Convergent insulin and TGF-ß signalling drives cancer cachexia by promoting aberrant fat body ECM accumulation in a Drosophila tumour model.

In this study, we found that in the adipose tissue of wildtype animals, insulin and TGF-ß signalling converge via a BMP antagonist short gastrulation (sog) to regulate ECM remodelling. In tumour bearing animals, Sog also modulates TGF-ß signalling to regulate ECM accumulation in the fat body. TGF-ß signalling causes ECM retention in the fat body and subsequently depletes muscles of fat body-derived ECM proteins. Activation of insulin signalling, inhibition of TGF-ß signalling, or modulation of ECM levels via SPARC, Rab10 or Collagen IV in the fat body, is able to rescue tissue wasting in the presence of tumour. Together, our study highlights the importance of adipose ECM remodelling in the context of cancer cachexia.

Autocrine IL-6 drives cell and extracellular matrix anisotropy in scar fibroblasts.

Fibrosis is associated with dramatic changes in extracellular matrix (ECM) architecture of unknown etiology. Here we exploit keloid scars as a paradigm to understand fibrotic ECM organization. We reveal that keloid patient fibroblasts uniquely produce a globally aligned ECM network in 2-D culture as observed in scar tissue. ECM anisotropy develops after rapid initiation of a fibroblast supracellular actin network, suggesting that cell alignment initiates ECM patterning. Keloid fibroblasts produce elevated levels of IL-6, and autocrine IL-6 production is both necessary and sufficient to induce cell and ECM alignment, as evidenced by ligand stimulation of normal dermal fibroblasts and treatment of keloid fibroblasts with the function blocking IL-6 receptor monoclonal antibody, tocilizumab. Downstream of IL-6, supracellular organization of keloid fibroblasts is controlled by activation of cell-cell adhesion. Adhesion formation inhibits contact-induced cellular overlap leading to nematic organization of cells and an alignment of focal adhesions. Keloid fibroblasts placed on isotropic ECM align the pre-existing matrix, suggesting that focal adhesion alignment leads to active anisotropic remodeling. These results show that IL-6-induced fibroblast cooperativity can control the development of a nematic ECM, highlighting both IL-6 signaling and cell-cell adhesions as potential therapeutic targets to inhibit this common feature of fibrosis.

Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration.

Cell migration is important for development and its aberrant regulation contributes to many diseases. The Scar/WAVE complex is essential for Arp2/3 mediated lamellipodia formation during mesenchymal cell migration and several coinciding signals activate it. However, so far, no direct negative regulators are known. Here we identify Nance-Horan Syndrome-like 1 protein (NHSL1) as a direct binding partner of the Scar/WAVE complex, which co-localise at protruding lamellipodia. This interaction is mediated by the Abi SH3 domain and two binding sites in NHSL1. Furthermore, active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1. Surprisingly, NHSL1 inhibits cell migration through its interaction with the Scar/WAVE complex. Mechanistically, NHSL1 may reduce cell migration efficiency by impeding Arp2/3 activity, as measured in cells using a Arp2/3 FRET-FLIM biosensor, resulting in reduced F-actin density of lamellipodia, and consequently impairing the stability of lamellipodia protrusions.

Rapid Homeostatic Turnover of Embryonic ECM during Tissue Morphogenesis.

The extracellular matrix (ECM) is a polymer network hypothesized to form a stable cellular scaffold. While the ECM can undergo acute remodeling during embryogenesis, it is experimentally difficult to determine whether basal turnover is also important. Most studies of homeostatic turnover assume an initial steady-state balance of production and degradation and measure half-life by quantifying the rate of decay after experimental intervention (e.g., pulse labeling). Here, we present an intervention-free approach to mathematically model basal ECM turnover during embryogenesis by exploiting our ability to live image de novo ECM development in Drosophila to quantify production from initiation to homeostasis. This reveals rapid turnover (half-life ∼7-10 h), which we confirmed by in vivo pulse-chase experiments. Moreover, ECM turnover is partially dependent on proteolysis and network interactions, and slowing turnover affects tissue morphogenesis. These data demonstrate that embryonic ECM undergoes constant replacement, which is likely necessary to maintain network plasticity to accommodate growth and morphogenesis.

Persistent and polarized global actin flow is essential for directionality during cell migration.

Cell migration is hypothesized to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here, we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This approach reveals that edge fluctuations during random motility are not persistent and are weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organization and asymmetry in the cell-wide flowfield that strongly correlates with cell directionality. This organization is regulated by a gradient of actin network compression and destruction, which is controlled by myosin contraction and cofilin-mediated disassembly. It is this stable actin-flow polarity, which integrates rapid fluctuations of the leading edge, that controls inherent cellular persistence.

Heterotypic contact inhibition of locomotion can drive cell sorting between epithelial and mesenchymal cell populations.

Interactions between different cell types can induce distinct contact inhibition of locomotion (CIL) responses that are hypothesised to control population-wide behaviours during embryogenesis. However, our understanding of the signals that lead to cell-type specific repulsion and the precise capacity of heterotypic CIL responses to drive emergent behaviours is lacking. Using a new model of heterotypic CIL, we show that fibrosarcoma cells, but not fibroblasts, are actively repelled by epithelial cells in culture. We show that knocking down EphB2 or ERK in fibrosarcoma cells specifically leads to disruption of the repulsion phase of CIL in response to interactions with epithelial cells. We also examine the population-wide effects when these various cell combinations are allowed to interact in culture. Unlike fibroblasts, fibrosarcoma cells completely segregate from epithelial cells and inhibiting their distinct CIL response by knocking down EphB2 or ERK family proteins also disrupts this emergent sorting behaviour. These data suggest that heterotypic CIL responses, in conjunction with processes such as differential adhesion, may aid the sorting of cell populations.

Tools Allowing Independent Visualization and Genetic Manipulation of Drosophila melanogaster Macrophages and Surrounding Tissues.

Drosophila melanogaster plasmatocytes, the phagocytic cells among hemocytes, are essential for immune responses, but also play key roles from early development to death through their interactions with other cell types. They regulate homeostasis and signaling during development, stem cell proliferation, metabolism, cancer, wound responses, and aging, displaying intriguing molecular and functional conservation with vertebrate macrophages. Given the relative ease of genetics in Drosophila compared to vertebrates, tools permitting visualization and genetic manipulation of plasmatocytes and surrounding tissues independently at all stages would greatly aid a fuller understanding of these processes, but are lacking. Here, we describe a comprehensive set of transgenic lines that allow this. These include extremely brightly fluorescing mCherry-based lines that allow GAL4-independent visualization of plasmatocyte nuclei, the cytoplasm, or the actin cytoskeleton from embryonic stage 8 through adulthood in both live and fixed samples even as heterozygotes, greatly facilitating screening. These lines allow live visualization and tracking of embryonic plasmatocytes, as well as larval plasmatocytes residing at the body wall or flowing with the surrounding hemolymph. With confocal imaging, interactions of plasmatocytes and inner tissues can be seen in live or fixed embryos, larvae, and adults. They permit efficient GAL4-independent Fluorescence-Activated Cell Sorting (FACS) analysis/sorting of plasmatocytes throughout life. To facilitate genetic studies of reciprocal signaling, we have also made a plasmatocyte-expressing QF2 line that, in combination with extant GAL4 drivers, allows independent genetic manipulation of both plasmatocytes and surrounding tissues, and GAL80 lines that block GAL4 drivers from affecting plasmatocytes, all of which function from the early embryo to the adult.

A Moving Source of Matrix Components Is Essential for De Novo Basement Membrane Formation.

The basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3-5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6-9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection.

Mycobacterium tuberculosis remains a global threat to human health, yet the molecular mechanisms regulating immunity remain poorly understood. Cytokines can promote or inhibit mycobacterial survival inside macrophages and the underlying mechanisms represent potential targets for host-directed therapies. Here we show that cytokine-STAT signalling promotes mycobacterial survival within macrophages by deregulating lipid droplets via ATG2 repression. In Drosophila infected with Mycobacterium marinum, mycobacterium-induced STAT activity triggered by unpaired-family cytokines reduces Atg2 expression, permitting deregulation of lipid droplets. Increased Atg2 expression or reduced macrophage triglyceride biosynthesis, normalizes lipid deposition in infected phagocytes and reduces numbers of viable intracellular mycobacteria. In human macrophages, addition of IL-6 promotes mycobacterial survival and BCG-induced lipid accumulation by a similar, but probably not identical, mechanism. Our results reveal Atg2 regulation as a mechanism by which cytokines can control lipid droplet homeostasis and consequently resistance to mycobacterial infection in Drosophila.

Mechanisms and in vivo functions of contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a process whereby a cell ceases motility or changes its trajectory upon collision with another cell. CIL was initially characterized more than half a century ago and became a widely studied model system to understand how cells migrate and dynamically interact. Although CIL fell from interest for several decades, the scientific community has recently rediscovered this process. We are now beginning to understand the precise steps of this complex behaviour and to elucidate its regulatory components, including receptors, polarity proteins and cytoskeletal elements. Furthermore, this process is no longer just in vitro phenomenology; we now know from several different in vivo models that CIL is essential for embryogenesis and in governing behaviours such as cell dispersion, boundary formation and collective cell migration. In addition, changes in CIL responses have been associated with other physiological processes, such as cancer cell dissemination during metastasis.

A direct interaction between fascin and microtubules contributes to adhesion dynamics and cell migration.

Fascin is an actin-binding and bundling protein that is highly upregulated in most epithelial cancers. Fascin promotes cell migration and adhesion dynamics in vitro and tumour cell metastasis in vivo. However, potential non-actin bundling roles for fascin remain unknown. Here, we show for the first time that fascin can directly interact with the microtubule cytoskeleton and that this does not depend upon fascin-actin bundling. Microtubule binding contributes to fascin-dependent control of focal adhesion dynamics and cell migration speed. We also show that fascin forms a complex with focal adhesion kinase (FAK, also known as PTK2) and Src, and that this signalling pathway lies downstream of fascin-microtubule association in the control of adhesion stability. These findings shed light on new non actin-dependent roles for fascin and might have implications for the design of therapies to target fascin in metastatic disease.

Inter-cellular forces orchestrate contact inhibition of locomotion.

Contact inhibition of locomotion (CIL) is a multifaceted process that causes many cell types to repel each other upon collision. During development, this seemingly uncoordinated reaction is a critical driver of cellular dispersion within embryonic tissues. Here, we show that Drosophila hemocytes require a precisely orchestrated CIL response for their developmental dispersal. Hemocyte collision and subsequent repulsion involves a stereotyped sequence of kinematic stages that are modulated by global changes in cytoskeletal dynamics. Tracking actin retrograde flow within hemocytes in vivo reveals synchronous reorganization of colliding actin networks through engagement of an inter-cellular adhesion. This inter-cellular actin-clutch leads to a subsequent build-up in lamellar tension, triggering the development of a transient stress fiber, which orchestrates cellular repulsion. Our findings reveal that the physical coupling of the flowing actin networks during CIL acts as a mechanotransducer, allowing cells to haptically sense each other and coordinate their behaviors.

Fascin promotes filopodia formation independent of its role in actin bundling.

Fascin is an evolutionarily conserved actin-binding protein that plays a key role in forming filopodia. It is widely thought that this function involves fascin directly bundling actin filaments, which is controlled by an N-terminal regulatory serine residue. In this paper, by studying cellular processes in Drosophila melanogaster that require fascin activity, we identify a regulatory residue within the C-terminal region of the protein (S289). Unexpectedly, although mutation (S289A) of this residue disrupted the actin-bundling capacity of fascin, fascin S289A fully rescued filopodia formation in fascin mutant flies. Live imaging of migrating macrophages in vivo revealed that this mutation restricted the localization of fascin to the distal ends of filopodia. The corresponding mutation of human fascin (S274) similarly affected its interaction with actin and altered filopodia dynamics within carcinoma cells. These data reveal an evolutionarily conserved role for this regulatory region and unveil a function for fascin, uncoupled from actin bundling, at the distal end of filopodia.

Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo.

Drosophila melanogaster macrophages are highly migratory cells that lend themselves beautifully to high resolution in vivo imaging experiments. By expressing fluorescent probes to reveal actin and microtubules, we can observe the dynamic interplay of these two cytoskeletal networks as macrophages migrate and interact with one another within a living organism. We show that before an episode of persistent motility, whether responding to developmental guidance or wound cues, macrophages assemble a polarized array of microtubules that bundle into a compass-like arm that appears to anticipate the direction of migration. Whenever cells collide with one another, their microtubule arms transiently align just before cell-cell repulsion, and we show that forcing depolymerization of microtubules by expression of Spastin leads to their defective polarity and failure to contact inhibit from one another. The same is true in orbit/clasp mutants, indicating a pivotal role for this microtubule-binding protein in the assembly and/or functioning of the microtubule arm during polarized migration and contact repulsion.

Prioritization of competing damage and developmental signals by migrating macrophages in the Drosophila embryo.

The function of immune cells is critically dependent on their capacity to respond to a complex series of navigational cues that enable them to home to various organ sites in the body or to respond to inflammatory cues such as those released at sites of tissue damage. From early embryonic stages, immune cells are faced with a barrage of signals that will not all be directing the cell to do the same thing. Here we use the Drosophila embryo to investigate how hemocytes (Drosophila macrophages), are able to prioritize key guidance signals and ignore others so that they are not pulled every which way. We identify the immediate wound attractant signal as H(2)O(2) and investigate how Drosophila macrophages respond to competing guidance cues-those emanating from a wound-versus standard developmental guidance cues, as well as those signals drawing cells toward neighboring dying cells. We reveal a hierarchy of responsiveness to attractant cues that varies over time and we identify why there is a wound refractile period early in embryonic development when macrophages cannot be distracted from their developmental migratory pathway to a site of tissue damage.

Fascin is required for blood cell migration during Drosophila embryogenesis.

Fascin is well characterized in vitro as an actin-bundling protein and its increased expression is correlated with the invasiveness of various cancers. However, the actual roles and regulation of Fascin in vivo remain elusive. Here we show that Fascin is required for the invasive-like migration of blood cells in Drosophila embryos. Fascin expression is highly regulated during embryonic development and, within the blood lineage, is specific to the motile subpopulation of cells, which comprises macrophage-like plasmatocytes. We show that Fascin is required for plasmatocyte migration, both as these cells undergo developmental dispersal and during an inflammatory response to epithelial wounding. Live analyses further demonstrate that Fascin localizes to, and is essential for the assembly of, dynamic actin-rich microspikes within plasmatocyte lamellae that polarize towards the direction of migration. We show that a regulatory serine of Fascin identified from in vitro studies is not required for in vivo cell motility, but is crucial for the formation of actin bundles within epithelial bristles. Together, these results offer a first glimpse into the mechanisms regulating Fascin function during normal development, which might be relevant for understanding the impact of Fascin in cancers.

Gene induction following wounding of wild-type versus macrophage-deficient Drosophila embryos.

By using a microarray screen to compare gene responses after sterile laser wounding of wild-type and 'macrophageless' serpent mutant Drosophila embryos, we show the wound-induced programmes that are independent of a pathogenic response and distinguish which of the genes are macrophage dependent. The evolutionarily conserved nature of this response is highlighted by our finding that one such new inflammation-associated gene, growth arrest and DNA damage-inducible gene 45 (GADD45), is upregulated in both Drosophila and murine repair models. Comparison of unwounded wild-type and serpent mutant embryos also shows a portfolio of 'macrophage-specific' genes, which suggest analogous functions with vertebrate inflammatory cells. Besides identifying the various classes of wound- and macrophage-related genes, our data indicate that sterile injury per se, in the absence of pathogens, triggers induction of a 'pathogen response', which might prime the organism for what is likely to be an increased risk of infection.

How the cornea heals: cornea-specific repair mechanisms affecting surgical outcomes.

In mammals, penetrating injuries typically heal by deposition of fibrotic "repair tissue" that fills and seals wounds but does not restore normal function. Excessive deposition of fibrotic repair tissue can lead to pathologies involving excessive scarring and contracture. In the cornea, fibrotic repair presents special challenges affecting both clarity and shape of the cornea. With the increasing popularity of surgical techniques that alter corneal refractive errors, understanding of cornea repair mechanisms has acquired new significance. The cornea has unique anatomic, cellular, molecular, and functional features that lead to important mechanistic differences in the process of repair in comparison with what occurs in skin and other organs. Moreover, corneal function calls for special outcomes. This review addresses these features from the viewpoint of the authors' research on factors of importance to understanding and improving surgical outcomes.

Activity and expression of Xenopus laevis matrix metalloproteinases: identification of a novel role for the hormone prolactin in regulating collagenolysis in both amphibians and mammals.

Prolactin (PRL) has long been implicated in Xenopus metamorphosis as an anti-metamorphic and/or juvenilizing hormone. Numerous studies showed that PRL could prevent effects of either endogenous or exogenous thyroid hormone (TH; T(3)). It has been shown that expression of matrix metalloproteinases (MMPs) is induced by TH during Xenopus metamorphosis. Direct in vivo evidence, however, for such anti-TH effects by PRL with respect to MMPs has not been available for the early phase of Xenopus development or metamorphosis. To understand the functional role of PRL, we investigated effects of PRL on Xenopus collagenase-3 (XCL3) and collagenase-4 (XCL4) expression in a cultured Xenopus laevis cell line, XL-177. Northern blot analysis demonstrated that XCL3 and XCL4 expression were not detected in control or T(3)-treated cells, but were differentially induced by PRL in a dose- and time-dependent fashion. Moreover, treatment with IL-1alpha as well as phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, or H8, a protein kinase A (PKA) inhibitor, augmented PRL-induced collagenase expression, suggesting that multiple protein kinase pathways and cytokines may participate in PRL-induced collagenase expression. Interestingly, XCL3 expression could be induced in XL-177 cells by T(3), but only when co-cultured with prometamorphic Xenopus tadpole tails (stage 54/55), suggesting that the tails secrete a required intermediate signaling molecule(s) for T(3)-induced XCL3 expression. Taken together, these data demonstrate that XCL3 and XCL4 can be differentially induced by PRL and T(3) and further suggest that PRL is a candidate regulator of TH-independent collagenase expression during the organ/tissue remodeling which occurs in Xenopus development.

Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes.

PURPOSE: Incisional or ablation injury to the corneal stroma is repaired by deposition of a fibrotic tissue produced by activated keratocytes, whereas cells lost from the underlying stroma after epithelial abrasion are simply replaced by keratocyte replication without expression of fibrotic markers. The purpose of this study was to investigate mechanisms that determine this differential keratocyte response. METHODS: A penetrating keratectomy rabbit model was adapted for mice to study the fibrotic repair response. A mouse epithelial abrasion model was applied to study the stromal cell replacement response. A primary rabbit corneal cell culture model and an organotypic culture model were also used. RESULTS: When the epithelium was prevented from resurfacing the cornea after penetrating keratectomy, expression of fibrotic markers was considerably reduced. TGF-beta2 was determined to be a major substance produced by corneal epithelial cells capable of inducing the fibrotic phenotype. In the intact mouse cornea, TGF-beta2 was confined to the uninjured epithelium, but was released into the stroma during fibrotic repair. By contrast, TGF-beta1 was never found in the epithelium. When epithelial cells were cultured on a basement-membrane-like gel or allowed to deposit their own basement membrane in organotypic culture, TGF-beta2 production was reduced. Return of a basement membrane after wounding in vivo correlated with loss of the fibrotic phenotype. In the epithelial debridement injury model in which the basement membrane was left intact, TGF-beta2 remained confined to the corneal epithelium, consistent with the absence of a fibrotic phenotype. CONCLUSIONS: These data suggest that integrity of the basement membrane is a deciding factor in determining the regenerative character of corneal repair.