Cell fragmentation in mouse preimplantation embryos induced by ectopic activation of the polar body extrusion pathway.

Cell fragmentation is commonly observed in human preimplantation embryos and is associated with poor prognosis during assisted reproductive technology (ART) procedures. However, the mechanisms leading to cell fragmentation remain largely unknown. Here, light sheet microscopy imaging of mouse embryos reveals that inefficient chromosome separation due to spindle defects, caused by dysfunctional molecular motors Myo1c or dynein, leads to fragmentation during mitosis. Extended exposure of the cell cortex to chromosomes locally triggers actomyosin contractility and pinches off cell fragments. This process is reminiscent of meiosis, during which small GTPase-mediated signals from chromosomes coordinate polar body extrusion (PBE) by actomyosin contraction. By interfering with the signals driving PBE, we find that this meiotic signaling pathway remains active during cleavage stages and is both required and sufficient to trigger fragmentation. Together, we find that fragmentation happens in mitosis after ectopic activation of actomyosin contractility by signals emanating from DNA, similar to those observed during meiosis. Our study uncovers the mechanisms underlying fragmentation in preimplantation embryos and, more generally, offers insight into the regulation of mitosis during the maternal-zygotic transition.

In vitro testing and efficacy of poly-lactic acid coating incorporating antibiotic loaded coralline bioceramic on Ti6Al4V implant against Staphylococcus aureus.

Biofilm formation on an implant surface is most commonly caused by the human pathogenic bacteria Staphylococcus aureus, which can lead to implant related infections and failure. It is a major problem for both implantable orthopedic and maxillofacial devices. The current antibiotic treatments are typically delivered orally or in an injectable form. They are not highly effective in preventing or removing biofilms, and they increase the risk of antibiotic resistance of bacteria and have a dose-dependent negative biological effect on human cells. Our aim was to improve current treatments via a localized and controlled antibiotic delivery-based implant coating system to deliver the antibiotic, gentamicin (Gm). The coating contains coral skeleton derived hydroxyapatite powders (HAp) that act as antibiotic carrier particles and have a biodegradable poly-lactic acid (PLA) thin film matrix. The system is designed to prevent implant related infections while avoiding the deleterious effects of high concentration antibiotics in implants on local cells including primary human adipose derived stem cells (ADSCs). Testing undertaken in this study measured the rate of S. aureus biofilm formation and determined the growth rate and proliferation of ADSCs. After 24 h, S. aureus biofilm formation and the percentage of live cells found on the surfaces of all 5%-30% (w/w) PLA-Gm-(HAp-Gm) coated Ti6Al4V implants was lower than the control samples. Furthermore, Ti6Al4V implants coated with up to 10% (w/w) PLA-Gm-(HAp-Gm) did not have noticeable Gm related adverse effect on ADSCs, as assessed by morphological and surface attachment analyses. These results support the use and application of the antibacterial PLA-Gm-(HAp-Gm) thin film coating design for implants, as an antibiotic release control mechanism to prevent implant-related infections.

Testing 3D printed biological platform for advancing simulated microgravity and space mechanobiology research.

The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

Timothy Grass Pollen Induces Spatial Reorganisation of F-Actin and Loss of Junctional Integrity in Respiratory Cells.

Grass pollens have been identified as mediators of respiratory distress, capable of exacerbating respiratory diseases including epidemic thunderstorm asthma (ETSA). It is hypothesised that during thunderstorms, grass pollen grains swell to absorb atmospheric water, rupture, and release internal protein content to the atmosphere. The inhalation of atmospheric grass pollen proteins results in deadly ETSA events. We sought to identify the underlying cellular mechanisms that may contribute towards the severity of ETSA in temperate climates using Timothy grass (Phleum pratense). Respiratory cells exposed to Timothy grass pollen protein extract (PPE) caused cells to undergo hypoxia ultimately triggering the subcellular re-organisation of F-actin from the peri junctional belt to cytoplasmic fibre assembly traversing the cell body. This change in actin configuration coincided with the spatial reorganisation of microtubules and importantly, decreased cell compressibility specifically at the cell centre. Further to this, we find that the pollen-induced reorganisation of the actin cytoskeleton prompting secretion of the pro-inflammatory cytokine, interleukin-8. In addition, the loss of peri-junctional actin following exposure to pollen proteins was accompanied by the release of epithelial transmembrane protein, E-cadherin from cell-cell junctions resulting in a decrease in epithelial barrier integrity. We demonstrate that Timothy grass pollen regulates F-actin dynamics and E-cadherin localisation in respiratory cells to mediate cell-cell junctional integrity highlighting a possible molecular pathway underpinning ETSA events.

How Do Mechanics Guide Fibroblast Activity? Complex Disruptions during Emphysema Shape Cellular Responses and Limit Research.

The emphysema death toll has steadily risen over recent decades, causing the disease to become the third most common cause of death worldwide in 2019. Emphysema is currently incurable and could be due to a genetic condition (Alpha-1 antitrypsin deficiency) or exposure to pollutants/irritants, such as cigarette smoke or poorly ventilated cooking fires. Despite the growing burden of emphysema, the mechanisms behind emphysematous pathogenesis and progression are not fully understood by the scientific literature. A key aspect of emphysematous progression is the destruction of the lung parenchyma extracellular matrix (ECM), causing a drastic shift in the mechanical properties of the lung (known as mechanobiology). The mechanical properties of the lung such as the stiffness of the parenchyma (measured as the elastic modulus) and the stretch forces required for inhalation and exhalation are both reduced in emphysema. Fibroblasts function to maintain the structural and mechanical integrity of the lung parenchyma, yet, in the context of emphysema, these fibroblasts appear incapable of repairing the ECM, allowing emphysema to progress. This relationship between the disturbances in the mechanical cues experienced by an emphysematous lung and fibroblast behaviour is constantly overlooked and consequently understudied, thus warranting further research. Interestingly, the failure of current research models to integrate the altered mechanical environment of an emphysematous lung may be limiting our understanding of emphysematous pathogenesis and progression, potentially disrupting the development of novel treatments. This review will focus on the significance of emphysematous lung mechanobiology to fibroblast activity and current research limitations by examining: (1) the impact of mechanical cues on fibroblast activity and the cell cycle, (2) the potential role of mechanical cues in the diminished activity of emphysematous fibroblasts and, finally, (3) the limitations of current emphysematous lung research models and treatments as a result of the overlooked emphysematous mechanical environment.

Tobramycin and Colistin display anti-inflammatory properties in CuFi-1 cystic fibrosis cell line.

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and 1H NMR. HRMS showed pairing of Ibuprofen and Tobramycin, further confirmed by 1H NMR whilst no pairing was observed for either Ibuprofen:Colistin or Ibuprofen:Tadim combinations. The anti-bacterial activity of the combinations against Pseudomonas aeruginosa showed that neither paired nor non-paired Ibuprofen:antibiotic therapies altered the anti-bacterial activity. The anti-inflammatory efficacy of the combination therapies was next determined at two different concentrations (Low and High) using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines. Differential response in the anti-inflammatory efficacy of Ibuprofen:Tobramycin combination was observed between the two concentrations due to changes in the structural conformation of the paired Ibuprofen:Tobramycin complex at High concentration, confirmed by 1H NMR. In contrast, the non-pairing of the Ibuprofen:Colistin and Ibuprofen:Tadim combinations showed a significant decrease in IL-8 secretion at both the concentrations. Importantly, all antibiotics alone showed anti-inflammatory properties, highlighting the inherent anti-inflammatory properties of these antibiotics.

Tropomyosin 2.1 collaborates with fibronectin to promote TGF-ß1-induced contraction of human lung fibroblasts.

Many lung diseases are characterized by fibrosis, leading to impaired tissue patency and reduced lung function. Development of fibrotic tissue depends on two-way interaction between the cells and the extra-cellular matrix (ECM). Concentration-dependent increased stiffening of the ECM is sensed by the cells, which in turn increases intracellular contraction and pulling on the matrix causing matrix reorganization and further stiffening. It is generally accepted that the inflammatory cytokine growth factor ß1 (TGF-ß1) is a major driver of lung fibrosis through the stimulation of ECM production. However, TGF-ß1 also regulates the expression of members of the tropomyosin (Tm) family of actin associating proteins that mediate ECM reorganization through intracellular-generated forces. Thus, TGF-ß1 may mediate the bi-directional signaling between cells and the ECM that promotes tissue fibrosis. Using combinations of cytokine stimulation, mRNA, protein profiling and cellular contractility assays with human lung fibroblasts, we show that concomitant induction of key Tm isoforms and ECM by TGF-ß1, significantly accelerates fibrotic phenotypes. Knocking down Tpm2.1 reduces fibroblast-mediated collagen gel contraction. Collectively, the data suggest combined ECM secretion and actin cytoskeleton contractility primes the tissue for enhanced fibrosis. Our study suggests that Tms are at the nexus of inflammation and tissue stiffening. Small molecules targeting specific Tm isoforms have recently been designed; thus targeting Tpm2.1 may represent a novel therapeutic target in lung fibrosis.

Development and in vitro characterization of a novel pMDI diclofenac formulation as an inhalable anti-inflammatory therapy for cystic fibrosis.

Anti-inflammatory treatment options for cystic fibrosis (CF) patients are currently limited and as such, there is an imperative need to develop new anti-inflammatory agents to reduce the persistent inflammation present within CF lungs. This study explored the potential of Diclofenac (DICLO) as a novel inhaled anti-inflammatory drug for CF treatment. The anti-inflammatory activity of DICLO on an air-liquid interface (ALI) cell culture model of healthy (NuLi-1) and CF (CuFi-1) airways showed a significant reduction in the secretion of pro-inflammatory cytokines, IL-6 and IL-8. Therefore, pressurized metered dose inhaler (pMDI) DICLO formulations were developed to allow targeted DICLO delivery to CF airways. As such, two pMDI DICLO formulations with varying ethanol concentrations: 5% (w/w) equating to 150 µg of DICLO per dose (Low dose), and 15% (w/w) equating to 430 µg of DICLO per dose (High dose) were developed and characterized to determine the optimum formulation. The Low dose pMDI DICLO formulation showed a significantly smaller particle diameter with uniform distribution resulting in a greater aerosol performance when compared to High dose formulation. Consequently, the Low dose pMDI DICLO formulation was further evaluated in terms of in vitro transport characteristics and anti-inflammatory activity. Importantly, the DICLO pMDI displayed anti-inflammatory activity in both healthy and CF in vitro models, highlighting the potential of an aerosolized low-dose DICLO formulation as a promising inhaled anti-inflammatory therapy for CF treatment.

Real-time quantitative monitoring of in vitro nasal drug delivery by a nasal epithelial mucosa-on-a-chip model.

OBJECTIVES: A human nasal epithelial mucosa (NEM) on-a-chip is developed integrated with a novel carbon nanofibers-modified carbon electrode for real-time quantitative monitoring of in vitro nasal drug delivery. The integration of platinum electrodes in the chip also enables real-time measurement of transepithelial electrical resistance (TEER). METHODS: The air-liquid interface culture of nasal epithelial RPMI 2650 cells in the NEM-on-a-chip was optimized to mimic the key functional characteristics of the human nasal mucosa. The epithelial transport of ibuprofen in the NEM-on-a-chip was electrochemically monitored in real-time under static and physiologically realistic dynamic flow conditions. RESULTS: The NEM-on-a-chip mimics the mucus production and nasal epithelial barrier function of the human nasal mucosa. The real-time drug quantification by the NEM-on-a-chip was validated versus the high-performance liquid chromatography method. The drug transport rate monitored in the NEM-on-a-chip was influenced by the flow in the bottom compartment of the chip, highlighting the importance of emulating the dynamic in vivo condition for nasal drug transport studies. CONCLUSION: This novel NEM-on-a-chip can be a low-cost and time-efficient alternative to the costly laborious conventional techniques for in vitro nasal drug transport assays. Importantly, its dynamic microenvironment enables conducting nasal drug transport tests under physiologically relevant dynamic conditions.

Modifying and Integrating in vitro and ex vivo Respiratory Models for Inhalation Drug Screening.

For the past 50 years, the route of inhalation has been utilized to administer therapies to treat a variety of respiratory and pulmonary diseases. When compared with other drug administration routes, inhalation offers a targeted, non-invasive approach to deliver rapid onset of drug action to the lung, minimizing systemic drug exposure and subsequent side effects. However, despite advances in inhaled therapies, there is still a need to improve the preclinical screening and the efficacy of inhaled therapeutics. Innovative in vitro models of respiratory physiology to determine therapeutic efficacy of inhaled compounds have included the use of organoids, micro-engineered lung-on-chip systems and sophisticated bench-top platforms to enable a better understanding of pulmonary mechanisms at the molecular level, rapidly progressing inhaled therapeutic candidates to the clinic. Furthermore, the integration of complementary ex vivo models, such as precision-cut lung slices (PCLS) and isolated perfused lung platforms have further advanced preclinical drug screening approaches by providing in vivo relevance. In this review, we address the challenges and advances of in vitro models and discuss the implementation of ex vivo inhaled drug screening models. Specifically, we address the importance of understanding human in vivo pulmonary mechanisms in assessing strategies of the preclinical screening of drug efficacy, toxicity and delivery of inhaled therapeutics.

An in vitro model for assessing drug transport in cystic fibrosis treatment: Characterisation of the CuFi-1 cell line.

Cystic fibrosis (CF) is a disease that most commonly affects the lungs and is characterized by mucus retention and a continuous cycle of bacterial infection and inflammation. Current CF treatment strategies are focused on targeted drug delivery to the lungs. Novel inhalable drug therapies require an in vitro CF model that appropriately mimics the in vivo CF lung environment to better understand drug delivery and transport across the CF epithelium, and predict drug therapeutic efficacy. Therefore, the aim of this research was to determine the appropriate air-liquid interface (ALI) culture method of the CuFi-1 (CF cell line) compared to the NuLi-1 (healthy cell line) cells to be used as in vitro models of CF airway epithelia. Furthermore, drug transport on both CuFi-1 and NuLi-1 was investigated to determine whether these cell lines could be used to study transport of drugs used in CF treatment using Ibuprofen (the only anti-inflammatory drug currently approved for CF) as a model drug. Differentiating characteristics specific to airway epithelia such as mucus production, inflammatory response and tight junction formation at two seeding densities (Low and High) were assessed throughout an 8-week ALI culture period. This study demonstrated that both the NuLi-1 and CuFi-1 cell lines fully differentiate in ALI culture with significant mucus secretion, IL-6 and IL-8 production, and functional tight junctions at week 8. Additionally, the High seeding density was found to alter the phenotype of the NuLi-1 cell line. For the first time, this study identifies that ibuprofen is transported via the paracellular pathway in ALI models of NuLi-1 and CuFi-1 cell lines. Overall, these findings highlight that NuLi-1 and CuFi-1 as promising in vitro ALI models to investigate the transport properties of novel inhalable drug therapies for CF treatment.

Properties of rapamycin solid lipid nanoparticles for lymphatic access through the lungs & part I: the effect of size.

Background: Lymphangioleiomyomatosis (LAM) is characterized by growth of smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Current oral rapamycin treatment is limited by low bioavailability of approximately 15%. Aim: The effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLNs) size on its penetration through the lymphatics. Method: Three Rapa-SLN formulations (200-1000 nm) were produced and assessed for particle characteristics and further for toxicity and performance in vitro. Results: Rapa-SLNs of 200 nm inhibited proliferation in TSC2-negative mouse embryonic fibroblast cells and penetrated the respiratory epithelium and lymphatic endothelium significantly faster compared with free rapamycin and larger Rapa-SLNs. Conclusion: Rapa-SLN approximately 200 nm allows efficient entry of rapamycin into the lymphatic system and is therefore a promising treatment for LAM patients.

Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease.

A lack of gravity experienced during space flight has been shown to have profound effects on human physiology including muscle atrophy, reductions in bone density and immune function, and endocrine disorders. At present, these physiological changes present major obstacles to long-term space missions. What is not clear is which pathophysiological disruptions reflect changes at the cellular level versus changes that occur due to the impact of weightlessness on the entire body. This review focuses on current research investigating the impact of microgravity at the cellular level including cellular morphology, proliferation, and adhesion. As direct research in space is currently cost prohibitive, we describe here the use of microgravity simulators for studies at the cellular level. Such instruments provide valuable tools for cost-effective research to better discern the impact of weightlessness on cellular function. Despite recent advances in understanding the relationship between extracellular forces and cell behavior, very little is understood about cellular biology and mechanotransduction under microgravity conditions. This review will examine recent insights into the impact of simulated microgravity on cell biology and how this technology may provide new insight into advancing our understanding of mechanically driven biology and disease.

Prostaglandin E2, but not cAMP nor ß2-agonists, induce tristetraprolin (TTP) in human airway smooth muscle cells.

Tristetraprolin (TTP) is an anti-inflammatory molecule known to post-transcriptionally regulate cytokine production and is, therefore, an attractive drug target for chronic respiratory diseases driven by inflammation, such as asthma and chronic obstructive pulmonary disease. Our recent in vitro studies in primary human airway smooth (ASM) cells have confirmed the essential anti-inflammatory role played by TTP as a critical partner in a cytokine regulatory network. However, several unanswered questions remain. While prior in vitro studies have suggested that TTP is regulated in a cAMP-mediated manner, raising the possibility that this may be one of the ways in which ß2-agonists achieve beneficial effects beyond bronchodilation, the impact of ß2-agonists on ASM cells is unknown. Furthermore, the effect of prostaglandin E2 (PGE2) on TTP expression in ASM cells has not been reported. We address this herein and reveal, for the first time, that TTP is not regulated by cAMP-activating agents nor following treatment with long-acting ß2-agonists. However, PGE2 does induce TTP mRNA expression and protein upregulation in ASM cells. Although the underlying mechanism of action remains undefined, we can confirm that PGE2-induced TTP upregulation is not mediated via cAMP, or EP2/EP4 receptor activation, and occurred in a manner independent of the p38 MAPK-mediated pathway. Taken together, these data confirm that ß2-agonists do not upregulate TTP in human ASM cells and indicate that another way in which PGE2 may achieve beneficial effects in asthma and COPD may be via upregulation of the master controller of inflammation-TTP.

EP2 and EP4 receptor antagonists: Impact on cytokine production and ß2 -adrenergic receptor desensitization in human airway smooth muscle.

Prostaglandin E2 (PGE2 ) is a key prostanoid known to have both proinflammatory and anti-inflammatory impact in the context of chronic respiratory diseases. We hypothesize that these opposing effects may be the result of different prostanoid E (EP) receptor-mediated signaling pathways. In this study, we focus on two of the four EP receptors, EP2 and EP4 , as they are known to induce cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Using primary human airway smooth muscle (ASM) cells, we first focussed on the PGE2 -induced production of two cAMP-dependent proinflammatory mediators: interleukin 6 (IL-6) and cyclo-oxygenase 2 production. We show that PGE2 -induced IL-6 protein secretion occurs via an EP2 -mediated pathway, in a manner independent of receptor-mediated effects on messenger RNA (mRNA) expression and temporal activation kinetics of the transcription factor cAMP response element binding. Moreover, stimulation of ASM with PGE2 did not establish a positive, receptor-mediated, feedback loop, as mRNA expression for EP2 and EP4 receptors were not upregulated and receptor antagonists were without effect. Our studies revealed that the EP2 , but not the EP4 , receptor is responsible for ß2 -adrenergic desensitization induced by PGE2 . We demonstrate that PGE2 -induced heterologous receptor desensitization responsible for tachyphylaxis to short- (salbutamol) or long- (formoterol) ß2 -agonists (measured by cAMP release) can be reversed by the EP2 receptor antagonist PF-04418948. Importantly, this study highlights that inhibiting the EP2 receptor restores ß2 -adrenergic receptor function in vitro and offers an attractive novel therapeutic target for treating infectious exacerbations in people suffering from chronic respiratory diseases in the future.

Repurposing of statins via inhalation to treat lung inflammatory conditions.

Despite many therapeutic advancements over the past decade, the continued rise in chronic inflammatory lung diseases incidence has driven the need to identify and develop new therapeutic strategies, with superior efficacy to treat these diseases. Statins are one class of drug that could potentially be repurposed as an alternative treatment for chronic lung diseases. They are currently used to treat hypercholesterolemia by inhibiting the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, that catalyses the rate limiting step in the mevalonate biosynthesis pathway, a key intermediate in cholesterol metabolism. Recent research has identified statins to have other protective pleiotropic properties including anti-inflammatory, anti-oxidant, muco-inhibitory effects that may be beneficial for the treatment of chronic inflammatory lung diseases. However, clinical studies have yielded conflicting results. This review will summarise some of the current evidences for statins pleiotropic effects that could be applied for the treatment of chronic inflammatory lung diseases, their mechanisms of actions, and the potential to repurpose statins as an inhaled therapy, including a detailed discussion on their different physical-chemical properties and how these characteristics could ultimately affect treatment efficacies. The repurposing of statins from conventional anti-cholesterol oral therapy to inhaled anti-inflammatory formulation is promising, as it provides direct delivery to the airways, reduced risk of side effects, increased bioavailability and tailored physical-chemical properties for enhanced efficacy.

The focal adhesion targeting domain of p130Cas confers a mechanosensing function.

Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.

Src kinase determines the dynamic exchange of the docking protein NEDD9 (neural precursor cell expressed developmentally down-regulated gene 9) at focal adhesions.

Dynamic exchange of molecules between the cytoplasm and integrin-based focal adhesions provides a rapid response system for modulating cell adhesion. Increased residency time of molecules that regulate adhesion turnover contributes to adhesion stability, ultimately determining migration speed across two-dimensional surfaces. In the present study we test the role of Src kinase in regulating dynamic exchange of the focal adhesion protein NEDD9/HEF1/Cas-L. Using either chemical inhibition or fibroblasts genetically null for Src together with fluorescence recovery after photobleaching (FRAP), we find that Src significantly reduces NEDD9 exchange at focal adhesions. Analysis of NEDD9 mutant constructs with the two major Src-interacting domains disabled revealed the greatest effects were due to the NEDD9 SH2 binding domain. This correlated with a significant change in two-dimensional migratory speed. Given the emerging role of NEDD9 as a regulator of focal adhesion stability, the time of NEDD9 association at the focal adhesions is key in modulating rates of migration and invasion. Our study suggests that Src kinase activity determines NEDD9 exchange at focal adhesions and may similarly modulate other focal adhesion-targeted Src substrates to regulate cell migration.

Tyrosine Y189 in the substrate domain of the adhesion docking protein NEDD9 is conserved with p130Cas Y253 and regulates NEDD9-mediated migration and focal adhesion dynamics.

The focal adhesion docking protein NEDD9/HEF1/Cas-L regulates cell migration and cancer invasion. NEDD9 is a member of the Cas family of proteins that share conserved overall protein-protein interaction domain structure, including a substrate domain that is characterized by extensive tyrosine (Y) phosphorylation. Previous studies have suggested that phosphorylation of Y253 in the substrate domain of the Cas family protein p130Cas is specifically required for p130Cas function in cell migration. While it is clear that tyrosine phosphorylation of the NEDD9 substrate domain is similarly required for the regulation of cell motility, whether individual NEDD9 tyrosine residues have discrete function in regulating motility has not previously been reported. In the present study we have used a global sequence alignment of Cas family proteins to identify a putative NEDD9 equivalent of p130Cas Y253. We find that NEDD9 Y189 aligns with p130Cas Y253 and that it is conserved among NEDD9 vertebrate orthologues. Expression of NEDD9 in which Y189 is mutated to phenylalanine results in increased rates of cell migration and is correlated with increased disassembly of GFP.NEDD9 focal adhesions. Conversely, mutation to Y189D significantly inhibits cell migration. Our previous data has suggested that NEDD9 stabilizes focal adhesions and the present data therefore suggests that phosphorylation of Y189 NEDD9 is required for this function. These findings indicate that the individual tyrosine residues of the NEDD9 substrate domain may serve discrete functional roles. Given the important role of this protein in promoting cancer invasion, greater understanding of the function of the individual tyrosine residues is important for the future design of approaches to target NEDD9 to arrest cancer cell invasion.

Occupy tissue: the movement in cancer metastasis.

The critical role of migration and invasion in cancer metastasis warrants new therapeutic approaches targeting the machinery regulating cell migration and invasion. While 2-dimensional (2D) models have helped identify a range of adhesion molecules, cytoskeletal components and regulators that are potentially important for cell migration, the use of models that better mimic the 3-dimensional (3D) environment has yielded new insights into the physiology of cell movement. For example, studying cells in 3D models has revealed that invading cancer cells may switch between heterogeneous invasion modes and thus evade pharmacological inhibition of invasion. Here we summarize published data in which the role of cell adhesion molecules in 2D vs. 3D migration have been directly compared and discuss mechanisms that regulate migration speed and persistence in 2D and 3D. Finally we discuss limits of 3D culture models to recapitulate the in vivo situation.