both cell fusion and endoreplication, resulting in a giant polyploid syncytium. Here, we identify the integrin focal adhesion complex as an activator of wound-induced polyploidization. Both integrin and focal adhesion kinase are upregulated in the wound-induced polyploid cells and are required for Yorkie-induced endoreplication and cell fusion. As a result, wound healing is perturbed when focal adhesion genes are knocked down. These findings show that conserved focal adhesion signaling is required to initiate wound-induced polyploid cell growth.

A Drosophila Model to Study Wound-induced Polyploidization.

Polyploidy is a frequent phenomenon whose impact on organismal health and disease is still poorly understood. A cell is defined as polyploid if it contains more than the diploid copy of its chromosomes, which is a result of endoreplication or cell fusion. In tissue repair, wound-induced polyploidization (WIP) has been found to be a conserved healing strategy from fruit flies to vertebrates. WIP has several advantages over cell proliferation, including resistance to oncogenic growth and genotoxic stress. The challenge has been to identify why polyploid cells arise and how these unique cells function. Provided is a detailed protocol to study WIP in the adult fruit fly epithelium where polyploid cells are generated within 2 days after a puncture wound. Taking advantage of D. melanogaster's extensive genetic tool kit, the genes required to initiate and regulate WIP, including Myc, have begun to be identified. Continued studies using this method can reveal how other genetic and physiological variables including sex, diet, and age regulate and influence WIP's function.

Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage.

Tissue repair usually requires either polyploid cell growth or cell division, but the molecular mechanism promoting polyploidy and limiting cell division remains poorly understood. Here, we find that injury to the adult Drosophila epithelium causes cells to enter the endocycle through the activation of Yorkie-dependent genes (Myc and E2f1). Myc is even sufficient to induce the endocycle in the uninjured post-mitotic epithelium. As result, epithelial cells enter S phase but mitosis is blocked by inhibition of mitotic gene expression. The mitotic cell cycle program can be activated by simultaneously expressing the Cdc25-like phosphatase String (stg), while genetically depleting APC/C E3 ligase fizzy-related (fzr). However, forcing cells to undergo mitosis is detrimental to wound repair as the adult fly epithelium accumulates DNA damage, and mitotic errors ensue when cells are forced to proliferate. In conclusion, we find that wound-induced polyploidization enables tissue repair when cell division is not a viable option.

Wound-Induced Polyploidy Is Required for Tissue Repair.

Significance: All organs suffer wounds to some extent during an animal's lifetime and to compensate for cell loss, tissues often rely on cell division. However, many organs are made up of differentiated cells with only a limited capacity to divide. It is not well understood how cells are replaced in the absence of cell division. Recent Advances: Recent studies in the model organism Drosophila melanogaster have proven that wound-induced polyploidy (WIP) is an essential mechanism to replace tissue mass and restore tissue integrity in the absence of cell division. In this repair mechanism, preexisting differentiated cells increase their DNA content and cell size by becoming polyploid. Critical Issues: Cells within mammalian organs such as the liver, heart, and cornea have also been observed to increase their DNA ploidy in response to injury, suggesting that WIP may be an evolutionarily conserved mechanism to compensate for cell loss. Future Directions: The Hippo signal transduction pathway is required for differentiated cells to initiate WIP in Drosophila. Continued studies in Drosophila will help to identify other signaling pathways required for WIP as well as the conserved mechanisms that polyploid cells may play during wound repair in all organisms.

Wound-Induced Polyploidization: Regulation by Hippo and JNK Signaling and Conservation in Mammals.

Tissue integrity and homeostasis often rely on the proliferation of stem cells or differentiated cells to replace lost, aged, or damaged cells. Recently, we described an alternative source of cell replacement- the expansion of resident, non-dividing diploid cells by wound-induced polyploidization (WIP). Here we show that the magnitude of WIP is proportional to the extent of cell loss using a new semi-automated assay with single cell resolution. Hippo and JNK signaling regulate WIP; unexpectedly however, JNK signaling through AP-1 limits rather than stimulates the level of Yki activation and polyploidization in the Drosophila epidermis. We found that polyploidization also quantitatively compensates for cell loss in a mammalian tissue, mouse corneal endothelium, where increased cell death occurs with age in a mouse model of Fuchs Endothelial Corneal Dystrophy (FECD). Our results suggest that WIP is an evolutionarily conserved homeostatic mechanism that maintains the size and synthetic capacity of adult tissues.

Polyploidization and cell fusion contribute to wound healing in the adult Drosophila epithelium.

BACKGROUND: Reestablishing epithelial integrity and biosynthetic capacity is critically important following tissue damage. The adult Drosophila abdominal epithelium provides an attractive new system to address how postmitotic diploid cells contribute to repair. RESULTS: Puncture wounds to the adult Drosophila epidermis close initially by forming a melanized scab. We found that epithelial cells near the wound site fuse to form a giant syncytium, which sends lamellae under the scab to re-epithelialize the damaged site. Other large cells arise more peripherally by initiating endocycles and becoming polyploid, or by cell fusion. Rac GTPase activity is needed for syncytium formation, while the Hippo signaling effector Yorkie modulates both polyploidization and cell fusion. Large cell formation is functionally important because when both polyploidization and fusion are blocked, wounds do not re-epithelialize. CONCLUSIONS: Our observations indicate that cell mass lost upon wounding can be replaced by polyploidization instead of mitotic proliferation. We propose that large cells generated by polyploidization or cell fusion are essential because they are better able than diploid cells to mechanically stabilize wounds, especially those containing permanent acellular structures, such as scar tissue.

The Legionella pneumophila EnhC protein interferes with immunostimulatory muramyl peptide production to evade innate immunity.

Successful pathogens have evolved to evade innate immune recognition of microbial molecules by pattern recognition receptors (PRR), which control microbial growth in host tissues. Upon Legionella pneumophila infection of macrophages, the cytosolic PRR Nod1 recognizes anhydro-disaccharide-tetrapeptide (anhDSTP) generated by soluble lytic transglycosylase (SltL), the predominant bacterial peptidoglycan degrading enzyme, to activate NF-κB-dependent innate immune responses. We show that L. pneumophila periplasmic protein EnhC, which is uniquely required for bacterial replication within macrophages, interferes with SltL to lower anhDSTP production. L. pneumophila mutant strains lacking EnhC (ΔenhC) increase Nod1-dependent NF-κB activation in host cells, while reducing SltL activity in a ΔenhC strain restores intracellular bacterial growth. Further, L. pneumophila ΔenhC is specifically rescued in Nod1- but not Nod2-deficient macrophages, arguing that EnhC facilitates evasion from Nod1 recognition. These results indicate that a bacterial pathogen regulates peptidoglycan degradation to control the production of PRR ligands and evade innate immune recognition.

Drosophila stem cell niches: a decade of discovery suggests a unified view of stem cell regulation.

The past decade of research on Drosophila stem cells and niches has provided key insights. Fly stem cells do not occupy a special "state" based on novel "stem cell genes" but resemble transiently arrested tissue progenitors. Moreover, individual stem cells and downstream progenitors are highly dynamic and dispensable, not tissue bulwarks. Niches, rather than fixed cell lineages, ensure tissue health by holding stem cells and repressing cell differentiation inside, but not outside. We review the five best-understood adult Drosophila stem cells and argue that the fundamental biology of stem cells and niches is conserved between Drosophila and mice.

LnaB: a Legionella pneumophila activator of NF-kappaB.

Legionella pneumophila possesses a large arsenal of type IV translocated substrates. Over 100 such proteins have been identified, but the functions of most are unknown. Previous studies have demonstrated that L. pneumophila activates NF-kappaB, a master transcriptional regulator of the mammalian innate immune response. Activation of NF-kappaB is dependent on the Legionella Icm/Dot type IV protein translocation system, consistent with the possibility that translocated bacterial proteins contribute to this response. To test this hypothesis, an expression library of 159 known and putative translocated substrates was created to evaluate whether ectopic production of a single L. pneumophila protein could activate NF-kappaB in mammalian cells. Expression of two of these proteins, LnaB (Legionella NF-kappaB activator B) and LegK1, resulted in approximately 150-fold induction of NF-kappaB activity in HEK293T cells, levels similar to the strong induction that occurs with ectopic expression of the known activator Nod1. LnaB is a substrate of the Icm/Dot system, and in the absence of this protein, a partial reduction of NF-kappaB activation in host cells occurs after challenge by post-exponential phase bacteria. These data indicate that LnaB is an Icm/Dot substrate that contributes to NF-kappaB activation during L. pneumophila infection in host cells.

A hemidominant Naip5 allele in mouse strain MOLF/Ei-derived macrophages restricts Legionella pneumophila intracellular growth.

Mouse-derived macrophages have the unique ability to restrict or permit Legionella pneumophila intracellular growth. The common inbred mouse strain C57BL/6J (B6) restricts L. pneumophila growth, whereas macrophages derived from A/J mice allow >10(3)-fold bacterial growth within three days. This phenotypic difference was mapped to the mouse Naip5 allele. The B6 restrictive Naip5 allele is dominant, and six amino acid changes in its product were predicted to control permissiveness. By using the wild-derived mouse strain MOLF/Ei, we found that MOLF/Ei-derived macrophages also restrict L. pneumophila growth, yet the Naip5 protein is identical to the A/J Naip5 at the six-amino-acid signature. The MOLF/Ei restrictive trait, unlike that of B6-derived macrophages, was not dominant over the A/J trait. In spite of this phenotypic difference, the L. pneumophila growth restriction in MOLF/Ei macrophages was mapped to the Naip5 region as well, indicating that the originally predicted change in the A/J Naip5 allele may not be critical for restriction. In the product of the A/J Naip5 permissive allele, there are four unique amino acid changes that map to a NACHT-like domain. Similar misregulating mutations have been identified in the NACHT domains of Nod-like receptor (NLR) proteins. Therefore, one of these mutations may be critical for restriction of L. pneumophila intracellular growth, and this parallels results found with human NLR variants with defects in the innate immune response.

NF-kappaB translocation prevents host cell death after low-dose challenge by Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, grows within macrophages and manipulates target cell signaling. Formation of a Legionella-containing replication vacuole requires the function of the bacterial type IV secretion system (Dot/Icm), which transfers protein substrates into the host cell cytoplasm. A global microarray analysis was used to examine the response of human macrophage-like U937 cells to low-dose infections with L. pneumophila. The most striking change in expression was the Dot/Icm-dependent up-regulation of antiapoptotic genes positively controlled by the transcriptional regulator nuclear factor kappaB (NF-kappaB). Consistent with this finding, L. pneumophila triggered nuclear localization of NF-kappaB in human and mouse macrophages in a Dot/Icm-dependent manner. The mechanism of activation at low-dose infections involved a signaling pathway that occurred independently of the Toll-like receptor adaptor MyD88 and the cytoplasmic sensor Nod1. In contrast, high multiplicity of infection conditions caused a host cell response that masked the unique Dot/Icm-dependent activation of NF-kappaB. Inhibition of NF-kappaB translocation into the nucleus resulted in premature host cell death and termination of bacterial replication. In the absence of one antiapoptotic protein, plasminogen activator inhibitor-2, host cell death increased in response to L. pneumophila infection, indicating that induction of antiapoptotic genes is critical for host cell survival.

Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system.

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.