Basement membrane dynamics in living animals: Insights and pitfalls.

Recent studies with fluorophore-tagged basement membrane (BM) components have led to remarkable discoveries about BMs but also inconsistent interpretations. Here, we review types of BM dynamics, discuss how we conduct and interpret fluorophore-tagged BM studies, and highlight experimental conditions that are important to consider.

The extracellular matrix in tissue morphogenesis: No longer a backseat driver.

The forces driving tissue morphogenesis are thought to originate from cellular activities. While it is appreciated that extracellular matrix (ECM) may also be involved, ECM function is assumed to be simply instructive in modulating the cellular behaviors that drive changes to tissue shape. However, there is increasing evidence that the ECM may not be the passive player portrayed in developmental biology textbooks. In this review we highlight examples of embryonic ECM dynamics that suggest cell-independent activity, along with developmental processes during which localized ECM alterations and ECM-autonomous forces are directing changes to tissue shape. Additionally, we discuss experimental approaches to unveil active ECM roles during tissue morphogenesis. We propose that it may be time to rethink our general definition of morphogenesis as a cellular-driven phenomenon and incorporate an underappreciated, and surprisingly dynamic ECM.

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.

Extracellular matrix assembly stress initiates Drosophila central nervous system morphogenesis.

Forces controlling tissue morphogenesis are attributed to cellular-driven activities, and any role for extracellular matrix (ECM) is assumed to be passive. However, all polymer networks, including ECM, can develop autonomous stresses during their assembly. Here, we examine the morphogenetic function of an ECM before reaching homeostatic equilibrium by analyzing de novo ECM assembly during Drosophila ventral nerve cord (VNC) condensation. Asymmetric VNC shortening and a rapid decrease in surface area correlate with the exponential assembly of collagen IV (Col4) surrounding the tissue. Concomitantly, a transient developmentally induced Col4 gradient leads to coherent long-range flow of ECM, which equilibrates the Col4 network. Finite element analysis and perturbation of Col4 network formation through the generation of dominant Col4 mutations that affect assembly reveal that VNC morphodynamics is partially driven by a sudden increase in ECM-driven surface tension. These data suggest that ECM assembly stress and associated network instabilities can actively participate in tissue morphogenesis.

A workflow for rapid unbiased quantification of fibrillar feature alignment in biological images.

Measuring the organisation of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fibre segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT-Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighbourhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighbourhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighbourhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

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.

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.

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.

Cells on film - the past and future of cinemicroscopy.

Movie making is now a ubiquitous experimental tool that biologists use alongside more traditional techniques such as molecular biology and biochemistry. It is no longer just cell biologists, but scientists from many other disciplines, such as immunology and neuroscience, that utilise movies to dissect their processes of interest. When did filming become such a standard laboratory technique? Who developed the use of the movie as an experimental tool? The Wellcome Library has recently restored and digitized a number of original 16-mm films from two pioneering cinemicroscopists, Ronald Canti and Michael Abercrombie, which are now freely available to the scientific community. In light of these films, this Essay will give a brief history of the early cinemicroscopists and discuss what is driving the use of movies in the laboratory today.

Unraveling tissue repair immune responses in flies.

Drosophila melanogaster has emerged as a powerful model to understand innate immune responses to infection (note the 2011 Nobel Prize in Physiology or Medicine), and in recent years this system has begun to inform on the role and regulation of immune responses during tissue injury. Due to the speed and complexity of inflammation signals upon damage, a complete understanding of the immune responses during repair requires a combination of live imaging at high temporal resolution and genetic dissection, which is possible in a number of different injury models in the fly. Here we discuss the range of wound-induced immune responses that can be modeled in flies. These wound models have revealed the most immediate signals leading to immune cell activation, and highlighted a number of complex signaling cascades required for subsequent injury-associated inflammatory responses. What has emerged from this system are a host of both local acting signals, and surprisingly, more systemic tissue repair immune responses.

Emergence of embryonic pattern through contact inhibition of locomotion.

The pioneering cell biologist Michael Abercrombie first described the process of contact inhibition of locomotion more than 50 years ago when migrating fibroblasts were observed to rapidly change direction and migrate away upon collision. Since then, we have gleaned little understanding of how contact inhibition is regulated and only lately observed its occurrence in vivo. We recently revealed that Drosophila macrophages (haemocytes) require contact inhibition for their uniform embryonic dispersal. Here, to investigate the role that contact inhibition plays in the patterning of haemocyte movements, we have mathematically analysed and simulated their contact repulsion dynamics. Our data reveal that the final pattern of haemocyte distribution, and the details and timing of its formation, can be explained by contact inhibition dynamics within the geometry of the Drosophila embryo. This has implications for morphogenesis in general as it suggests that patterns can emerge, irrespective of external cues, when cells interact through simple rules of contact repulsion.

Live imaging of Drosophila melanogaster embryonic hemocyte migrations.

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

The inflammation-fibrosis link? A Jekyll and Hyde role for blood cells during wound repair.

The healing of a skin wound is a complex process involving many cell lineages. In adult tissues, repair is always accompanied by a robust inflammatory response, which is necessary to counter the potential for infection at any site where the skin barrier is breached. Unlike embryonic tissues that can repair perfectly without a remnant scar at the wound site, adult tissue repair always leads to formation of a fibrotic scar where the wound has healed. In recent years, it has become clear that the wound inflammatory response may be, at least in part, responsible for fibrosis at sites of tissue repair. In this review, we consider the beneficial vs the detrimental functions of inflammatory cells during the repair response and compare data from other tissues, the lung, and liver, where fibrosis and its resolution may be related to a damage-triggered inflammatory response. We also consider how it may be possible to molecularly disentangle the potentially good from the bad influences of inflammatory cells during tissue repair and how fundamental studies in inflammatory cell biology may prove the way forward for development of drug targets in this respect.

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.

Selective reduction of fibrotic markers in repairing corneas of mice deficient in Smad3.

The cytokine transforming growth factor-beta (TGF-beta) is a key mediator of fibrosis in all organs. Expression of fibrotic markers in repairing cutaneous wounds is reduced in mice lacking Smad3, a downstream cytoplasmic mediator of TGF-beta signaling (Ashcroft et al., 1999, Nat Cell Biol 1(5):260-266). This is correlated with a reduction in inflammation, and thus in the blood elements thought to be a significant source of TGF-beta at the wound site, the principle form being TGF-beta1. Since the major cellular source of TGF-beta in corneal wounds is the epithelium, and the principal isoform is TGF-beta2, we investigated whether Smad3 deficiency has similar anti-fibrotic effects on corneal repair. In contrast to the situation of cutaneous repair, expression of the fibrotic marker, fibronectin, was equivalent in corneal repair tissue of Smad3-/- mice as compared to their +/- littermates, even though expression of a second fibrotic marker not previously examined in cutaneous wounds, alpha-smooth muscle (sm) actin, was reduced. Also unlike in cutaneous wounds, the inflammatory response was unaffected. These differences between corneal and cutaneous repair correlated with the lack of apparent change in the levels of corneal TGF-beta2. There was a significant reduction of alpha-sm actin expression in stromal cell cultures established from Smad3-/- mice as compared to their +/- littermates, but the rate of cell proliferation stimulated by TGF-beta, as well as expression of fibronectin, was unaffected. Therefore, a deficiency in Smad3 has different effects on corneal and cutaneous repair, probably due to the difference in cellular source and principal isoform of the TGF-beta involved.

Uncoupling keratocyte loss of corneal crystallin from markers of fibrotic repair.

PURPOSE: Corneal crystallins are lost from resident cells of the corneal stroma during wound repair, and this is associated with a loss of cell transparency. The goal of this study was to identify factors inducing loss of the corneal crystallin transketolase (TKT). METHODS: A cell culture model of freshly isolated rabbit corneal keratocytes was used. Fibrotic markers included cell proliferation, adoption of a "fibroblastic" spindle-shaped morphology associated with cytoskeletal rearrangement, loss of TKT, and expression of alpha-smooth muscle actin (alpha-sm actin), a marker for the myofibroblast. RESULTS: When freshly isolated keratocytes were cultured in the continuous presence of 10% calf serum, the high level of intracellular TKT protein was reduced dramatically within 24 to 48 hours. In contrast, TKT protein was retained in cells maintained in the absence of serum. When cells were prevented from proliferating by exposure to serum for <24 hours or by continuously exposing to serum at a contact-inhibiting plating density, TKT loss was inhibited. TKT loss was induced by treatment of serum-free cultures with the serum cytokines platelet-derived growth factor or basic fibroblast growth factor, both of which also stimulated keratocyte proliferation, although not other changes associated with fibrosis. However, TKT loss was not induced by treatment of serum-free cultures with a third serum cytokine, transforming growth factor- (TGF)-beta, even though TGF-beta stimulated cell proliferation at low doses and induced the fibroblastic spindle-shape and express alpha-sm actin at high doses. CONCLUSIONS: TKT loss in corneal keratocytes can be induced by PDGF or bFGF and this loss can be uncoupled from other fibrotic markers. Targeting these cytokines or the signaling pathways that they activate could enable retention of corneal crystallin in stromal cells during repair, a more regenerative outcome. The result would be enhanced clarity of the cornea.