Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility.

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) inhibitor-induced changes of traction forces, fiber alignment, and fiber density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

PTU, a novel ureido-fatty acid, inhibits MDA-MB-231 cell invasion and dissemination by modulating Wnt5a secretion and cytoskeletal signaling.

Migration and invasion promote tumor cell metastasis, which is the leading cause of cancer death. At present there are no effective treatments. Epidemiological studies have suggested that ω-3 polyunsaturated fatty acids (PUFA) may decrease cancer aggressiveness. In recent studies epoxide metabolites of ω-3 PUFA exhibited anti-cancer activity, although increased in vivo stability is required to develop useful drugs. Here we synthesized novel stabilized ureido-fatty acid ω-3 epoxide isosteres and found that one analogue - p-tolyl-ureidopalmitic acid (PTU) - inhibited migration and invasion by MDA-MB-231 breast cancer cells in vitro and in vivo in xenografted nu/nu mice. From proteomics analysis of PTU-treated cells major regulated pathways were linked to the actin cytoskeleton and actin-based motility. The principal finding was that PTU impaired the formation of actin protrusions by decreasing the secretion of Wnt5a, which dysregulated the Wnt/planar cell polarity (PCP) pathway and actin cytoskeletal dynamics. Exogenous Wnt5a restored invasion and Wnt/PCP signalling in PTU-treated cells. PTU is the prototype of a novel class of agents that selectively dysregulate the Wnt/PCP pathway by inhibiting Wnt5a secretion and actin dynamics to impair MDA-MB-231 cell migration and invasion.

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.

Forcing a growth factor response - tissue-stiffness modulation of integrin signaling and crosstalk with growth factor receptors.

Research throughout the 90s established that integrin crosstalk with growth factor receptors stimulates robust growth factor signaling. These insights were derived chiefly from comparing adherent versus suspension cell cultures. Considering the new understanding that mechanosensory inputs tune adhesion signaling, it is now timely to revisit this crosstalk in different mechanical environments. Here, we present a brief historical perspective on integrin signaling against the backdrop of the mechanically diverse extracellular microenvironment, then review the evidence supporting the mechanical regulation of integrin crosstalk with growth factor signaling. We discuss early studies revealing distinct signaling consequences for integrin occupancy (binding to matrix) and aggregation (binding to immobile ligand). We consider how the mechanical environments encountered in vivo intersect with this diverse signaling, focusing on receptor endocytosis. We discuss the implications of mechanically tuned integrin signaling for growth factor signaling, using the epidermal growth factor receptor (EGFR) as an illustrative example. We discuss how the use of rigid tissue culture plastic for cancer drug screening may select agents that lack efficacy in the soft in vivo tissue environment. Tuning of integrin signaling via external mechanical forces in vivo and subsequent effects on growth factor signaling thus has implications for normal cellular physiology and anti-cancer therapies.

Cytotoxic T cells swarm by homotypic chemokine signalling.

Cytotoxic T lymphocytes (CTLs) are thought to arrive at target sites either via random search or following signals by other leukocytes. Here, we reveal independent emergent behaviour in CTL populations attacking tumour masses. Primary murine CTLs coordinate their migration in a process reminiscent of the swarming observed in neutrophils. CTLs engaging cognate targets accelerate the recruitment of distant T cells through long-range homotypic signalling, in part mediated via the diffusion of chemokines CCL3 and CCL4. Newly arriving CTLs augment the chemotactic signal, further accelerating mass recruitment in a positive feedback loop. Activated effector human T cells and chimeric antigen receptor (CAR) T cells similarly employ intra-population signalling to drive rapid convergence. Thus, CTLs recognising a cognate target can induce a localised mass response by amplifying the direct recruitment of additional T cells independently of other leukocytes.

Immune cells known as cytotoxic T lymphocytes, or CTLs for short, move around the body searching for infected or damaged cells that may cause harm. Once these specialised killer cells identify a target, they launch an attack, removing the harmful cell from the body. CTLs can also recognise and eliminate cancer cells, and can be infused into cancer patients as a form of treatment called adoptive cell transfer immunotherapy. Unfortunately, this kind of treatment does not yet work well on solid tumours because the immune cells often do not infiltrate them sufficiently. It is thought that CTLs arrive at their targets either by randomly searching or by following chemicals secreted by other immune cells. However, the methods used to map the movement of these killer cells have made it difficult to determine how populations of CTLs coordinate their behaviour independently of other cells in the immune system. To overcome this barrier, Galeano Niño, Pageon, Tay et al. employed a three-dimensional model known as a tumouroid embedded in a matrix of proteins, which mimics the tissue environment of a real tumour in the laboratory. These models were used to track the movement of CTLs extracted from mice and humans, as well as human T cells engineered to recognise cancer cells. The experiments showed that when a CTL identifies a tumour cell, it releases chemical signals known as chemokines, which attract other CTLs and recruit them to the target site. Further experiments and computer simulations revealed that as the number of CTLs arriving at the target site increases, this amplifies the chemokine signal being secreted, resulting in more and more CTLs being attracted to the tumour. Other human T cells that had been engineered to recognize cancer cells were also found to employ this method of mass recruitment, and collectively 'swarm' towards targeted tumours. These findings shed new light on how CTLs work together to attack a target. It is possible that exploiting the mechanism used by CTLs could help improve the efficiency of tumour-targeting immunotherapies. However, further studies are needed to determine whether these findings can be applied to solid tumours in cancer patients.

Collective forces of tumor spheroids in three-dimensional biopolymer networks.

We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that our method is accurate up to strains of 50% and remains valid even for irregularly shaped tissue samples when considering only the deformations in the far field. Finally, we demonstrate that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1 hr after seeding, while collective forces on longer timescales are guided by mechanical feedback from the extracellular matrix.

Five Piconewtons: The Difference between Osteogenic and Adipogenic Fate Choice in Human Mesenchymal Stem Cells.

The ability of mesenchymal stem cells to sense nanoscale variations in extracellular matrix (ECM) compositions in their local microenvironment is crucial to their survival and their fate; however, the underlying molecular mechanisms defining how such fates are temporally modulated remain poorly understood. In this work, we have utilized self-assembled block copolymer surfaces to present nanodomains of an adhesive peptide found in many ECM proteins at different lateral spacings (from 30 to 60 nm) and studied the temporal response (2 h to 14 days) of human mesenchymal stem cells (hMSCs) using a panel of real-time localization and activity biosensors. Our findings revealed that within the first 4 to 24 h postadhesion and spreading, hMSCs on smaller nanodomain spacings recruit more activated FAK and Src proteins to produce larger, longer-lived, and increased numbers of focal adhesions (FAs). The adhesions formed on smaller nanospacings rapidly recruit higher amounts of nonmuscle myosin IIA and vinculin and experience tension forces (by >5 pN/FA) significantly higher than those observed on larger nanodomain spacings. The transmission of higher levels of tension into the cytoskeleton at short times was accompanied by higher Rac1, cytosolic ß-catenin, and nuclear localization of YAP/TAZ and RUNX2, which together biased the commitment of hMSCs to an osteogenic fate. This investigation provides mechanistic insights to confirm that smaller lateral spacings of adhesive nanodomains alter hMSC mechanosensing and biases mechanotransduction at short times via differential coupling of FAK/Src/Rac1/myosin IIA/YAP/TAZ signaling pathways to support longer-term changes in stem cell differentiation and state.

Tropomyosin Tpm 2.1 loss induces glioblastoma spreading in soft brain-like environments.

INTRODUCTION: The brain is a very soft tissue. Glioblastoma (GBM) brain tumours are highly infiltrative into the surrounding healthy brain tissue and invasion mechanisms that have been defined using rigid substrates therefore may not apply to GBM dissemination. GBMs characteristically lose expression of the high molecular weight tropomyosins, a class of actin-associating proteins and essential regulators of the actin stress fibres and focal adhesions that underpin cell migration on rigid substrates. METHODS: Here, we investigated how loss of the high molecular weight tropomyosins affects GBM on soft matrices that recapitulate the biomechanical architecture of the brain. RESULTS: We find that Tpm 2.1 is down-regulated in GBM grown on soft substrates. We demonstrate that Tpm 2.1 depletion by siRNA induces cell spreading and elongation in soft 3D hydrogels, irrespective of matrix composition. Tpm 1.7, a second high molecular weight tropomyosin is also down-regulated when cells are cultured on soft brain-like surfaces and we show that effects of this isoform are matrix dependent, with Tpm 1.7 inducing cell rounding in 3D collagen gels. Finally, we show that the absence of Tpm 2.1 from primary patient-derived GBMs correlates with elongated, mesenchymal invasion. CONCLUSIONS: We propose that Tpm 2.1 down-regulation facilitates GBM colonisation of the soft brain environment. This specialisation of the GBM actin cytoskeleton organisation that is highly suited to the soft brain-like environment may provide novel therapeutic targets for arresting GBM invasion.

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac-mediated multicellular invasion.

The migration and invasion of cells through tissues in the body is facilitated by a dynamic actin cytoskeleton. The actin-associating protein, tropomyosin Tpm3.1 has emerged to play important roles in cell migration and invasion. To date, investigations have focused on single cell migration and invasion where Tpm3.1 expression is inversely associated with Rac GTPase-mediated cell invasion. While single cell and collective cell invasion have many features in common, collective invasion is additionally impacted by cell-cell adhesion, and the role of Tpm3.1 in collective invasion has not been established. In the present study we have modelled multicellular invasion using neuroblastoma spheroids embedded in 3D collagen and analysed the function of Tpm3.1 using recently established compounds that target the Tpm3.1 C-terminus. The major findings from our study reveal that combined Rac inhibition and Tpm3.1 targeting result in greater inhibition of multicellular invasion than either treatment alone. Together, the data suggest that Tpm3.1 disruption sensitises neuroblastoma cells to inhibition of Rac-mediated multicellular invasion.

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.

Rac GTPase regulation of 3D invasion in neuroblastomas lacking MYCN amplification.

Neuroblastomas are highly invasive tumors that occur in pediatric patients and treatment of invasive disease remains a challenge. The study of cells invading in 3-dimensional (3D) hydrogels has revealed morphologically distinct modes of invasion by which cancer cells adapt to the local tissue environment in order to invade local tissue. Specifically, the small G protein Rac GTPase has been implicated as regulating the elongated/mesenchymal mode of cell invasion. In the present study we demonstrate an inverse association between Rac expression and amplification of MYCN, a well-established prognostic indicator in neuroblastoma. Moreover, the association further tracks with previously described morphological variants of neuroblastoma. Importantly, while MYCN amplification is associated with universally poor prognosis, the clinical course of patients whose tumors lack MYCN amplification are more difficult to predict. Therefore, we analyzed the role that Rac plays in regulating the invasive behavior of neuroblastoma cells lacking MYCN amplification. Using siRNA targeting Rac in single cell suspensions in 3D collagen gels and Rac inhibition of multicellular spheroids (MCS) embedded in collagen gels, we find that the high Rac-expressing lines differ in their morphological response to Rac depletion and inhibition. Live cell imaging of embedded MCS reveals distinct individual and collective modes of invasion between the cell lines. Critically, Rac inhibition blocked both individual and collective invasion in 2 of the 3 high Rac expressing cell lines. Our study suggests that Rac activity may be an important determinant of metastatic capability in subsets of neuroblastoma cells lacking MYCN amplification.

Cooperative cell invasion: matrix metalloproteinase-mediated incorporation between cells.

Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S- and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion.

Thymosin-ß4 is a determinant of drug sensitivity for Fenretinide and Vorinostat combination therapy in neuroblastoma.

Retinoids are an important component of neuroblastoma therapy at the stage of minimal residual disease, yet 40-50% of patients treated with 13-cis-retinoic acid (13-cis-RA) still relapse, indicating the need for more effective retinoid therapy. Vorinostat, or Suberoylanilide hydroxamic acid (SAHA), is a potent inhibitor of histone deacetylase (HDAC) classes I & II and has antitumor activity in vitro and in vivo. Fenretinide (4-HPR) is a synthetic retinoid which acts on cancer cells through both nuclear retinoid receptor and non-receptor mechanisms. In this study, we found that the combination of 4-HPR + SAHA exhibited potent cytotoxic effects on neuroblastoma cells, much more effective than 13-cis-RA + SAHA. The 4-HPR + SAHA combination induced caspase-dependent apoptosis through activation of caspase 3, reduced colony formation and cell migration in vitro, and tumorigenicity in vivo. The 4-HPR and SAHA combination significantly increased mRNA expression of thymosin-beta-4 (Tß4) and decreased mRNA expression of retinoic acid receptor α (RARα). Importantly, the up-regulation of Tß4 and down-regulation of RARα were both necessary for the 4-HPR + SAHA cytotoxic effect on neuroblastoma cells. Moreover, Tß4 knockdown in neuroblastoma cells increased cell migration and blocked the effect of 4-HPR + SAHA on cell migration and focal adhesion formation. In primary human neuroblastoma tumor tissues, low expression of Tß4 was associated with metastatic disease and predicted poor patient prognosis. Our findings demonstrate that Tß4 is a novel therapeutic target in neuroblastoma, and that 4-HPR + SAHA is a potential therapy for the disease.

Role of dynamin in elongated cell migration in a 3D matrix.

The use of 3-dimensional (3D) collagen gels has yielded new insights into the migratory behaviour of cancer cells. While the large GTPase dynamin has emerged as an important regulator of cancer cell migration and invasion under 2D conditions, its role in 3D migration is unclear. We have used a potent dynamin modulator, a bis-tyrphostin derivative, Ryngo® 1-23, to investigate the role of dynamin in 3D migration in 3 different cell lines. The compound specifically inhibits persistent, elongated 3D migration in U87MG and SMA-560 cells. Treated U87MG cells adopt a rounded morphology that is not due to apoptosis, loss of matrix metalloprotease activity or inhibition of clathrin-mediated endocytosis. Given that Ryngo 1-23 is known to regulate dynamin oligomerisation and actin dynamics at the leading edge, we analysed actin filament distribution. Ryngo 1-23 induced a switch in actin filament organization in 3D cultures resulting in the generation of multiple short actin-rich microspikes. Correlated with the change in actin filament distribution, cells displayed reduced collagen gel contraction. Since acto-myosin force transmission to the extra-cellular matrix underpins persistent, elongated migration, our results suggest that Ryngo 1-23 modulates this process in 3D migration via dynamin-mediated regulation of acto-myosin force transmission to the extra-cellular matrix.

Tropomyosin Tm5NM1 spatially restricts src kinase activity through perturbation of Rab11 vesicle trafficking.

In order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules.

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.

Insights into adhesion biology using single-molecule localization microscopy.

Focal adhesions are complex multi-protein structures that mediate cell adhesion and cell migration in multicellular organisms. Most of the protein components involved in focal adhesion formation have been identified, but a major challenge remains: determination of the spatial and temporal dynamics of adhesion proteins in order to understand the molecular mechanisms of adhesion assembly, maturation, signal regulation, and disassembly. Progress in this field has been hampered by the limited resolution of fluorescence microscopy. Recent advances have led to the development of super-resolution techniques including single-molecule localization microscopy (SMLM). Here, we discuss how the application of these techniques has revealed important new insights into focal adhesion structure and dynamics, including the first description of the three-dimensional nano-architecture of focal adhesions and of the dynamic exchange of integrins in focal adhesions. Hence, SMLM has contributed to the refinement of existing models of adhesions as well as the establishment of novel models, thereby opening new research directions. With current improvements in SMLM instrumentation and analysis, it has become possible to study cellular adhesions at the single-molecule level.

NEDD9 regulates 3D migratory activity independent of the Rac1 morphology switch in glioma and neuroblastoma.

UNLABELLED: Metastasizing tumor cells must transmigrate the dense extracellular matrix that surrounds most organs. The use of three-dimensional (3D) collagen gels has revealed that many cancer cells can switch between different modes of invasion that are characterized by distinct morphologies (e.g., rounded vs. elongated). The adhesion protein NEDD9 has the potential to regulate the switch between elongated and rounded morphologies; therefore, its role was interrogated in the invasion switch of glioblastoma and neuroblastoma tumors that similarly derive from populations of neural crest cells. Interestingly, siRNA-mediated depletion of NEDD9 failed to induce cell rounding in glioma or neuroblastoma cells, contrasting the effects that have been described in other tumor model systems. Given that Rac1 GTPase has been suggested to mediate the switch between elongated and rounded invasion, the functionality of the Rac1 morphology switch was evaluated in the glioma and neuroblastoma cells. Using both dominant-negative Rac1 and Rac1-specific siRNA, the presence of this morphologic switch was confirmed in the neuroblastoma, but not in the glioma cells. However, in the absence of a morphologic change following NEDD9 depletion, a significant decrease in the cellular migration rate was observed. Thus, the data reveal that NEDD9 can regulate 3D migration speed independent of the Rac1 morphology switch. IMPLICATIONS: NEDD9 targeting is therapeutically viable as it does not stimulate adaptive changes in glioma and neuroblastoma invasion.

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.