Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment.

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.

Nuclear transport of the serum response factor coactivator MRTF-A is downregulated at tensional homeostasis.

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.

The Nance-Horan syndrome protein encodes a functional WAVE homology domain (WHD) and is important for co-ordinating actin remodelling and maintaining cell morphology.

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.

Cell motility: insights from the backstage.

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.

Changes in tarsal plate fibrillar collagens and elastic fibre phenotype in floppy eyelid syndrome.

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, 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. 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 < 0.001) was increased. No significant difference in the abundance of CD3, CD20 and CD68 expressing cells was identified. CONCLUSIONS: 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.

Annexin A2 at the interface between F-actin and membranes enriched in phosphatidylinositol 4,5,-bisphosphate.

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)P2) 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)P2 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)P2. 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)P2 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)P2 containing vesicles. These observations are consistent with a role for annexin A2 as an actin nucleator on PI(4,5)P2-enriched membranes.

A Tenon's capsule/bulbar conjunctiva interface biomimetic to model fibrosis and local drug delivery.

Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Rab27a co-ordinates actin-dependent transport by controlling organelle-associated motors and track assembly proteins.

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.

Ena/VASP family: new partners, bigger enigma.

Linking external signals to remodeling of the cytoskeleton is essential to multiple processes during animal development and physiology. Two new studies have uncovered a new family of proteins that can regulate actin dynamics both locally at the membrane interface and globally throughout the cell.

Connecting cell adhesion to the actin polymerization machinery: vinculin as the missing link?

Cell migration is essential to many physiological and pathological processes such as embryogenesis, wound healing or metastasis. This complex process involves a tight coordination between three essential steps - protrusion, adhesion and retraction. Although historically protrusion and adhesion have been linked through structural protein-protein interactions, a direct functional link between the two has long eluded biologists. Recent work from the Burridge laboratory now suggests that vinculin, a cytoskeletal protein involved in the building of the adhesion scaffold, could be the missing link that connects early adhesion sites to the actin-driven protrusive machinery.

Macrophages promote a profibrotic phenotype in orbital fibroblasts through increased hyaluronic acid production and cell contractility.

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.

Polarised migration: cofilin holds the front.

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.

Assessment of solid microneedle rollers to enhance transmembrane delivery of doxycycline and inhibition of MMP activity.

Many chronic wounds exhibit high matrix metalloproteinase (MMP) activity that impedes the normal wound healing process. Intradermal delivery (IDD) of sub-antimicrobial concentrations of doxycycline, as an MMP inhibitor, could target early stages of chronic wound development and inhibit further wound progression. To deliver doxycycline intradermally, the skin barrier must be disrupted. Microneedle rollers offer a minimally invasive technique to penetrate the skin by creating multiple microchannels that act as temporary conduits for drugs to diffuse through. In this study, an innovative and facile approach for delivery of doxycycline across Strat-MTM membrane was investigated using microneedle rollers. The quantity and rate of doxycycline diffusing through the micropores directly correlated with increasing microneedle lengths (250, 500 and 750 µm). Treatment of Strat-MTM with microneedle rollers resulted in a reduction in fibroblast-mediated collagen gel contraction and MMP activity compared with untreated Strat-MTM. Our results show that treatment of an epidermal mimetic with microneedle rollers provides sufficient permeabilization for doxycycline diffusion and inhibition of MMP activity. We conclude that microneedle rollers are a promising, clinically ready tool suitable for delivery of doxycycline intradermally to treat chronic wounds.

An Ilomastat-CD Eye Drop Formulation to Treat Ocular Scarring.

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-ß-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.

Local delivery of novel MRTF/SRF inhibitors prevents scar tissue formation in a preclinical model of fibrosis.

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 < 0.0005) and 33% (P < 0.01), respectively, and significantly decreased fibrosis and scarring histologically. Unlike mitomycin-C, neither CCG-222740 nor CCG-203971 caused any detectable epithelial toxicity or systemic side effects with very low drug levels measured in the aqueous, vitreous, and serum. We conclude that inhibitors of MRTF/SRF-regulated gene transcription such as CCG-222740, potentially represent a new therapeutic strategy to prevent scar tissue formation in the eye and other tissues.

Fibroblasts profiling in scarring trachoma identifies IL-6 as a functional component of a fibroblast-macrophage pro-fibrotic and pro-inflammatory feedback loop.

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.

Current and future prospects for the prevention of ocular fibrosis.

Significant advances have been made in developing new treatments and refining existing treatments for the prevention of scarring after disease, trauma, or surgical prevention. The advent of new technologies in addition to traditional chemical drugs such as dendrimers, antibodies, aptamers, ribozymes, gene therapy with viral vectors, and RNA interference, opens the door to a whole new generation of therapies to prevent fibrosis in the eye. The ability to control fibrotic processes in the eye offers many tantalizing prospects, including prevention of corneal blindness from scarring to "20/5 vision" with perfect corneal wound healing after wavefront refractive surgery prevention of PCO to fully accommodative lens implants, 100% success of glaucoma surgery with pressure at approximately 10 mmHg associated with < 5% progression over a decade, to no failure of retinal detachment surgery and minimal visual loss from AMD. Finally, most exciting is the prospect that neutralizing the fibrotic response to disease and injury will allow us to revert to the "fetal" mode when regeneration is the normal process, such as shown in the recent report that demonstrated that induction of bcl-2 gene expression together with downregulation of gliosis results in axonal regeneration in mice.

Eyelid and Sternum Fibroblasts Differ in Their Contraction Potential and Responses to Inflammatory Cytokines.

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-ß (P < 0.01) and interleukin-1ß (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.