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2.  Functional Characterization of KIN-32, the Caenorhabditis elegans Homolog of Focal Adhesion Kinase 
We have identified the single Caenorhabditis elegans focal adhesion kinase (FAK) homolog KIN-32, which has the signature FAK structure including an N-terminal Four.1-Ezrin-Radixin-Moesin (FERM) domain followed by a tyrosine kinase domain and a C-terminal domain with weak homology to the focal adhesion targeting domain. The functional requirements for KIN-32 were examined using RNA interference depletion experiments and analysis of a deletion allele, kin-32(ok166), in which a large segment of the FERM domain is missing. Our results show that reduced levels of expression or absence of the FERM domain do not affect viability, fertility, or anatomy in C. elegans. Expression of an analogous FERM deletion in mouse FAK showed kinase activity in vitro and supported normal focal adhesion localization in cell culture. Thus, the FERM domain of KIN-32, and possibly KIN-32 activity in general, appears to be dispensable for normal C. elegans physiology.
doi:10.1002/dvdy.21457
PMCID: PMC3937857  PMID: 18297732
FERM; kinase; nematode; RNAi; FAK
3.  Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells 
Experimental cell research  2012;318(15):1820-1831.
Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture.
doi:10.1016/j.yexcr.2012.06.009
PMCID: PMC3582329  PMID: 22710062
extracellular matrix; fibronectin; integrin; self-renewal; embryonic stem cell
4.  mig-38, a novel gene that regulates distal tip cell turning during gonadogenesis in C. elegans hermaphrodites 
Developmental biology  2012;368(2):404-414.
In Caenorhabditis elegans gonad morphogenesis, the final U-shapes of the two hermaphrodite gonad arms are determined by migration of the distal tip cells (DTCs). These somatic cells migrate in opposite directions on the ventral basement membrane until specific extracellular cues induce turning from ventral to dorsal and then centripetally toward the midbody region on the dorsal basement membrane. To dissect the mechanism of DTC turning, we examined the role of a novel gene, F40F11.2/mig-38, whose depletion by RNAi results in failure of DTC turning so that DTCs continue their migration away from the midbody region. mig-38 is expressed in the gonad primordium, and expression continues throughout DTC migration where it acts cell-autonomously to control DTC turning. RNAi depletion of both mig-38 and ina-1, which encodes an integrin adhesion receptor, enhanced the loss of turning phenotype indicating a genetic interaction between these genes. Furthermore, the integrin-associated protein MIG-15/Nck-interacting kinase (NIK) works with MIG-38 to direct DTC turning as shown by mig-38 RNAi with the mig-15(rh80) hypomorph. These results indicate that MIG-38 enhances the role of MIG-15 in integrin-dependent DTC turning. Knockdown of talin, a protein that is important for integrin activation, causes the DTCs to stop migration prematurely. When both talin and MIG-38 were depleted by RNAi treatment, the premature stop phenotype was suppressed. This suppression effect was reversed upon additional depletion of MIG-15 or its binding partner NCK-1. These results suggest that both talin and the MIG-15/NCK-1 complex promote DTC motility and that MIG-38 may act as a negative regulator of the complex. We propose a model to explain the dual role of MIG-38 in motility and turning.
doi:10.1016/j.ydbio.2012.06.011
PMCID: PMC3697129  PMID: 22732572
Cell migration; Distal tip cell; C. elegans; Hermaphrodite gonadogenesis; mig-38; Integrin
5.  Fibronectins, Their Fibrillogenesis, and In Vivo Functions 
Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.
Formation of fibrillar fibronectin matrices involves interactions with cell surface integrins that induce fibronectin conformational changes required for self-association. Cell culture and embryonic model systems have provided key insights into the mechanisms that initiate and regulate the fibronectin assembly process.
doi:10.1101/cshperspect.a005041
PMCID: PMC3119908  PMID: 21576254
6.  Assembly of Fibronectin Extracellular Matrix 
In the process of matrix assembly, multivalent extracellular matrix (ECM) proteins are induced to self-associate and to interact with other ECM proteins to form fibrillar networks. Matrix assembly is usually initiated by ECM glycoproteins binding to cell surface receptors, such as fibronectin (FN) dimers binding to α5β1 integrin. Receptor binding stimulates FN self-association mediated by the N-terminal assembly domain and organizes the actin cytoskeleton to promote cell contractility. FN conformational changes expose additional binding sites that participate in fibril formation and in conversion of fibrils into a stabilized, insoluble form. Once assembled, the FN matrix impacts tissue organization by contributing to the assembly of other ECM proteins. Here, we describe the major steps, molecular interactions, and cellular mechanisms involved in assembling FN dimers into fibrillar matrix while highlighting important issues and major questions that require further investigation.
doi:10.1146/annurev-cellbio-100109-104020
PMCID: PMC3628685  PMID: 20690820
integrins; conformational change; insolubility; fibrillar; type I collagen; microfibrils
7.  Temporal and spatial regulation of integrins during development 
Current opinion in cell biology  2008;20(5):520-524.
Summary
Integrin receptors for extracellular matrix (ECM) are critical determinants of biological processes. Regulation of integrin expression is one way for cells to respond to changes in the ECM, to integrate intracellular signals, and to obtain appropriate adhesion for cell motility, proliferation, and differentiation. Transcriptional and post-translational mechanisms for changing the integrin repertoire at the cell surface have recently been described. These mechanisms work through transcriptional regulation that alters the proportions of one integrin relative to another, referred to as integrin switching, or through localized regulation of integrin-ECM interactions, thus providing exquisite control over cell rearrangements during tissue morphogenesis and remodeling. These integrin regulatory pathways may also be important targets in such emerging fields as tissue engineering and regenerative medicine.
doi:10.1016/j.ceb.2008.05.010
PMCID: PMC2572561  PMID: 18603422
8.  Gonad morphogenesis and distal tip cell migration in the Caenorhabditis elegans hermaphrodite 
Cell migration and morphogenesis are key events in tissue development and organogenesis. In Caenorhabditis elegans, the migratory path of the distal tip cells determines the morphology of the hermaphroditic gonad. The distal tip cells undergo a series of migratory phases interspersed with turns to form the gonad. A wide variety of genes have been identified as crucial to this process, from genes that encode components and modifiers of the extracellular matrix to signaling proteins and transcriptional regulators. The connections between extracellular and transmembrane protein functions and intracellular pathways are essential for distal tip cell migration, and the integration of this information governs gonad morphogenesis and determines gonad size and shape.
doi:10.1002/wdev.45
PMCID: PMC3614366  PMID: 23559979
9.  Fibronectin Expression Modulates Mammary Epithelial Cell Proliferation during Acinar Differentiation 
Cancer research  2008;68(9):3185-3192.
The mammary gland consists of a polarized epithelium surrounded by a basement membrane matrix that forms a series of branching ducts ending in hollow, sphere-like acini. Essential roles for the epithelial basement membrane during acinar differentiation, in particular laminin and its integrin receptors, have been identified using mammary epithelial cells cultured on a reconstituted basement membrane. Contributions from fibronectin, which is abundant in the mammary gland during development and tumorigenesis, have not been fully examined. Here, we show that fibronectin expression by mammary epithelial cells is dynamically regulated during the morphogenic process. Experiments with synthetic polyacrylamide gel substrates implicate both specific extracellular matrix components, including fibronectin itself, and matrix rigidity in this regulation. Alterations in fibronectin levels perturbed acinar organization. During acinar development, increased fibronectin levels resulted in overproliferation of mammary epithelial cells and increased acinar size. Addition of fibronectin to differentiated acini stimulated proliferation and reversed growth arrest of mammary epithelial cells negatively affecting maintenance of proper acinar morphology. These results show that expression of fibronectin creates a permissive environment for cell growth that antagonizes the differentiation signals from the basement membrane. These effects suggest a link between fibronectin expression and epithelial cell growth during development and oncogenesis in the mammary gland.
doi:10.1158/0008-5472.CAN-07-2673
PMCID: PMC2748963  PMID: 18451144
10.  Analysis of Cell Migration Using Caenorhabditis elegans as a Model System 
The nematode Caenorhabditis elegans is an excellent model system in which to study long-distance cell migration in vivo. This chapter describes methods used to study a subset of migratory cells in the hermaphrodite nematode, the distal tip cells. These methods take advantage of the organism’s transparent body and the expression of green fluorescent protein to observe cell migration and behavior. Additionally, the availability of nematode mutants and gene knockdown techniques that affect cell migration allow the analysis and comparison of wild-type and aberrant migratory paths. Methods for nematode growth and maintenance, strain acquisition, observation and live imaging, gene knockdown, and analysis of cell migration defects are covered.
doi:10.1007/978-1-61779-207-6_16
PMCID: PMC3601446  PMID: 21748680
C. elegans; Cell migration; Mutants; RNAi; Distal tip cells; Live imaging; Green fluorescent protein
11.  Endoplasmic spreading requires coalescence of vimentin intermediate filaments at force-bearing adhesions 
Molecular Biology of the Cell  2013;24(1):21-30.
Interaction of vimentin filaments (vIFs) and force-bearing adhesions is essential for endoplasm spreading. For adhesions to be connected to a contractile network involved in endoplasm spreading, vIFs are needed. Thus endoplasm spreading and microtubule stabilization in the periphery require a multicomponent actin network anchored at adhesions.
For cells to develop long-range forces and carry materials to the periphery, the microtubule and organelle-rich region at the center of the cell—the endoplasm—needs to extend to near the cell edge. Depletion of the actin cross-linking protein filamin A (FlnA) causes a collapse of the endoplasm into a sphere around the nucleus of fibroblasts and disruption of matrix adhesions, indicating that FlnA is involved in endoplasmic spreading and adhesion growth. Here, we report that treatment with the calpain inhibitor N-[N-(N-acetyl-l-leucyl)-l-leucyl]-l-norleucine (ALLN) restores endoplasmic spreading as well as focal adhesion (FA) growth on fibronectin-coated surfaces in a Fln-depleted background. Addback of calpain-uncleavable talin, not full-length talin, achieves a similar effect in Fln-depleted cells and indicates a crucial role for talin in endoplasmic spreading. Because FA maturation involves the vimentin intermediate filament (vIF) network, we also examined the role of vIFs in endoplasmic spreading. Wild-type cells expressing a vimentin variant incapable of polymerization exhibit deficient endoplasmic spreading as well as defects in FA growth. ALLN treatment restores FA growth despite the lack of vIFs but does not restore endoplasmic spreading, implying that vIFs are essential for endoplasm spreading. Consistent with that hypothesis, vIFs are always displaced from adhesions when the endoplasm does not spread. In Fln-depleted cells, vIFs extend beyond adhesions, nearly to the cell edge. Finally, inhibiting myosin II–mediated contraction blocks endoplasmic spreading and adhesion growth. Thus we propose a model in which myosin II–mediated forces and coalescence of vIFs at mature FAs are required for endoplasmic spreading.
doi:10.1091/mbc.E12-05-0377
PMCID: PMC3530776  PMID: 23115305
12.  Fibronectins, Their Fibrillogenesis, and In Vivo Functions 
Cold Spring Harbor perspectives in biology  2011;3(7):10.1101/cshperspect.a005041 a005041.
Fibronectin (FN) is a multidomain protein with the ability to bind simultaneously to cell surface receptors, collagen, proteoglycans, and other FN molecules. Many of these domains and interactions are also involved in the assembly of FN dimers into a multimeric fibrillar matrix. When, where, and how FN binds to its various partners must be controlled and coordinated during fibrillogenesis. Steps in the process of FN fibrillogenesis including FN self-association, receptor activities, and intracellular pathways have been under intense investigation for years. In this review, the domain organization of FN including the extra domains and variable region that are controlled by alternative splicing are described. We discuss how FN–FN and cell–FN interactions play essential roles in the initiation and progression of matrix assembly using complementary results from cell culture and embryonic model systems that have enhanced our understanding of this process.
doi:10.1101/cshperspect.a005041
PMCID: PMC3119908  PMID: 21576254
13.  Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly 
The Journal of Cell Biology  2007;179(5):999-1009.
Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate/chloride antiporter whose function is impaired in several human chondrodysplasias. We show that DTDST is upregulated by dexamethasone stimulation of HT1080 fibrosarcoma cells and is required for fibronectin (FN) extracellular matrix deposition by these cells. DTDST imports sulfate for the modification of glycosaminoglycans. We find that N-sulfation of these chains is important for FN matrix assembly and that sulfation of cell surface proteoglycans is reduced in the absence of DTDST. Of the candidate HT1080 cell surface proteoglycans, only loss of syndecan-2 compromises FN assembly, as shown by syndecan-2 small interfering RNA knockdown. DTDST is both necessary and sufficient to induce FN matrix assembly in HT1080 cells. Knockdown of DTDST ablates FN matrix, whereas its overexpression increases assembly without dexamethasone stimulation. These results identify a previously unrecognized regulatory pathway for matrix assembly via modulation of a sulfate transporter and proteoglycan sulfation. These data raise the possibility that FN assembly defects contribute to chondrodysplasias.
doi:10.1083/jcb.200707150
PMCID: PMC2099202  PMID: 18056413
14.  A novel fibronectin binding site required for fibronectin fibril growth during matrix assembly 
The Journal of Cell Biology  2001;154(5):1081-1088.
Fibronectin (FN) assembly into a fibrillar extracellular matrix is a stepwise process requiring participation from multiple FN domains. Fibril formation is regulated in part by segments within the first seven type III repeats (III1–7). To define the specific function(s) of this region, recombinant FNs (recFNs) containing an overlapping set of deletions were tested for the ability to assemble into fibrils. Surprisingly, recFN lacking type III repeat III1 (FNΔIII1), which contains a cryptic FN binding site and has been suggested to be essential for fibril assembly, formed a matrix identical in all respects to a native FN matrix. Similarly, displacement of the cell binding domain in repeats III9–10 to a position close to the NH2-terminal assembly domain, as well as a large deletion spanning repeats III4–7, had no effect on assembly. In contrast, two deletions that included repeat III2, ΔIII1–2 and ΔIII2–5, caused significant reductions in fibril elongation, although binding of FN to the cell surface and initiation of assembly still proceeded. Using individual repeats in binding assays, we show that III2 but not III1 contains an FN binding site. Thus, these results pinpoint repeat III2 as an important module for FN–FN interactions during fibril growth.
doi:10.1083/jcb.200102034
PMCID: PMC2196193  PMID: 11535624
fibronectin; matrix assembly; type III repeats; RGD sequence; self-association
15.  Nanomaterials can Dynamically Steer Cell Responses to Biological Ligands 
Traditional tissue regeneration approaches to activate cell behaviours on biomaterials rely on the use of extracellular matrix based or soluble growth factor cues. In this article, we highlight a novel approach to dynamically steer cellular phenomena such as cell motility based on nanoscale substratum features of biological ligands. Albumin derived nanocarriers (ANCs) of variable nanoscale size features were functionalized with fibronectin III9–10 matrix ligand and effects on primary human keratinocyte activation were investigated. The display of fibronectin fragment from ANCs significantly enhanced cell migration compared to free ligands at equivalent concentrations. Notably, cell migration was influenced by the size of underlying ANCs even for variably sized ANCs presenting comparable levels of fibronectin fragment. For equivalent ligand concentrations, cell migration on the smaller-sized ANCs (30 nm and 50 nm) was significantly more enhanced compared to that on larger-sized ANCs (75 nm and 100 nm). In contrast, the enhancement of cell migration on nanocarriers was abolished by the use of immobilized biofunctionalized ANCs, indicating that “dynamic” nanocarrier internalization events underlie the role of nanocarrier geometry on the differential regulation of cell migration kinetics. Uptake studies using fluorescent ANCs indicated that larger-sized ANCs showed delayed endocytic kinetics and hence could present barriers for internalization during the cell adhesion and motility processes. Motile cells exhibited diminished migration upon exposure to clathrin-inhibitors, but not caveolin-inhibitors, suggesting the role of clathrin-mediated endocytosis in facilitating cell migratory responsiveness to the nanocarriers. Overall, a monotonic relationship was found between the degree of nanocarrier cytointernalization rate and cell migration rate, suggesting the possibility of designing biointerfacial features for dynamic control of cell migration. Thus, the major findings of this study are that (a) the presentation of a biorelevant ligand on a mobile nanocarrier can be used to sensitize cellular motility activation to the adhesion ligands; and (b) such nanocarrier interfaces can dynamically attune cell migration kinetics by “modulating” the uptake of the ligand-nanocarrier complex via nanocarrier size.
doi:10.1002/smll.201001518
PMCID: PMC3335745  PMID: 21213389
nanocomposites; cell motility; nanobiotechnology; biological interfaces
16.  The Role of the Exocyst in Matrix Metalloproteinase Secretion and Actin Dynamics during Tumor Cell Invadopodia Formation 
Molecular Biology of the Cell  2009;20(16):3763-3771.
Invadopodia are actin-rich membrane protrusions formed by tumor cells that degrade the extracellular matrix for invasion. Invadopodia formation involves membrane protrusions driven by Arp2/3-mediated actin polymerization and secretion of matrix metalloproteinases (MMPs) at the focal degrading sites. The exocyst mediates the tethering of post-Golgi secretory vesicles at the plasma membrane for exocytosis and has recently been implicated in regulating actin dynamics during cell migration. Here, we report that the exocyst plays a pivotal role in invadopodial activity. With RNAi knockdown of the exocyst component Exo70 or Sec8, MDA-MB-231 cells expressing constitutively active c-Src failed to form invadopodia. On the other hand, overexpression of Exo70 promoted invadopodia formation. Disrupting the exocyst function by siEXO70 or siSEC8 treatment or by expression of a dominant negative fragment of Exo70 inhibited the secretion of MMPs. We have also found that the exocyst interacts with the Arp2/3 complex in cells with high invasion potential; blocking the exocyst-Arp2/3 interaction inhibited Arp2/3-mediated actin polymerization and invadopodia formation. Together, our results suggest that the exocyst plays important roles in cell invasion by mediating the secretion of MMPs at focal degrading sites and regulating Arp2/3-mediated actin dynamics.
doi:10.1091/mbc.E08-09-0967
PMCID: PMC2777935  PMID: 19535457
17.  BPAG1e Maintains Keratinocyte Polarity through β4 Integrin–mediated Modulation of Rac 1 and Cofilin Activities 
Molecular Biology of the Cell  2009;20(12):2954-2962.
α6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.
doi:10.1091/mbc.E09-01-0051
PMCID: PMC2695802  PMID: 19403692
18.  Mislocalized Scaffolding by the Na-H Exchanger NHE1 Dominantly Inhibits Fibronectin Production and TGF-β Activation 
Molecular Biology of the Cell  2009;20(8):2327-2336.
Secretion and assembly of the extracellular matrix protein fibronectin regulates a number of normal cell and tissue functions and is dysregulated in disease states such as fibrosis, diabetes, and cancer. We found that mislocalized scaffolding by the plasma membrane Na-H exchanger NHE1 suppresses fibronectin expression, secretion, and assembly. In fibroblasts, wild-type NHE1 localizes to the distal margin of membrane protrusions or lamellipodia but a mutant NHE1-KRA2 lacking binding sites for PI(4,5)P2 and the ERM proteins ezrin, radixin, and moesin is mislocalized and found uniformly along the plasma membrane. Although NHE1 regulates intracellular pH homeostasis, fibronectin production is not regulated by changes in intracellular pH, nor is it attenuated in NHE1-deficient cells, indicating fibronectin expression is independent of NHE1 activity. However, fibronectin production is nearly absent in cells expressing NHE1-KRA2 because scaffolding by NHE1 is mislocalized. Additionally, secretion of active but not latent TGF-β is reduced and exogenous TGF-β restores fibronectin secretion and assembly. Our data indicate that scaffolding by NHE1-KRA2 dominantly suppresses fibronectin synthesis and TGF-β activation, and they suggest that NHE1-KRA2 can be used for obtaining a mechanistic understanding of how fibronectin production is regulated and speculatively for therapeutic control of dysregulated production in pathological conditions.
doi:10.1091/mbc.E08-08-0842
PMCID: PMC2669038  PMID: 19225158
19.  Tetraspanin Proteins Regulate Membrane Type-1 Matrix Metalloproteinase-dependent Pericellular Proteolysis 
Molecular Biology of the Cell  2009;20(7):2030-2040.
Membrane type-1 matrix metalloproteinase (MT1-MMP) supports tumor cell invasion through extracellular matrix barriers containing fibrin, collagen, fibronectin, and other proteins. Here, we show that simultaneous knockdown of two or three members of the tetraspanin family (CD9, CD81, and TSPAN12) markedly decreases MT1-MMP proteolytic functions in cancer cells. Affected functions include fibronectin proteolysis, invasion and growth in three-dimensional fibrin and collagen gels, and MMP-2 activation. Tetraspanin proteins (CD9, CD81, and TSPAN2) selectively coimmunoprecipitate and colocalize with MT1-MMP. Although tetraspanins do not affect the initial biosynthesis of MT1-MMP, they do protect the newly synthesized protein from lysosomal degradation and support its delivery to the cell surface. Interfering with MT1-MMP-tetraspanin collaboration may be a useful therapeutic approach to limit cancer cell invasion and metastasis.
doi:10.1091/mbc.E08-11-1149
PMCID: PMC2663921  PMID: 19211836
20.  A Shared Mechanism of Adhesion Modulation for Tenascin-C and Fibulin-1 
Molecular Biology of the Cell  2009;20(4):1141-1149.
Adhesion modulatory proteins are important effectors of cell–matrix interactions during tissue remodeling and regeneration. They comprise a diverse group of matricellular proteins that confer antiadhesive properties to the extracellular matrix (ECM). We compared the inhibitory effects of two adhesion modulatory proteins, fibulin-1 and tenascin-C, both of which bind to the C-terminal heparin-binding (HepII) domain of fibronectin (FN) but are structurally distinct. Here, we report that, like tenascin-C, fibulin-1 inhibits fibroblast spreading and cell-mediated contraction of a fibrin–FN matrix. These proteins act by modulation of focal adhesion kinase and extracellular signal-regulated kinase signaling. The inhibitory effects were bypassed by lysophosphatidic acid, an activator of RhoA GTPase. Fibroblast response to fibulin-1, similar to tenascin-C, was dependent on expression of the heparan sulfate proteoglycan syndecan-4, which also binds to the HepII domain. Therefore, blockade of HepII-mediated signaling by competitive binding of fibulin-1 or tenascin-C represents a shared mechanism of adhesion modulation among disparate modulatory proteins.
doi:10.1091/mbc.E08-06-0621
PMCID: PMC2642747  PMID: 19109427
21.  Fibrillin Assembly Requires Fibronectin 
Molecular Biology of the Cell  2009;20(3):846-858.
Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and nonelastic extracellular matrices. Proper assembly mechanisms are central to the formation and function of these microfibrils, and their properties are often compromised in pathological circumstances such as in Marfan syndrome and in other fibrillinopathies. Here, we have used human dermal fibroblasts to analyze the assembly of fibrillin-1 in dependence of other matrix-forming proteins. siRNA knockdown experiments demonstrated that the assembly of fibrillin-1 is strictly dependent on the presence of extracellular fibronectin fibrils. Immunolabeling performed at the light and electron microscopic level showed colocalization of fibrillin-1 with fibronectin fibrils at the early stages of the assembly process. Protein-binding assays demonstrated interactions of fibronectin with a C-terminal region of fibrillin-1, -2, and -3 and with an N-terminal region of fibrillin-1. The C-terminal half of fibrillin-2 and -3 had propensities to multimerize, as has been previously shown for fibrillin-1. The C-terminal of all three fibrillins interacted strongly with fibronectin as multimers, but not as monomers. Mapping studies revealed that the major binding interaction between fibrillins and fibronectin involves the collagen/gelatin-binding region between domains FNI6 and FNI9.
doi:10.1091/mbc.E08-08-0830
PMCID: PMC2633374  PMID: 19037100
22.  Epidermal Growth Factor–induced Enhancement of Glioblastoma Cell Migration in 3D Arises from an Intrinsic Increase in Speed But an Extrinsic Matrix- and Proteolysis-dependent Increase in Persistence 
Molecular Biology of the Cell  2008;19(10):4249-4259.
Epidermal growth factor (EGF) receptor-mediated cell migration plays a vital role in invasion of many tumor types. EGF receptor ligands increase invasiveness in vivo, but it remains unclear how consequent effects on intrinsic cell motility behavior versus effects on extrinsic matrix properties integrate to result in net increase of translational speed and/or directional persistence of migration in a 3D environment. Understanding this convolution is important for therapeutic targeting of tumor invasion, as key regulatory pathways for intrinsic versus extrinsic effects may not be coincident. Accordingly, we have undertaken a quantitative single-cell imaging study of glioblastoma cell movement in 3D matrices and on 2D substrata across a range of collagen densities with systematic variation of protease-mediated matrix degradation. In 3D, EGF induced a mild increase in cell speed and a strong increase in directional persistence, the latter depending heavily on matrix density and EGF-stimulated protease activity. In contrast, in 2D, EGF induced a similarly mild increase in speed but conversely a decrease in directional persistence (both independent of protease activity). Thus, the EGF-enhanced 3D tumor cell migration results only partially from cell-intrinsic effects, with override of cell-intrinsic persistence decrease by protease-mediated cell-extrinsic reduction of matrix steric hindrance.
doi:10.1091/mbc.E08-05-0501
PMCID: PMC2555959  PMID: 18632979
23.  Caenorhabditis elegans Teneurin, ten-1, Is Required for Gonadal and Pharyngeal Basement Membrane Integrity and Acts Redundantly with Integrin ina-1 and Dystroglycan dgn-1 
Molecular Biology of the Cell  2008;19(9):3898-3908.
The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1 deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants. We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and affects the function of somatic gonad precursor cells.
doi:10.1091/mbc.E08-01-0028
PMCID: PMC2526705  PMID: 18632986
24.  HIGH-YIELD ACTIVATION OF SCAFFOLD POLYMER SURFACES TO ATTACH CELL ADHESION MOLECULES 
Zirconium tetra(tert-butoxide) reacts with surface amide groups of polyamide nylon 6/6 to give (η2-amidate)zirconium complexes in high yield. These surface complexes react to bond the cell-adhesive peptide arginine-glycine-aspartic acid (RGD) to the polymer surface. A surface loading of 0.18 nmol/cm2 of RGD is achieved, which is 20−1000 times higher than previously reported attainable on natural or synthetic polymers by other strategies. Approximately 40% of the nylon surface is covered by the RGD which gives a surface that is both stable to hydrolysis and highly active for cell adhesion and spreading in vitro.
doi:10.1021/ja065217t
PMCID: PMC2569827  PMID: 17199287
25.  Differences in Regulation of Drosophila and Vertebrate Integrin Affinity by Talin 
Molecular Biology of the Cell  2008;19(8):3589-3598.
Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talin's role in regulating Drosophila αPS2βPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting αPS2βPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of αPS2βPS with those of human αIIbβ3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of αPS2βPS with those of αIIbβ3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type αIIbβ3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talin's integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila αPS2βPS affinity because of structural features inherent in the αPS2βPS extracellular and/or transmembrane domains.
doi:10.1091/mbc.E08-01-0085
PMCID: PMC2488300  PMID: 18508915

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