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Graves’ orbitopathy (GO) is an autoimmune inflammatory disease affecting the orbit. Orbital fibroblasts are a key component in GO pathogenesis, which includes inflammation, adipogenesis, hyaluronic acid (HA) secretion, and fibrosis. Macrophages are thought to participate in the immunological stage of GO, but whether they can directly affect the fibroblasts phenotype and modulate disease progression is unknown. We previously showed that GO adipogenic and fibrotic phenotypes could be modelled in a pseudo-physiological 3D environment in vitro. Here, we introduced macrophages in this 3D culture model to investigate role for macrophages in modulating adipogenesis, HA production, and contractility in orbital fibroblasts. Macrophages had a minimal effect on lipid droplet formation in fibroblasts, but significantly increased HA production and cell contractility, suggesting that they may promote the fibrotic phenotype. This effect was found to be mediated at least in part through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activation and linked to an increase in actin polymerization and protrusive activity in fibroblasts. Overall our work shows for the first time a direct role for macrophages in modulating the fibroblasts’ phenotype in GO, supporting a role for macrophages in the progression of the fibrotic phenotype through induction of HA production and stimulation of the contractile phenotype in orbital fibroblasts.

© The Author(s) 2020., Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm., This work was supported by a Medical Research Council New Investigator Award to A.N.H. (grant reference G1100063), Wellcome Trust Grant Awards (grant reference 108429/Z/15/Z to E.V.S. and 204843/Z/16/Z to A.N.H.), a Biotechnology and Biological Sciences Research Council (grant reference BB/F016956/1) and University of Nottingham funded PhD studentship awarded to C.L.R. D.A.B. was funded by Biochemical Society Vacation Studentship. L.M. is funded through the Spanish Ministry of Economy. Industry and Competitiveness (MINECO) [BIO2015-70978-R]. E.K. and T.W. are funded by DFG SPP1464, KE 447/10-2” and DFG KE 447/18-1. F.S. is funded by DFG Research Training Group, GRK 2174 Award. N.A. is supported by a PhD studentship from Taibah University, Medina, Kingdom of Saudi Arabia. W.W.K. and E.W.T. are funded by The Royal Society (Newton International Fellowship grant NF161582 to W.W.K.), European Commission (Marie Sklodowska Curie Individual Fellowship grant 752165 to W.W.K.), and Cancer Research UK (C29637/A20183 to E.W.T.). A.K.M. acknowledges financial support of the John and Lucille van Geest Foundation.

Purpose The purpose of this study was to develop a topical matrix metalloproteinase inhibitor preparation for antiscarring therapy. Methods The broad spectrum matrix metalloproteinase inhibitor ilomastat was formulated using 2-hydroxypropyl-β-cyclodextrin in aqueous solution. In vitro activity of ilomastat-cyclodextrin (ilomastat-CD) was examined using fibroblasts seeded in collagen. Permeation of ilomastat-CD eye drop through pig eye conjunctiva was confirmed using Franz diffusion cells. Ilomastat-CD eye drop was applied to rabbit eyes in vivo, and the distribution of ilomastat in ocular tissues and fluids was determined by liquid chromatography-mass spectroscopy. Results The aqueous solubility of ilomastat-CD was ∼1000 μg/mL in water and 1400 μg/mL in PBS (pH 7.4), which is greater than ilomastat alone (140 and 160 μg/mL in water and PBS, respectively). The in vitro activity of ilomastat-CD to inhibit collagen contraction in the presence of human Tenon fibroblast cells was unchanged compared to uncomplexed ilomastat. Topically administered ilomastat-CD in vivo to rabbit eyes resulted in a therapeutic concentration of ilomastat being present in the sclera and conjunctiva and within the aqueous humor. Conclusions Ilomastat-CD has the potential to be formulated as an eye drop for use as an antifibrotic, which may have implications for the prevention of scarring in many settings, for example glaucoma filtration surgery.

Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that motor myosin-Va, works with dynamic actin tracks, to drive long-range organelle dispersion in microtubule depleted cells. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here we show that SPIRE1/2 and formin-1 (FMN1) proteins generate actin tracks required for myosin-Va-dependent transport in melanocytes. Moreover we show that, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIRE1/2 to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this we suggest a model in which organelles and force generators (motors and track assemblers) are linked forming a cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm.

The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway represents a promising therapeutic target to prevent fibrosis. We have tested the effects of new pharmacological inhibitors of MRTF/SRF signalling in a preclinical model of fibrosis. CCG-222740, a novel MRTF/SRF inhibitor, markedly decreased SRF reporter gene activity and showed a greater inhibitory effect on MRTF/SRF target genes than the previously described MRTF-A inhibitor CCG-203971. CCG-222740 was also five times more potent, with an IC50 of 5 μM, in a fibroblast-mediated collagen contraction assay, was less cytotoxic, and a more potent inhibitor of alpha-smooth muscle actin protein expression than CCG-203971. Local delivery of CCG-222740 and CCG-203971 in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery increased the long-term success of the surgery by 67% (P

Trachoma is a conjunctiva scarring disease, which is the leading infectious cause of blindness worldwide. Yet, the molecular mechanisms underlying progressive fibrosis in trachoma are unknown. To investigate the contribution of local resident fibroblasts to disease progression, we isolated conjunctival fibroblasts from patients with scarring trachoma and matching control individuals, and compared their gene expression profiles and functional properties in vitro. We show that scarring trachoma fibroblasts substantially differ from control counterparts, displaying pro-fibrotic and pro-inflammatory features matched by an altered gene expression profile. This pro-inflammatory signature was exemplified by increased IL-6 expression and secretion, and a stronger response to macrophage-mediated stimulation of contraction. We further demonstrate that scarring trachoma fibroblasts can promote Akt phosphorylation in macrophages in an IL-6 -dependent manner. Overall this work has uncovered a distinctive molecular fingerprint for scarring trachoma fibroblasts, and identified IL-6- as a potential contributor to the chronic conjunctival fibrosis, mediating reciprocal pro-fibrotic/pro-inflammatory interactions between macrophages and fibroblasts.

Here, we show that the local incorporation of osmotically active hyaluronan into previously compressed collagen constructs results in further rapid dehydration/compression of collagen layers, channel formation and generation of new interfaces; these novel structures, at the nano–micro (i.e. meso-scale) were formed within native collagen gels, in a highly predictable spatial manner and offer important new methods of fabricating scaffolds (e.g. tubes and open-spirals) with potential for use in tissue regeneration such as in peripheral nerves and small vessels. This paper tests the possibility that the local fluid content of a dense collagen network can be controlled by incorporation of an osmotically active (native) macromolecule—hyluronan. This is an exemplar physiological, osmotic swelling agent. Hyaluronan is commonly secreted by cells deep in connective tissues, so is a good candidate for this role in a cell-driven system balancing mechanical compaction of bulk tissue collagen. These constructs may have potential as functionalin vitromodels representing developmental and pathological processes.

Background: The aims of this study are to investigate the expression of the main structural components of the tarsal extracellular matrix (ECM) in floppy eyelid syndrome (FES) focusing on elastic fibres and collagen types I and III, and also to identify possible cell-mediated inflammatory mechanisms in the pathogenesis of this condition. Methods: A histopathological case control study was conducted using 30 upper lid specimens from patients with FES and 15 undiseased upper lid control specimens. Structural ECM components were assessed using a combination of immunctorial ataining ohistochemical and techniques including antibodies to collagens I and III, Verhoeff's iron haematoxylin, Gomori's aldehyde fuchsin and Lillie's oxidised aldehyde fuchsin. The contribution of different cellular components of the inflammatory response was investigated by immunohistochemical techniques using antibodies to CD3, CD20, CD68. Slide scoring was performed using a semiquantitative technique on an ordinal scale. Statistical analysis was performed using matched ordinal regression analysis. Results: FES tarsal plate tissue demonstrated a decreased abundance of mature elastic fibres (P ≤ 0.001) and an increased abundance of oxytalan fibres (P = 0.006). Intensity of staining for collagens I (P = 0.012) and III (P

Although fibroblasts play an essential part during the wound healing response, the mechanisms by which they mediate tissue remodelling and contraction are still unclear. Using live cell and matrix imaging within 3D free-floating fibroblast-populated collagen lattices as a model for tissue contraction, we compared the behaviour of a range of fibroblasts with low and high contraction abilities and analysed the effect of the broad spectrum MMP-inhibitor GM6001 on cell behaviour and matrix contraction. We identified two mechanisms underlying matrix contraction, one via direct cell-mediated contractile activity, the second through matrix degradation. These appear to be linked to cell morphology and regulated by the collagen concentration within the matrix. Cells with a rounded morphology proliferated in the matrix but did not remodel it efficiently, resulting in a poor ability to contract matrices. Cells with an elongated morphology showed higher levels of protrusive activity, leading to efficient matrix remodelling and contraction. GM6001 inhibited week-long matrix contraction to various extents with the different cell lines. However, quantitative analysis of the cell protrusive activity showed that GM6001 consistently decreased cell dynamics in 3D by about 20%, and this was correlated with a significant reduction in early matrix contraction. Overall our results suggest that although fibroblast-mediated matrix contraction depends on both cell dynamics and MMP-mediated matrix degradation, the efficiency of GM6001 treatment in preventing contraction might be linked to a direct effect on cell dynamics.

Nance-Horan syndrome (NHS) is an X-linked developmental disorder, characterized by bilateral congenital cataracts, dental anomalies, facial dysmorphism and mental retardation. Null mutations in a novel gene, NHS, cause the syndrome. The NHS gene appears to have multiple isoforms as a result of alternative transcription, but a cellular function for the NHS protein has yet to be defined. We describe NHS as a founder member of a new protein family (NHS, NHSL1 and NHSL2). Here, we demonstrate that NHS is a novel regulator of actin remodelling and cell morphology. NHS localizes to sites of cell-cell contact, the leading edge of lamellipodia and focal adhesions. The N-terminus of isoforms NHS-A and NHS-1A, implicated in the pathogenesis of NHS, have a functional WAVE homology domain that interacts with the Abi protein family, haematopoietic stem/progenitor cell protein 300 (HSPC300), Nap1 and Sra1. NHS knockdown resulted in the disruption of the actin cytoskeleton. We show that NHS controls cell morphology by maintaining the integrity of the circumferential actin ring and controlling lamellipod formation. NHS knockdown led to a striking increase in cell spreading. Conversely, ectopic overexpression of NHS inhibited lamellipod formation. Remodelling of the actin cytoskeleton and localized actin polymerization into branched actin filaments at the plasma membrane are essential for mediating changes in cell shape, migration and cell contact. Our data identify NHS as a new regulator of actin remodelling. We suggest that NHS orchestrates actin regulatory protein function in response to signalling events during development.

Vesicle rocketing has been used as a model system for understanding the dynamics of the membrane-associated F-actin cytoskeleton, but in many experimental systems is induced by persistent, non-physiological stimuli. Localised changes in the concentration of phosphatidylinositol 4,5-bisphosphate (PI (4,5)P(2)) in membranes stimulate the recruitment of actin-remodelling proteins to their sites of action, regulate their activity and favour vesicle rocketing. The calcium and anionic phospholipid-binding protein annexin A2 is necessary for macropinocytic rocketing and has been shown to bind both PI(4,5)P(2) and the barbed-ends of F-actin filaments. Here we show that annexin A2 localises to the comet tails which form constitutively in fibroblasts from patients with Lowe Syndrome. These fibroblasts are deficient in OCRL1, a phosphatidylinositol polyphosphate 5-phosphatase with specificity for PI(4,5)P(2). We show that upon depletion of annexin A2 from these cells vesicle rocketing is reduced, and that this is also dependent upon PI (4,5)P(2) formation. Annexin A2 co-localised with comet-tails induced by pervanadate and hyperosmotic shock in a basophilic cell line, and in an epithelial cell line upon activation of PKC. In vitro annexin A2 promoted comet formation in a bead-rocketing assay and was sufficient to link F-actin filaments to PI(4,5)P(2) containing vesicles. These observations are consistent with a role for annexin A2 as an actin nucleator on PI (4,5)P(2)-enriched membranes.

The glomerular podocyte is a highly specialized cell, with the ability to ultrafilter blood and support glomerular capillary pressures. However, little is known about either the genetic programs leading to this functionality or the final phenotype. We approached this question utilizing a human conditionally immortalized cell line, which differentiates from a proliferating epithelial phenotype to a differentiated form. We profiled gene expression during several time points during differentiation and grouped the regulated genes into major functional categories. A novel category of genes that was upregulated during differentiation was of smooth muscle-related molecules. We further examined the smooth muscle phenotype and showed that podocytes consistently express the differentiated smooth muscle markers smoothelin and calponin and the specific transcription factor myocardin, both in vitro and in vivo. The contractile contribution of the podocyte to the glomerular capillary is controversial. We demonstrated using two novel techniques that podocytes contract vigorously in vitro when differentiated and in real time were able to demonstrate that angiotensin II treatment decreases monolayer resistance, morphologically correlating with enhanced contractility. We conclude that the mature podocyte in vitro possesses functional apparatus of contractile smooth muscle cells, with potential implications for its in vivo ability to regulate glomerular dynamic and permeability characteristics.

Purpose Glaucoma is the first leading cause of irreversible blindness worldwide and fibrosis is the main cause of failure of glaucoma surgery. There are currently no available anti-fibrotic treatments in the eye. We have previously described how the MRTF/SRF transcription pathway is intricately linked to all the key pathways in ocular fibrosis. We thus hypothesised that inhibiting the MRTF/SRF pathway would be a good therapeutic target to prevent fibrosis in the eye. Methods MRTF and SRF expression were knocked down in human Tenon's fibroblasts using small interfering RNAs, and three-dimensional fibroblast-populated collagen gels were used to measure contraction in vitro. Cytotoxicity was assessed using the live-dead assay. Live cell imaging was used to study the cell protrusive behaviour during contraction and quantification was performed using a custom parameter, the dynamic index. Reflection confocal microscopy and DQ collagen were also used to measure matrix degradation. Real-time qPCR was used to compare the gene expression of key matrix metalloproteinases in contracting gels. Results MRTF and SRF silencing significantly decreased collagen matrix contraction by 80% and 87% respectively. MRTF silencing did not impair cell viability compared to controls. Knocking down MRTF markedly reduced the protrusive behaviour of human Tenon's fibroblasts (dynamic index = 0.19 as compared to 0.66 for controls). MRTF silencing also significantly decreased matrix degradation and the expression of MMP-1, MMP-2, MMP-9, and MMP-14 genes. Conclusions Our study is the first to show that inhibiting the MRTF/SRF pathway significantly decreases collagen matrix contraction, cell protrusive behaviour of fibroblasts, and matrix degradation in the conjunctiva, and thus represents a promising new therapeutic approach to prevent post-surgical fibrosis in glaucoma.

PURPOSE. Graves’ orbitopathy (GO) is a sight-threatening autoimmune disorder causing extraocular muscle fibrosis, upper lid retraction and eye bulging due to orbital fat expansion. These clinical features are mediated by aspects of orbital fibroblasts differentiation, including adipogenesis and fibrosis. Our previous work suggested that this dual phenotype might be a manifestation of mixed cell populations, partially linked to the expression of mesenchymal stem cell (MSC) marker CD90. Thus, we set out to determine whether GO orbital fibroblasts displayed MSC properties. METHODS. Control and GO orbital fibroblasts previously characterized for CD90 and CD45 expression were analyzed by flow cytometry for classical MSC positive (CD73, CD105) and negative (CD14, CD19, HLA-DR, and CD34) markers. Graves’ orbitopathy fibroblasts were tested further for their ability to undergo lineage specific differentiation following standard protocols. RESULTS. Control and GO fibroblasts strongly expressed CD73 and CD105, with a higher percentage of positive cells and stronger expression levels in GO. Neither cell type expresses CD14, CD19, and HLA-DR. Protein CD34 was expressed at low levels by 45% to 70% of the cells, with its expression significantly lower in GO cells. Graves’ orbitopathy fibroblasts displayed features of osteogenesis (calcium deposits, and osteocalcin [BGLAP] and osteonectin [SPARC] expression), chondrogenesis (glycosaminoglycan production; SOX9 and aggrecan [ACAN] expression), myogenesis (α-smooth muscle actin expression), and neurogenesis (β-III tubulin expression) upon differentiation. CONCLUSIONS. Our findings suggest that orbital fibroblasts contain a population of cells that fulfil the criteria defining MSC. This subpopulation may be increased in GO, possibly underlying the complex differentiation phenotype of the disease

BACKGROUND: Adverse skin scarring varies by anatomical site with, for example, presternal skin showing a greater hypertrophic response when compared with eyelid; such differences have traditionally been attributed to regional variations in skin tension, thickness, and Langer's lines. Fibroblasts are the main cell implicated in fibrosis, and they too are known to show anatomical variation in their expression, differentiation, and intercellular interactions. We, therefore, investigated whether intrinsic differences in skin fibroblasts derived from separate locations might contribute to the observed discrepancies in clinical scarring. METHODS: Primary in vitro cultures were established using matched eyelid and presternal skin from 3 healthy donors undergoing blepharoplasty surgery. We used an in vitro collagen gel model of fibroblast-mediated tissue contraction to compare the properties of the dermal fibroblasts from each site. Cell contractile force and matrix stiffness were assessed in 3-dimensional tissue constructs using an automated high-throughput device. RESULTS: Dermal fibroblasts isolated from eyelid and sternum differ both in their ability to contract a gel matrix and in their response to cytokine stimulation; despite having lower intrinsic contractile force (P < 0.01) and resting stiffness (P < 0.02), the presternal cells were more contractile (P < 0.001) following stimulation with serum, or inflammatory cytokines transforming growth factor-β (P < 0.01) and interleukin-1β (P < 0.05). CONCLUSIONS: The propensity to cutaneous scarring may, at least in part, result from intrinsic differences in the local fibroblasts' ability to contract and their sensitivity to inflammatory cytokines. Improved understanding of the underlying molecular pathways should prove useful in identifying new therapeutic targets for altering surgical and other scarring.

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that ``zipper{''} up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.} publishersversion published

The ability of epithelial cells to polarize requires cell-cell adhesion mediated by cadherin receptors. During cell-cell contact, the mechanism via which a flat, spread cell shape is changed into a tall, cuboidal epithelial morphology is not known. We found that cadherin-dependent adhesion modulates actin dynamics by triggering changes in actin organization both locally at junctions and within the rest of the cell. Upon induction of cell-cell contacts, two spatial actin populations are distinguishable: junctional actin and peripheral thin bundles. With time, the relative position of these two populations changes and becomes indistinguishable to form a cortical actin ring that is characteristic of mature, fully polarized epithelial cells. Junctional actin and thin actin bundles differ in their actin dynamics and mechanism of formation, and interestingly, have distinct roles during epithelial polarization. Whereas junctional actin stabilizes clustered cadherin receptors at cell-cell contacts, contraction of peripheral actin bundle is essential for an increase in the maximum height at the lateral domain during polarization (cuboidal morphology). Thus, both junctional actin and thin bundles are necessary, and cooperate with each other to generate a polarized epithelial morphology.

CC chemokines participate in the recruitment and activation of immune cells through CC chemokine receptors (CCRs). Here, we report that cross-talk between CCR1-mediated signaling pathway and FcepsilonRI-mediated signaling pathway affects degranulation positively but affects chemotaxis of mast cells adversely. Costimulation via FcepsilonRI engagement with IgE/antigen and CCR1 engagement with recombinant human CCL3 synergistically enhanced degranulation in rat basophilic leukemia-2H3 cells expressing human CCR1 (RBL-CCR1). Interestingly, FcepsilonRI engagement inhibited CCL3-mediated chemotaxis and membrane ruffling of RBL-CCR1 cells. Small GTP-binding proteins of the Rho family, Rac, Cdc42, and Rho control chemotaxis by mediating the reorganization of the actin cytoskeleton. Both a Rho inhibitor C3 exoenzyme and a Rho kinase (ROCK) inhibitor Y-27632 inhibited chemotaxis of RBL-CCR1 cells toward CCL3, indicating that activation of the Rho/ROCK signaling pathway is required for the CCL3-mediated chemotaxis of the cells. Costimulation with IgE/antigen and CCL3 enhanced Rac and Cdc42 activation but decreased ROCK activation in RBL-CCR1 cells compared with that in the cells stimulated with CCL3 alone. These results suggest that costimulation via FcepsilonRI and CCR1 engagements induced 1) inhibition of membrane ruffling, 2) decreased ROCK activation, and 3) reciprocal imbalance between Small GTP-binding proteins of the Rho family, which result in the inhibition of chemotaxis of RBL-CCR1 cells. The cross-talk between FcepsilonRI-mediated signaling pathway and CCR-mediated signaling pathway would induce optimal activation and arrested chemotaxis of mast cells, thus contributing to allergic inflammation.

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

Tissue contraction and fibrosis are major causes of morbidity in the human body. In the eye in particular, fibrosis and scarring are responsible for the pathogenesis or failure of treatment of all major blinding diseases, with postoperative wound healing responses posing a major problem for most ocular surgery on a worldwide scale. This is one of the largest areas of unmet need in ophthalmology, with currently no antifibrotic treatments available clinically. This review focuses on the ubiquitous myocardin-related transcription factor/serum response factor (MRTF-A/SRF) transcription pathway as a potential novel therapeutic target in fibrotic eye diseases. It describes how the MRTF-A/SRF pathway is intricately linked to all the key regulators and pathways in ocular fibrosis, and how it could potentially lead to a new avenue of antifibrotic therapies in the future.

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5–7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.

In vivo imaging of GFP-labeled metastatic tumor cells reveals cell orientation towards blood vessels. Orientation of tumor cells during chemotactic responses to ligands such as EGF begins with lamellipod extension. Evaluation of some of the downstream events in lamellipod extension indicates: (1) plasma membrane distribution of the EGF receptor is uniform but internalized receptor accumulates on the side of the cell closest to the source of EGF; (2) the alpha p110 isoform of PI-3 kinase is required; and (3) protrusion of the lamellipod relies upon the combined actions of the Arp2/3 complex and cofilin for generation of filamentous actin.

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.

Using both light and high resolution electron microscopy, we analyzed the spatial and temporal relationships between the Arp2/3 complex and the nucleation activity that is required for lamellipod extension in mammary carcinoma cells after epidermal growth factor stimulation. A rapid two- to fourfold increase in filament barbed end number occurs transiently after stimulation and remains confined almost exclusively to the extreme outer edge of the extending lamellipod (within 100–200 nm of the plasma membrane). This is accompanied by an increase in filament density at the leading edge and a general decrease in filament length, with a specific loss of long filaments. Concomitantly, the Arp2/3 complex is recruited with a 1.5-fold increase throughout the entire cortical filament network extending 1–1.5 μm in depth from the membrane at the leading edge. The recruitment of the Arp2/3 complex at the membrane of the extending lamellipod indicates that Arp2/3 may be involved in initial generation of growing filaments. However, only a small subset of the complex present in the cortical network colocalizes near free barbed ends. This suggests that the 100–200-nm submembraneous compartment at the leading edge of the extending lamellipod constitutes a special biochemical microenvironment that favors the generation and maintenance of free barbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the on-rate of capping protein. Our results are inconsistent with the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypothesis of an Arp2/3-mediated capture of actin oligomers that formed close to the membrane by other mechanisms such as severing. They also support pointed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.

The mammary adenocarcinoma cell line MTLn3 is chemotactic towards epidermal growth factor (EGF), and this induced motility is thought to promote breast cancer invasion and metastasis. Stimulation of MTLn3 cells with EGF results in the extension of a flat, thin structure filled with filamentous actin and termed a lamellipod. Lamellipod extension is dependent on actin polymerization and is localized to the border of adherent cells. The structure of EGF-stimulated lamellipods in MTLn3 cells is well suited to analysis of chemoattractant-stimulated protrusion. Actin polymerization occurs within 200 nm of the extending edge of the lamellipod. Although extension of the lamellipod is not dependent upon interaction with the substratum, stabilization of the extended lamellipod is dependent on an adhesive substratum. Dorsal ruffling is suppressed during lamellipod extension. Tyrosine phosphorylation is reduced in preexisting focal contacts compared to new contacts induced by EGF stimulation. The coordination of turnover of focal contacts with lamellipod extension is proposed to result in polarized cell motility in response to gradients of chemoattractants. Microsc. Res. Tech. 43:433–443, 1998. © 1998 Wiley-Liss, Inc.

Stimulation of metastatic MTLn3 cells with EGF causes the rapid extension of lamellipods, which contain a zone of F-actin at the leading edge. In order to establish the mechanism for accumulation of F-actin at the leading edge and its relationship to lamellipod extension in response to EGF, we have studied the kinetics and location of EGF-induced actin nucleation activity in MTLn3 cells and characterized the actin dynamics at the leading edge by measuring the changes at the pointed and barbed ends of actin filaments upon EGF stimulation of MTLn3 cells. The major result of this study is that stimulation of MTLn3 cells with EGF causes a transient increase in actin nucleation activity resulting from the appearance of free barbed ends very close to the leading edge of extending lamellipods. In addition, cytochalasin D causes a significant decrease in the total F-actin content in EGF-stimulated cells, indicating that both actin polymerization and depolymerization are stimulated by EGF. Pointed end incorporation of rhodamine-labeled actin by the EGF stimulated cells is 2.12+/−0.47 times higher than that of control cells. Since EGF stimulation causes an increase in both barbed and pointed end incorporation of rhodamine-labeled actin in the same location, the EGF-stimulated nucleation sites are more likely due either to severing of pre-existing filaments or de novo nucleation of filaments at the leading edge thereby creating new barbed and pointed ends. The timing and location of EGF-induced actin nucleation activity in MTLn3 cells can account for the observed accumulation of F-actin at the leading edge and demonstrate that this F-actin rich zone is the primary actin polymerization zone after stimulation.

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells.

Background Glaucoma is the leading cause of irreversible blindness and it affects over 70 million people worldwide. Existing strategies to prevent postsurgical fibrosis in glaucoma are inadequate and have complications that could potentially lead to blindness. Despite recent evidence that the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway has a pivotal role in fibroblast activation, there are currently no published studies investigating its role in conjunctival fibrosis. Methods We prospectively collected 30 non-fibrotic and fibrotic conjunctival tissues from patients with glaucoma at the time of surgery at Moorfields Eye Hospital, London, UK, according to local ethics approval. We analysed the profibrotic marker expression on whole tissue sections using in-situ tissue immunohistochemistry, and compared the contractile properties between primary cultures of non-fibrotic and fibrotic human conjunctival fibroblasts. We also studied the effect of silencing the MRTF/SRF pathway on the function of fibrotic conjunctival fibroblasts and analysed the group differences using Student's t test. Findings The fibrotic human conjunctiva expressed increased markers of fibrosis such as α smooth muscle actin, tenascin C, vimentin, and fibronectin. The fibrotic conjunctival fibroblasts significantly increased matrix contraction (from 28% [SD 16] to 76% [25], p=0·0024), and the cell force (0·25 mN [SD 0 ·08] to 0·56 mN [0·05], p=0·0044) compared with the non-fibrotic fibroblasts. MRTF and SRF silencing with siRNAs markedly decreased matrix contraction in fibrotic conjunctival fibroblasts by 80% and 87%, respectively. Knocking down MRTF significantly reduced the fibroblast dynamic index from 0·66 to 0·19 (p MRTF silencing decreased matrix degradation from 0·87 to 0·33 (p=0·006) and downregulated the expression of key matrix metalloproteinases genes such as MMP1, MMP2, MMP9 , and MMP14 (p Interpretation Our study is the first, to our knowledge, to show that silencing the MRTF/SRF pathway significantly decreases matrix contraction, fibroblast protrusive behaviour, matrix degradation, and MMP gene expression in fibrotic human conjunctival fibroblasts. Our study suggests that inhibiting the MRTF/SRF pathway could become a potential new therapeutic approach to prevent conjunctival fibrosis in glaucoma. Funding NIHR Biomedical Research Centre at Moorfields Eye Hospital and UCL Institute of Ophthalmology, Francis Crick Institute.

Persistent cell locomotion is a key feature of eukaryotic cells responding to diverse physiological cues. New work now directly implicates ADF/cofilin proteins as essential regulators of polarised cell migration.

AimsThe pathogenesis of thyroid-associated orbitopathy (TAO) remains unclear. The aim of this study is to elucidate the gene expression profile of orbital fat from patients with active, but untreated, TAO.MethodsA case–control gene expression study was conducted using test samples of orbital fat from TAO patients and control orbital fat specimens; apart from drugs to control thyrotoxicosis, the TAO patients had received no treatment for orbital disease. cDNA expression analysis was performed using the Affymetrix GeneChip Human Genome U133 Plus 2.0 platform and validated using quantitative PCR.ResultsThe highest-ranked differentially expressed genes were dominated by IGF-1 signalling genes. These include IGF-1, IGF-1 receptor binding/signalling genes, such as SOCS3 and IRS2, and downstream signalling and transcriptional regulators, such as SGK (PDK/Akt signalling) and c-JUN. Our microarray data also demonstrate dysregulation of wingless-type MMTV (Wnt) signalling gene expression, including Wnt5a, sFRPs and DKK.ConclusionAltered Wnt signalling confirms previous array findings. Further investigation of the role of Wnt signalling in TAO pathogenesis is warranted. These data also provide the first evidence of dysregulation of IGF-1 pathway genes in TAO tissue, further strengthening the evidence for the role of IGF-1 signalling in the pathogenesis and potential treatment of TAO.

In the true spirit of Michael Abercrombie's pioneering studies on cell locomotion, the Fifth Abercrombie's Symposium on Cell Behaviour--held in St Catherine's College at Oxford University (September 15-18, 2002)--celebrated the intricate beauty of cell motility with an explosion of new technologies that Abercrombie could only have dreamed of. Building on the complementary approaches of quantitative cell biology, biochemistry and genetics, the meeting provided new insights into the ever-growing complexity of the signal transduction pathways involved in cell movement.

Maintenance of stable E-cadherin–dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell–cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac–PAK1–Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell–cell contacts and stabilizes preassembled cadherin complexes.

The aims of this study are to investigate the expression of the main structural components of the tarsal extracellular matrix (ECM) in floppy eyelid syndrome (FES) focusing on elastic fibres and collagen types I and III, and also to identify possible cell-mediated inflammatory mechanisms in the pathogenesis of this condition.A histopathological case control study was conducted using 30 upper lid specimens from patients with FES and 15 undiseased upper lid control specimens. Structural ECM components were assessed using a combination of immunctorial ataining ohistochemical and techniques including antibodies to collagens I and III, Verhöeff's iron haematoxylin, Gomori's aldehyde fuchsin and Lillie's oxidised aldehyde fuchsin. The contribution of different cellular components of the inflammatory response was investigated by immunohistochemical techniques using antibodies to CD3, CD20, CD68. Slide scoring was performed using a semiquantitative technique on an ordinal scale. Statistical analysis was performed using matched ordinal regression analysis.FES tarsal plate tissue demonstrated a decreased abundance of mature elastic fibres (P ≤ 0.001) and an increased abundance of oxytalan fibres (P = 0.006). Intensity of staining for collagens I (P = 0.012) and III (P0.001) was increased. No significant difference in the abundance of CD3, CD20 and CD68 expressing cells was identified.The findings of altered elastic fibre phenotype and collagen accumulation are consistent with an adaptive response to cyclic mechanical loading of the tarsal plate, rather than an aetiological feature. These findings are important in understanding how the tarsal ECM responds to mechanical loading.

The serum response factor (SRF) coactivator myocardin-related transcription factor A (MAL/MKL1/MRTF-A), the nuclear transport and activity of which is regulated by monomeric actin, has been implicated in tension-based regulation of SRF-mediated transcriptional activity. However, the mechanisms involved remain unclear. We used fibroblasts grown within collagen matrices to explore whether MRTF-A transport is regulated by tissue tension. We show that MRTF-A nuclear accumulation following stimulation with serum, actin drugs or acute mechanical stress is prevented within mechanically loaded, anchored matrices at tensional homeostasis. This is accompanied by a higher G/F actin ratio, defective nuclear import and increased cofilin expression. We propose that tension regulates MRTF-A/SRF activity through cofilin-mediated modulation of actin dynamics.

Previous studies have suggested that increased malignant potential might be related to increased genomic instability, but this issue still remains controversial. We tested this hypothesis in a human tumour spontaneous metastasis model, using six clones and variants isolated from the parental poorly metastatic M4Be melanoma cell line, and expressing various metastatic abilities. The spontaneous rates of mutation to ouabain resistance measured in these cells by Luria and Delbrück fluctuation analysis correlated with the metastatic ability of the cells: moderately and highly metastatic cells showed spontaneous mutation rates 10 to 50 times higher than those of poorly metastatic cells. Genomic instability at the chromosome level was assessed by searching for accumulated structural abnormalities in the moderately and highly metastatic cell lines. All the cell lines appeared hypertriploid, and showed comparable modal numbers and great chromosome dispersion. Unstable DNA amplification in the form of double minute chromosomes was shown in one of the four poorly metastatic cell lines, and in a significantly higher proportion of the cells of two of the three metastatic cell lines. Abnormal chromosomes were demonstrated in all cell lines, with markers involving specific rearrangements of chromosomes 1, 6, 7, 8, 9, 11, 14 and 15, as frequently observed in human melanoma cells. Clonal markers were present in all cell lines, documenting the common origin of all variants and clones, and specific marker amplification was noticed in highly metastatic cells compared to poorly metastatic lines. These results suggest that human tumour progression might be accompanied both by an increase in genomic instability and by accumulation of karyotypic abnormalities.

In the present report we describe the characteristics of 2 clones, E2 and C5, isolated from the human colon adenocarcinoma cell line LoVo. When grafted to immunosuppressed newborn rats, these clones formed tumors that varied with regard to differentiation rate, basement-membrane organization and lung metastatic potential. Production and distribution of laminin by E2, C5 and related tumors was studied by immunohistochemistry with an anti-laminin monoclonal antibody 4C12 (MAb 4C12). In lowly metastatic E2-derived tumors, strong regular stainings were observed which were strictly peri-tumoral and corresponded to the basal lamina. Since the antibody interacted with human laminin (the graft) but not with rat laminin (the host), this result indicated that basement-membrane laminin was supplied mainly by tumor-cell synthesis. In highly metastatic C5-derived tumors, the staining obtained with MAb 4C12 was peri-cellular and unorganized. Laminin synthesis by E2 and C5 cells in sub-cultures or soon after dissociation from explanted tumors was studied by metabolic labelling with 35S-methionine under steady-state conditions followed by immunoprecipitation and SDS-PAGE. High-molecular-weight laminin comprised by disulfide-linked A and B chains, i.e., heterotrimeric laminin, was found in cell lysates and in the secretion medium of cell lines and tumor cells. In addition, B1B2 dimers and free B chains were observed in cell lysates. Quantitatively, laminin expression by E2 and C5 clones or tumor cells was not significantly different. These findings suggest that basement-membrane defects in invasive clone LoVo C5 were not due to laminin under-expression.

Publisher Summary Damage and degeneration of tissues in and around the eye frequently leads to visual impairment because of the anatomy and physiology of the eye. Consequently, repair processes play a role in most major blinding eye conditions or the failure of treatment. Cataract is the most important cause of world blindness, and degenerative processes with a repair response play a part in both the formation of cataract and the after cataract in capsule sparing surgery. Lid contraction and corneal scarring result in blindness in trachoma, and conjunctival fibrosis blinds after burns or autoimmune diseases such as cicatricial pemphigoid. Following glaucoma surgery, post-operative sub-conjunctival scarring results in sub-optimal pressure control and disease progression, and similar scarring restricts the predictability of strabismus surgery and contributes to motility problems in thyroid eye disease. Retinal scarring in proliferative vitreoretinopathy, and specifically age related macular degeneration, results in more loss of vision than any other disease in the developed world. Treatments and surgical approaches have been developed to successfully modulate the wound healing response. Anti-cancer agents such as mitomycin C inhibit fibroblast function and survival when applied locally. Steroids applied topically and systemically reduce inflammation and fibrosis. However, both these agents have significant side effects. This chapter reviews the agents used to modulate healing scarring.