Cell locomotion and focal adhesions are regulated by substrate flexibility

Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.

Stroma regulates increased epithelial lateral cell adhesion in 3D culture : a role for actin/cadherin dynamics

BACKGROUND: Cell shape and tissue architecture are controlled by changes to junctional proteins and the cytoskeleton. How tissues control the dynamics of adhesion and cytoskeletal tension is unclear. We have studied epithelial tissue architecture using 3D culture models and found that adult primary prostate epithelial cells grow into hollow acinus-like spheroids. Importantly, when co-cultured with stroma the epithelia show increased lateral cell adhesions. To investigate this mechanism further we aimed to: identify a cell line model to allow repeatable and robust experiments; determine whether or not epithelial adhesion molecules were affected by stromal culture; and determine which stromal signalling molecules may influence cell adhesion in 3D epithelial cell cultures. METHODOLOGY/PRINCIPAL FINDINGS: The prostate cell line, BPH-1, showed increased lateral cell adhesion in response to stroma, when grown as 3D spheroids. Electron microscopy showed that 9.4% of lateral membranes were within 20 nm of each other and that this increased to 54% in the presence of stroma, after 7 days in culture. Stromal signalling did not influence E-cadherin or desmosome RNA or protein expression, but increased E-cadherin/actin co-localisation on the basolateral membranes, and decreased paracellular permeability. Microarray analysis identified several growth factors and pathways that were differentially expressed in stroma in response to 3D epithelial culture. The upregulated growth factors TGFβ2, CXCL12 and FGF10 were selected for further analysis because of previous associations with morphology. Small molecule inhibition of TGFβ2 signalling but not of CXCL12 and FGF10 signalling led to a decrease in actin and E-cadherin co-localisation and increased paracellular permeability. CONCLUSIONS/SIGNIFICANCE: In 3D culture models, paracrine stromal signals increase epithelial cell adhesion via adhesion/cytoskeleton interactions and TGFβ2-dependent mechanisms may play a key role. These findings indicate a role for stroma in maintaining adult epithelial tissue morphology and integrity.

Impact of extracellular matrix derived from osteoarthritis subchondral bone osteoblasts on osteocytes : role of integrinβ1 and focal adhesion kinase signaling cues

INTRODUCTION: Our recent study indicated that subchondral bone pathogenesis in osteoarthritis (OA) is associated with osteocyte morphology and phenotypic abnormalities. However, the mechanism underlying this abnormality needs to be identified. In this study we investigated the effect of extracellular matrix (ECM) produced from normal and OA bone on osteocytic cells function. METHODS: De-cellularized matrices, resembling the bone provisional ECM secreted from primary human subchondral bone osteoblasts (SBOs) of normal and OA patients were used as a model to study the effect on osteocytic cells. Osteocytic cells (MLOY4 osteocyte cell line) cultured on normal and OA derived ECMs were analyzed by confocal microscopy, scanning electron microscopy (SEM), cell attachment assays, zymography, apoptosis assays, qRT-PCR and western blotting. The role of integrinβ1 and focal adhesion kinase (FAK) signaling pathways during these interactions were monitored using appropriate blocking antibodies. RESULTS: The ECM produced by OA SBOs contained less mineral content, showed altered organization of matrix proteins and matrix structure compared with the matrices produced by normal SBOs. Culture of osteocytic cells on these defective OA ECM resulted in a decrease of integrinβ1 expression and the de-activation of FAK cell signaling pathway, which subsequently affected the initial osteocytic cell's attachment and functions including morphological abnormalities of cytoskeletal structures, focal adhesions, increased apoptosis, altered osteocyte specific gene expression and increased Matrix metalloproteinases (MMP-2) and -9 expression. CONCLUSION: This study provides new insights in understanding how altered OA bone matrix can lead to the abnormal osteocyte phenotypic changes, which is typical in OA pathogenesis.

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln- N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

The mechanical rigidity of the extracellular matrix regulates the structure, motility, and proliferation of glioma cells

Glioblastoma multiforme (GBM) is a malignant astrocytoma of the central nervous system associated with a median survival time of 15 months, even with aggressive therapy. This rapid progression is due in part to diffuse infiltration of single tumor cells into the brain parenchyma, which is thought to involve aberrant interactions between tumor cells and the extracellular matrix (ECM). Here, we test the hypothesis that mechanical cues from the ECM contribute to key tumor cell properties relevant to invasion. We cultured a series of glioma cell lines (U373-MG, U87-MG, U251-MG, SNB19, C6) on fibronectin-coated polymeric ECM substrates of defined mechanical rigidity and investigated the role of ECM rigidity in regulating tumor cell structure, migration, and proliferation. On highly rigid ECMs, tumor cells spread extensively, form prominent stress fibers and mature focal adhesions, and migrate rapidly. As ECM rigidity is lowered to values comparable with normal brain tissue, tumor cells appear rounded and fail to productively migrate. Remarkably, cell proliferation is also strongly regulated by ECM rigidity, with cells dividing much more rapidly on rigid than on compliant ECMs. Pharmacologic inhibition of nonmuscle myosin II–based contractility blunts this rigidity-sensitivity and rescues cell motility on highly compliant substrates. Collectively, our results provide support for a novel model in which ECM rigidity provides a transformative, microenvironmental cue that acts through actomyosin contractility to regulate the invasive properties of GBM tumor cells.

Vimentin and epithelial-mesenchymal transition in human breast cancer : observations in vitro and in vivo

Breast cancer is a highly prevalent disease among women worldwide. While the expression of certain proteins within these tumours is used for prognosis and selection of therapies, there is a continuing need for additional markers to be identified. A considerable amount of current literature, based predominantly on cell culture systems, suggests that a major mechanism responsible for the progression of breast cancer is due to tumour cells losing their epithelial features and gaining mesenchymal properties. These events are proposed to be very similar to the epithelial-mesenchymal transition (EMT) process that has been well characterised in embryonic development. For the developmental and putative cancer EMT, the cell intermediate filament status changes from a keratin-rich network which connects to adherens junctions and hemidesmosomes, to a vimentin-rich network connecting to focal adhesions. This review summarises observations of vimentin expression in breast cancer model systems, and discusses the potential role of EMT in human breast cancer progression, and the prognostic usefulness of vimentin expression.

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397- transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.

Targeting and transport : how microtubules control focal adhesion dynamics

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge.

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.

Mathematical models of calcium and tight junctions in normal and reconstructed epidermis

This project investigated the calcium distributions of the skin, and the growth patterns of skin substitutes grown in the laboratory, using mathematical models. The research found that the calcium distribution in the upper layer of the skin is controlled by three different mechanisms, not one as previously thought. The research also suggests that tight junctions, which are adhesions between neighbouring skin cells, cannot be solely responsible for the differences in the growth patterns of skin substitutes and normal skin.

The cross-talk of LDL-cholesterol with cell migration: Insights from the Niemann Pick Type C1 mutation

Cholesterol is considered indispensible for the recruitment and functioning of integrins in focal adhesions for cell migration. However, the physiological cholesterol pools that control integrin trafficking and focal adhesion assembly remain unclear. Using Niemann Pick Type C1 (NPC) mutant cells, which accumulate Low Density lipoprotein (LDL)-derived cholesterol in late endosomes (LE), several recent studies indicate that LDL-cholesterol has multiple roles in regulating focal adhesion dynamics. Firstly, targeting of endocytosed LDL-cholesterol from LE to focal adhesions controls their formation at the leading edge of migrating cells. Other newly emerging literature suggests that this may be coupled to vesicular transport of integrins, Src kinase and metalloproteases from the LE compartment to focal adhesions. Secondly, our recent work identified LDL-cholesterol as a key factor that determines the distribution and ability of several Soluble NSF Attachment Protein (SNAP) Receptor (SNARE) proteins, key players in vesicle transport, to control integrin trafficking to the cell surface and extracellular matrix (ECM) secretion. Collectively, dietary, genetic and pathological changes in cholesterol metabolism may link with efficiency and speed of integrin and ECM cell surface delivery in metastatic cancer cells. This commentary will summarize how direct and indirect pathways enable LDL-cholesterol to modulate cell motility.

Role of Basement Membrane and Extracellular Matrix Proteins in the Adhesion and Spreading of Immortalized Human Corneal Epithelial Cells

The repair of corneal wounds requires both epithelial cell adhesion and migration. Basement membrane (BM) and extracellular matrix (ECM) proteins function in these processes via integrin and non-integrin receptors. We have studied the adhesion, spreading and migration of immortalized human corneal epithelial (HCE) cells and their interactions with the laminins (Lms), fibronectins and tenascins produced. Human corneal BM expresses Lms-332 and -511, while Lm-111 was not found in these experiments. HCE cells produced both processed and unprocessed Lm-332, whereas neither Lm-111 nor Lm-511 was produced. Because HCE cells did not produce Lm-511, although it was present in corneal BM, we suggest that Lm-511 is produced by stromal keratocytes. The adhesion of HCE cells to Lms-111, -332 and -511 was studied first by determining the receptor composition of HCE cells and then by using quantitative cell adhesion assays. Immunofluorescence studies revealed the presence of integrin α2, α3, α6, β1 and β4 subunits. Among the non-integrin receptors, Lutheran (Lu) was found on adhering HCE cells. The cells adhered via integrin α3β1 to both purified human Lms-332 and -511 as well as to endogenous Lm-332. However, only integrin β1 subunit functioned in HCE cell adhesion to mouse Lm-111. The adhesion of HCE cells to Lm-511 was also mediated by Lu. Since Lm-511 did not induce Lu into focal adhesions in HCE cells, we suggest that Lm-511 serves as an ECM ligand enabling cell motility. HCE cells produced extradomain-A fibronectin, oncofetal fibronectin and tenascin-C (Tn-C), which are also found during corneal wound healing. Monoclonal antibodies (MAbs) against integrins α5β1 and αvβ6 as well as the arginine-glycine-aspartic acid (RGD) peptide inhibited the adhesion of HCE cells to fibronectin. Although the cells did not adhere to Tn-C, they adhered to the fibronectin/Tn-C coat and were then more efficiently inhibited by the function-blocking MAbs and RGD peptide. During the early adhesion, HCE cells codeposited Lm-332 and the large subunit of tenascin-C (Tn-CL) beneath the cells via the Golgi apparatus and microtubules. Integrin β4 subunit, which is a hemidesmosomal component, did not mediate the early adhesion of HCE cells to Lm-332 or Lm-332/Tn-C. Based on these results, we suggest that the adhesion of HCE cells is initiated by Lm-332 and modulated by Tn-CL, as it has been reported to prevent the assembly of hemidesmosomes. Thereby, Tn-CL functions in the motility of HCE cells during wound healing. The different distribution of processed and unprocessed Lm-332 in adhering, spreading and migrating HCE cells suggests a distinct role for these isoforms. We conclude that the processed Lm-332 functions in cell adhesion, whereas the unprocessed Lm-332 participates in cell spreading and migration.

Mathematical models of calcium and tight junctions in normal and reconstructed epidermis

This project investigated the calcium distributions of the skin, and the growth patterns of skin substitutes grown in the laboratory, using mathematical models. The research found that the calcium distribution in the upper layer of the skin is controlled by three different mechanisms, not one as previously thought. The research also suggests that tight junctions, which are adhesions between neighbouring skin cells, cannot be solely responsible for the differences in the growth patterns of skin substitutes and normal skin.