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2.  Cell–cell and cell–matrix interactions 
PMCID: PMC3302741
3.  Novel role of the muskelin–RanBP9 complex as a nucleocytoplasmic mediator of cell morphology regulation 
The Journal of Cell Biology  2008;182(4):727-739.
The evolutionarily conserved kelch-repeat protein muskelin was identified as an intracellular mediator of cell spreading. We discovered that its morphological activity is controlled by association with RanBP9/RanBPM, a protein involved in transmembrane signaling and a conserved intracellular protein complex. By subcellular fractionation, endogenous muskelin is present in both the nucleus and the cytosol. Muskelin subcellular localization is coregulated by its C terminus, which provides a cytoplasmic restraint and also controls the interaction of muskelin with RanBP9, and its atypical lissencephaly-1 homology motif, which has a nuclear localization activity which is regulated by the status of the C terminus. Transient or stable short interfering RNA–based knockdown of muskelin resulted in protrusive cell morphologies with enlarged cell perimeters. Morphology was specifically restored by complementary DNAs encoding forms of muskelin with full activity of the C terminus for cytoplasmic localization and RanBP9 binding. Knockdown of RanBP9 resulted in equivalent morphological alterations. These novel findings identify a role for muskelin–RanBP9 complex in pathways that integrate cell morphology regulation and nucleocytoplasmic communication.
PMCID: PMC2518711  PMID: 18710924
4.  Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis 
Molecular Biology of the Cell  2007;18(11):4591-4602.
Recurrence of carcinomas due to cells that migrate away from the primary tumor is a major problem in cancer treatment. Immunohistochemical analyses of human carcinomas have consistently correlated up-regulation of the actin-bundling protein fascin with a clinically aggressive phenotype and poor prognosis. To understand the functional and mechanistic contributions of fascin, we undertook inducible short hairpin RNA (shRNA) knockdown of fascin in human colon carcinoma cells derived from an aggressive primary tumor. Fascin-depletion led to decreased numbers of filopodia and altered morphology of cell protrusions, decreased Rac-dependent migration on laminin, decreased turnover of focal adhesions, and, in vivo, decreased xenograft tumor development and metastasis. cDNA rescue of fascin shRNA-knockdown cells with wild-type green fluorescent protein-fascin or fascins mutated at the protein kinase C (PKC) phosphorylation site revealed that both the actin-bundling and active PKC-binding activities of fascin are required for the organization of filopodial protrusions, Rac-dependent migration, and tumor metastasis. Thus, fascin contributes to carcinoma migration and metastasis through dual pathways that impact on multiple subcellular structures needed for cell migration.
PMCID: PMC2043557  PMID: 17855511
5.  Molecular Phylogeny of a RING E3 Ubiquitin Ligase, Conserved in Eukaryotic Cells and Dominated by Homologous Components, the Muskelin/RanBPM/CTLH Complex 
PLoS ONE  2013;8(10):e75217.
Ubiquitination is an essential post-translational modification that regulates signalling and protein turnover in eukaryotic cells. Specificity of ubiquitination is driven by ubiquitin E3 ligases, many of which remain poorly understood. One such is the mammalian muskelin/RanBP9/CTLH complex that includes eight proteins, five of which (RanBP9/RanBPM, TWA1, MAEA, Rmnd5 and muskelin), share striking similarities of domain architecture and have been implicated in regulation of cell organisation. In budding yeast, the homologous GID complex acts to down-regulate gluconeogenesis. In both complexes, Rmnd5/GID2 corresponds to a RING ubiquitin ligase. To better understand this E3 ligase system, we conducted molecular phylogenetic and sequence analyses of the related components. TWA1, Rmnd5, MAEA and WDR26 are conserved throughout all eukaryotic supergroups, albeit WDR26 was not identified in Rhizaria. RanBPM is absent from Excavates and from some sub-lineages. Armc8 and c17orf39 were represented across unikonts but in bikonts were identified only in Viridiplantae and in O. trifallax within alveolates. Muskelin is present only in Opisthokonts. Phylogenetic and sequence analyses of the shared LisH and CTLH domains of RanBPM, TWA1, MAEA and Rmnd5 revealed closer relationships and profiles of conserved residues between, respectively, Rmnd5 and MAEA, and RanBPM and TWA1. Rmnd5 and MAEA are also related by the presence of conserved, variant RING domains. Examination of how N- or C-terminal domain deletions alter the sub-cellular localisation of each protein in mammalian cells identified distinct contributions of the LisH domains to protein localisation or folding/stability. In conclusion, all components except muskelin are inferred to have been present in the last eukaryotic common ancestor. Diversification of this ligase complex in different eukaryotic lineages may result from the apparently fast evolution of RanBPM, differing requirements for WDR26, Armc8 or c17orf39, and the origin of muskelin in opisthokonts as a RanBPM-binding protein.
PMCID: PMC3797097  PMID: 24143168
6.  The Thrombospondins 
Thrombospondins are glycoproteins that associate with the extracellular matrix and have roles in cell signaling and tissue remodeling.
Thrombospondins are evolutionarily conserved, calcium-binding glycoproteins that undergo transient or longer-term interactions with other extracellular matrix components. They share properties with other matrix molecules, cytokines, adaptor proteins, and chaperones, modulate the organization of collagen fibrils, and bind and localize an array of growth factors or proteases. At cell surfaces, interactions with an array of receptors activate cell-dependent signaling and phenotypic outcomes. Through these dynamic, pleiotropic, and context-dependent pathways, mammalian thrombospondins contribute to wound healing and angiogenesis, vessel wall biology, connective tissue organization, and synaptogenesis. We overview the domain organization and structure of thrombospondins, key features of their evolution, and their cell biology. We discuss their roles in vivo, associations with human disease, and ongoing translational applications. In many respects, we are only beginning to appreciate the important roles of these proteins in physiology and pathology.
PMCID: PMC3179333  PMID: 21875984
7.  Association of fascin-1 with mortality, disease progression and metastasis in carcinomas: a systematic review and meta-analysis 
BMC Medicine  2013;11:52.
Fascin-1 is an actin-bundling protein expressed in many human carcinomas, although absent from most normal epithelia. Fascin-1 promotes filopodia formation, migration and invasion in carcinoma cells; in mouse xenograft tumor models it contributes to metastasis. Fascin-1 is an interesting candidate biomarker for aggressive, metastatic carcinomas but data from individual studies of human tumors have not yet been pooled systematically.
This systematic review was conducted in accordance with PRISMA guidelines, using fixed and random effects models, as appropriate, to undertake meta-analysis.
A total of 26 immunohistochemical studies of 5 prevalent human carcinomas were identified for meta-analysis. Fascin-1 was associated with increased risk of mortality for breast (pooled hazard ratio, (HR) = 2.58; 95% confidence interval (CI) 1.48 to 4.52; P = 0.001), colorectal (HR = 1.60 (1.37 to 1.86; P <0.001) and esophageal carcinomas (HR = 1.35; CI 1.13 to 1.60; P = 0.001). There was no evidence of association of fascin-1 with mortality in gastric and lung carcinomas. Fascin-1 was associated with increased risk of disease progression in breast (HR = 2.48; CI 1.38 to 4.46; P = 0.002) and colorectal carcinomas (HR = 2.12; CI 1.00 to 4.47; P = 0.05), but not with progression of lung carcinomas (HR = 0.95; CI 0.49 to 1.85; P = 0.9). Fascin-1 was associated with increased risk of lymph node metastasis in colorectal (pooled risk ratio (RR) = 1.47; CI 1.26 to 1.71; P <0.001) and gastric carcinomas (RR = 1.43; CI 1.21 to 1.70; P <0.001). There was no evidence of association of fascin-1 with lymph node metastasis in lung or esophageal carcinomas. Fascin-1 was associated with increased risk of distant metastasis in colorectal (RR = 1.70; CI 1.18 to 2.45; P = 0.004) and gastric carcinomas (RR = 1.93; CI 1.21 to 3.33; P = 0.02). No association with distant metastasis in esophageal carcinomas was observed. Pooling across all the carcinomas provided strong evidence for association of fascin-1 with increased risk of mortality (HR = 1.44; CI 1.24 to 1.68; P <0.001; n = 3,645), lymph node metastasis (RR = 1.36; CI 1.18 to 1.55; P <0.001; n = 2,906) and distant metastasis (1.76; 1.34 to 2.32; P <0.001; n = 1,514).
Fascin-1 is associated consistently with increased risk of mortality in breast, colorectal and esophageal carcinomas and with metastasis in colorectal and gastric carcinomas. The results were stable to various sensitivity analyses and did not vary by predefined subgroups. These data will assist rational decision making for focusing investigations of fascin-1 as a biomarker or therapeutic target onto the most relevant carcinomas.
PMCID: PMC3635876  PMID: 23442983
Fascin-1; carcinoma; mortality; metastasis; meta-analysis
8.  A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration 
Biology Open  2012;2(2):217-226.
Thrombospondins are multimeric extracellular matrix glycoproteins that play important roles in development, synaptogenesis and wound healing in mammals. We previously identified four putative thrombospondins in the genome of the starlet sea anemone Nematostella vectensis. This study presents the first analysis of these thrombospondins, with the goals of understanding fundamental roles of thrombospondins in the Eumetazoa. Reverse transcriptase PCR showed that each of the N. vectensis thrombospondins (Nv85341, Nv22035, Nv168100 and Nv30790) is transcribed. Three of the four thrombospondins include an RGD or KGD motif in their thrombospondin type 3 repeats at sites equivalent to mammalian thrombospondins, suggesting ancient roles as RGD integrin ligands. Phylogenetic analysis based on the C-terminal regions demonstrated a high level of sequence diversity between N. vectensis thrombospondins. A full-length cDNA sequence was obtained for Nv168100 (NvTSP168100), which has an unusual domain organization. Immunohistochemistry with an antibody to NvTSP168100 revealed labeling of neuron-like cells in the mesoglea of the retractor muscles and the pharynx. In situ hybridization and quantitative PCR showed that NvTSP168100 is upregulated during regeneration. Immunohistochemistry of the area of regeneration identified strong immunostaining of the glycocalyx, the carbohydrate-rich matrix coating the epidermis, and electron microscopy identified changes in glycocalyx organization during regeneration. Thus, N. vectensis thrombospondins share structural features with thrombospondins from mammals and may have roles in the nervous system and in matrix reorganization during regeneration.
PMCID: PMC3575656  PMID: 23430283
Cnidaria; Mesoglea; Extracellular matrix; Glycocalyx; Regeneration; Nervous system
9.  A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability 
BMC Biology  2012;10:72.
Fascin-1 is an actin crosslinking protein that is important for the assembly of cell protrusions in neurons, skeletal and smooth muscle, fibroblasts, and dendritic cells. Although absent from most normal adult epithelia, fascin-1 is upregulated in many human carcinomas, and is associated with poor prognosis because of its promotion of carcinoma cell migration, invasion, and metastasis. Rac and Cdc42 small guanine triphosphatases have been identified as upstream regulators of the association of fascin-1 with actin, but the possible role of Rho has remained obscure. Additionally, experiments have been hampered by the inability to measure the fascin-1/actin interaction directly in intact cells. We investigated the hypothesis that fascin-1 is a functional target of Rho in normal and carcinoma cells, using experimental approaches that included a novel fluorescence resonance energy transfer (FRET)/fluorescence lifetime imaging (FLIM) method to measure the interaction of fascin-1 with actin.
Rho activity modulates the interaction of fascin-1 with actin, as detected by a novel FRET method, in skeletal myoblasts and human colon carcinoma cells. Mechanistically, Rho regulation depends on Rho kinase activity, is independent of the status of myosin II activity, and is not mediated by promotion of the fascin/PKC complex. The p-Lin-11/Isl-1/Mec-3 kinases (LIMK), LIMK1 and LIMK2, act downstream of Rho kinases as novel binding partners of fascin-1, and this complex regulates the stability of filopodia.
We have identified a novel activity of Rho in promoting a complex between fascin-1 and LIMK1/2 that modulates the interaction of fascin-1 with actin. These data provide new mechanistic insight into the intracellular coordination of contractile and protrusive actin-based structures. During the course of the study, we developed a novel FRET method for analysis of the fascin-1/actin interaction, with potential general applicability for analyzing the activities of actin-binding proteins in intact cells.
PMCID: PMC3488970  PMID: 22883572
11.  The Evolution of Thrombospondins and Their Ligand-Binding Activities 
Molecular Biology and Evolution  2010;27(9):2187-2197.
The extracellular matrix (ECM) is a complex, multiprotein network that has essential roles in tissue integrity and intercellular signaling in the metazoa. Thrombospondins (TSPs) are extracellular, calcium-binding glycoproteins that have biologically important roles in mammals in angiogenesis, vascular biology, connective tissues, immune response, and synaptogenesis. The evolution of these complex functional properties is poorly understood. We report here on the evolution of TSPs and their ligand-binding capacities, from comparative genomics of species representing the major phyla of metazoa and experimental analyses of the oligomerization properties of noncanonical TSPs of basal deuterostomes. Monomeric, dimeric, trimeric, and pentameric TSPs have arisen through separate evolutionary events involving gain, loss, or modification of a coiled-coil domain or distinct domains at the amino-terminus. The relative transience of monomeric forms under evolution implicates a biological importance for multivalency of the C-terminal region of TSPs. Most protostomes have a single TSP gene encoding a pentameric TSP. The pentameric form is also present in deuterostomes, and gene duplications at the origin of deuterostomes and gene loss and further gene duplication events in the vertebrate lineage gave rise to distinct forms and novel domain architectures. Parallel analysis of the major ligands of mammalian TSPs revealed that many binding activities are neofunctions representing either coevolutionary innovations in the deuterostome lineage or neofunctions of ancient molecules such as CD36. Contrasting widely conserved capacities include binding to heparan glycosaminoglycans, fibrillar collagen, or RGD-dependent integrins. These findings identify TSPs as fundamental components of the extracellular interaction systems of metazoa and thus impact understanding of the evolution of ECM networks. The widely conserved activities of TSPs in binding to ECM components or PS2 clade integrins will be relevant to use of TSPs in synthetic extracellular matrices or tissue engineering. In contrast, the neofunctions of vertebrate TSPs likely include interactions suitable for therapeutic targeting without general disruption of ECM.
PMCID: PMC3107593  PMID: 20427418
thrombospondins; integrins; extracellular matrix; coiled coil; collagen
12.  The Evolution of Extracellular Matrix 
Molecular Biology of the Cell  2010;21(24):4300-4305.
We present a perspective on the molecular evolution of the extracellular matrix (ECM) in metazoa that draws on research publications and data from sequenced genomes and expressed sequence tag libraries. ECM components do not function in isolation, and the biological ECM system or “adhesome” also depends on posttranslational processing enzymes, cell surface receptors, and extracellular proteases. We focus principally on the adhesome of internal tissues and discuss its origins at the dawn of the metazoa and the expansion of complexity that occurred in the chordate lineage. The analyses demonstrate very high conservation of a core adhesome that apparently evolved in a major wave of innovation in conjunction with the origin of metazoa. Integrin, CD36, and certain domains predate the metazoa, and some ECM-related proteins are identified in choanoflagellates as predicted sequences. Modern deuterostomes and vertebrates have many novelties and elaborations of ECM as a result of domain shuffling, domain innovations and gene family expansions. Knowledge of the evolution of metazoan ECM is important for understanding how it is built as a system, its roles in normal tissues and disease processes, and has relevance for tissue engineering, the development of artificial organs, and the goals of synthetic biology.
PMCID: PMC3002383  PMID: 21160071
13.  The thrombospondins 
Thrombospondin-1 (TSP-1) was studied in the 1980s as a major component of platelet α-granules released upon platelet activation and also as a cell adhesion molecule. In 1993, we published a short review that discussed the exciting identification by molecular cloning of four additional vertebrate gene products related to TSP-1 [Current Biology 3 (1993) 188]. We put forward a structurally based classification for the newly identified proteins and discussed the functional and evolutionary implications of the new gene family. Since that time, the depth and breadth of knowledge on vertebrate TSPs and their functions in cells and tissues in health and disease has expanded into important new areas. Of particular interest is the new knowledge on the complex, domain and cell-type specific effects of TSPs on cell-signaling and cell-adhesion behaviour, the roles of TSP-1 and TSP-2 as anti-angiogenic agents, the roles of TSP-1 and TSP-2 in wound-healing, and associations of point mutations and polymorphisms in TSP-1, TSP-4 and TSP-5/COMP with human genetic diseases. The TSP family also now includes invertebrate members. In this article, we give the 2004 view on TSPs and our perspectives on the significant challenges that remain. Other articles in this issue discuss the functions of vertebrate TSPs in depth.
PMCID: PMC2885884  PMID: 15094109
Thrombospondin-1; Extracellular glycoproteins; TSR
14.  Fascin-1 Promoter Activity Is Regulated by CREB and the Aryl Hydrocarbon Receptor in Human Carcinoma Cells 
PLoS ONE  2009;4(4):e5130.
Fascin is an actin-bundling protein that is absent from most normal epithelia yet is upregulated in multiple forms of human carcinoma, where its expression correlates clinically with a poor prognosis. How fascin-1 transcription is activated in carcinoma cells is largely unknown, although the hypothesis of regulation by β-catenin signaling has received attention. The question is important because of the clinical significance of fascin expression in human carcinomas.
Methodology/Principal Findings
Through comparative genomics we made an unbiased analysis of the DNA sequence of the fascin-1 promoter region from six mammalian species. We identified two regions in which highly conserved motifs are concentrated. Luciferase promoter reporter assays for the human fascin-1 promoter were carried out in fascin-positive and -negative human breast and colon carcinoma cells, and in human dermal fibroblasts that are constitutively fascin-positive. In all fascin-positive cells, the region −219/+114 that contains multiple highly conserved motifs had strong transcriptional activity. The region −2953/−1582 appeared to contain repressor activity. By examining the effects of single or multiple point mutations of conserved motifs within the −219/+114 region on transcriptional reporter activity, we identified for the first time that the conserved CREB and AhR binding motifs are major determinants of transcriptional activity in human colon carcinoma cells. Chromatin immunoprecipitations for CREB, AhR or β-catenin from extracts from fascin-positive or -negative human colon carcinoma cells identified that CREB and AhR specifically associate with the −219/+114 region of the FSCN1 promoter in fascin-positive colon carcinoma cells. An association of β-catenin was not specific to fascin-positive cells.
Upregulation of fascin-1 in aggressive human carcinomas appears to have a multi-factorial basis. The data identify novel roles for CREB and AhR as major, specific regulators of FSCN-1 transcription in human carcinoma cells but do not support the hypothesis that β-catenin signaling has a central role.
PMCID: PMC2661145  PMID: 19340314
15.  Association of loss of epithelial syndecan-1 with stage and local metastasis of colorectal adenocarcinomas: An immunohistochemical study of clinically annotated tumors 
BMC Cancer  2008;8:185.
Syndecan-1 is a transmembrane proteoglycan with important roles in cell-cell and cell-extracellular matrix adhesion and as a growth factor co-receptor. Syndecan-1 is highly expressed by normal epithelial cells and loss of expression has been associated with epithelial-mesenchymal transition and the transformed phenotype. Loss of epithelial syndecan-1 has been reported in human colorectal adenocarcinomas, but whether this has prognostic significance remains undecided. Here we have examined syndecan-1 expression and its potential prognostic value with reference to a clinically annotated tissue microarray for human colon adenocarcinomas.
Syndecan-1 expression was examined by immunohistochemistry of a tissue microarray containing cores from 158 colorectal adenocarcinomas and 15 adenomas linked to a Cleveland Clinic, IRB-approved database with a mean clinical follow-up of 38 months. The Kaplan-Meier method was used to analyze the relationship between syndecan-1 expression and patient survival. Potential correlations between syndecan-1 expression and the candidate prognostic biomarker fascin were examined.
Syndecan-1 is expressed at the basolateral borders of normal colonic epithelial cells. On adenocarcinoma cells, syndecan-1 was present around cell membranes and in cytoplasm. In 87% of adenocarcinomas, syndecan-1 was decreased or absent; only 13% of patients had stained for syndecan-1 on more than 75% of tumor cells. Decreased syndecan-1 correlated with a higher TNM stage and lymph node metastasis and was more common in males (p = 0.042), but was not associated with age, tumor location or Ki67 index. Reduced tumor syndecan-1 staining also correlated with upregulation of stromal fascin (p = 0.016). Stromal syndecan-1 was observed in 16.6% of tumors. There was no difference in survival between patients with low or high levels of either tumor or stromal syndecan-1.
Syndecan-1 immunoreactivity was decreased in the majority of human colon adenocarcinomas in correlation with TNM stage and metastasis to local lymph nodes. In a small fraction of adenocarcinomas, syndecan-1 was upregulated in the local stroma. Syndecan-1 expression status did not correlate with patient survival outcomes. Combined analysis of syndecan-1 in relation to a potential prognostic biomarker, fascin, identified that loss of tumor syndecan-1 correlated significantly with strong stromal fascin staining.
PMCID: PMC2459187  PMID: 18590537
16.  Neuronal expression of muskelin in the rodent central nervous system 
BMC Neuroscience  2007;8:28.
The kelch repeat protein muskelin mediates cytoskeletal responses to the extracellular matrix protein thrombospondin 1, (TSP1), that is known to promote synaptogenesis in the central nervous system (CNS). Muskelin displays intracellular localization and affects cytoskeletal organization in adherent cells. Muskelin is expressed in adult brain and has been reported to bind the Cdk5 activator p39, which also facilitates the formation of functional synapses. Since little is known about muskelin in neuronal tissues, we here analysed the tissue distribution of muskelin in rodent brain and analysed its subcellular localization using cultured neurons from multiple life stages.
Our data show that muskelin transcripts and polypeptides are expressed throughout the central nervous system with significantly high levels in hippocampus and cerebellum, a finding that resembles the tissue distribution of p39. At the subcellular level, muskelin is found in the soma, in neurite projections and the nucleus with a punctate distribution in both axons and dendrites. Immunostaining and synaptosome preparations identify partial localization of muskelin at synaptic sites. Differential centrifugation further reveals muskelin in membrane-enriched, rather than cytosolic fractions.
Our results suggest that muskelin represents a multifunctional protein associated with membranes and/or large protein complexes in most neurons of the central nervous system. These data are in conclusion with distinct roles of muskelin's functional interaction partners.
PMCID: PMC1876237  PMID: 17474996
17.  Prognostic significance of fascin expression in advanced colorectal cancer: an immunohistochemical study of colorectal adenomas and adenocarcinomas 
BMC Cancer  2006;6:241.
Fascin is an actin bundling protein with roles in the formation of cell protrusions and motility of mesenchymal and neuronal cells. Fascin is normally low or absent from epithelia, but is upregulated in several epithelial neoplasms where it may contribute to an invasive phenotype. Here, we report on the prevalence and potential clinical significance of fascin expression in relation to the progression of colorectal adenocarcinoma and to tumor cell proliferation as measured by Ki67 index.
Conventional tissue sections of 107 colorectal adenomas and 35 adenocarcinomas were analyzed by immunohistochemistry for fascin and Ki67 expression.
Fascin expression and Ki67 proliferation index were also investigated by use of a tissue microarray containing cores from a further 158 colorectal adenocarcinomas and 15 adenomas linked to a CCF, IRB-approved database with a mean of 38 months of clinical follow-up. Survival analysis was carried out by the Kaplan-Meier and Cox regression methods.
Fascin was not expressed by the normal colonic epithelium. In conventional sections, 16% of adenomas and 26% of adenocarcinomas showed fascin expression in greater than 10% of the tumor cells. In the clinically-annotated tumors, fascin immunoreactivity was more common in tumors located in the proximal colon (p = 0.009), but was not associated with age, gender, or TNM stage. Patients with stage III/IV adenocarcinomas (n = 62) with strong fascin immunoreactivity had a worse prognosis than patients with low or absent fascin, (3-year overall survival of 11% versus 43% for fascin-negative patients; p = 0.023). In adenomas, fascin and Ki67 tended to be inversely correlated at the cellular level; this trend was less apparent in adenocarcinomas.
Fascin is upregulated in a proportion of adenomas, where its expression is often focal. Strong and diffuse expression was seen in a subset of advanced colorectal adenocarcinomas that correlated with shorter survival in stage III and IV patients. Fascin may have prognostic value as an early biomarker for more aggressive colorectal adenocarcinomas.
PMCID: PMC1615879  PMID: 17029629
18.  Phylogenomic analysis of vertebrate thrombospondins reveals fish-specific paralogues, ancestral gene relationships and a tetrapod innovation 
Thrombospondins (TSPs) are evolutionarily-conserved, extracellular, calcium-binding glycoproteins with important roles in cell-extracellular matrix interactions, angiogenesis, synaptogenesis and connective tissue organisation. Five TSPs, designated TSP-1 through TSP-5, are encoded in the human genome. All but one have known roles in acquired or inherited human diseases. To further understand the roles of TSPs in human physiology and pathology, it would be advantageous to extend the repertoire of relevant vertebrate models. In general the zebrafish is proving an excellent model organism for vertebrate biology, therefore we set out to evaluate the status of TSPs in zebrafish and two species of pufferfish.
We identified by bioinformatics that three fish species encode larger numbers of TSPs than vertebrates, yet all these sequences group as homologues of TSP-1 to -4. By phylogenomic analysis of neighboring genes, we uncovered that, in fish, a TSP-4-like sequence is encoded from the gene corresponding to the tetrapod TSP-5 gene. Thus, all TSP genes show conservation of synteny between fish and tetrapods. In the human genome, the TSP-1, TSP-3, TSP-4 and TSP-5 genes lie within paralogous regions that provide insight into the ancestral genomic context of vertebrate TSPs.
A new model for TSP evolution in vertebrates is presented. The TSP-5 protein sequence has evolved rapidly from a TSP-4-like sequence as an innovation in the tetrapod lineage. TSP biology in fish is complicated by the presence of additional lineage- and species-specific TSP paralogues. These novel results give deeper insight into the evolution of TSPs in vertebrates and open new directions for understanding the physiological and pathological roles of TSP-4 and TSP-5 in humans.
PMCID: PMC1464143  PMID: 16620379
19.  Comparative genomics of the syndecans defines an ancestral genomic context associated with matrilins in vertebrates 
BMC Genomics  2006;7:83.
The syndecans are the major family of transmembrane proteoglycans in animals and are known for multiple roles in cell interactions and growth factor signalling during development, inflammatory response, wound-repair and tumorigenesis. Although syndecans have been cloned from several invertebrate and vertebrate species, the extent of conservation of the family across the animal kingdom is unknown and there are gaps in our knowledge of chordate syndecans. Here, we develop a new level of knowledge for the whole syndecan family, by combining molecular phylogeny of syndecan protein sequences with analysis of the genomic contexts of syndecan genes in multiple vertebrate organisms.
We identified syndecan-encoding sequences in representative Cnidaria and throughout the Bilateria. The C1 and C2 regions of the cytoplasmic domain are highly conserved throughout the animal kingdom. We identified in the variable region a universally-conserved leucine residue and a tyrosine residue that is conserved throughout the Bilateria. Of all the genomes examined, only tetrapod and fish genomes encode multiple syndecans. No syndecan-1 was identified in fish. The genomic context of each vertebrate syndecan gene is syntenic between human, mouse and chicken, and this conservation clearly extends to syndecan-2 and -3 in T. nigroviridis. In addition, tetrapod syndecans were found to be encoded from paralogous chromosomal regions that also contain the four members of the matrilin family. Whereas the matrilin-3 and syndecan-1 genes are adjacent in tetrapods, this chromosomal region appears to have undergone extensive lineage-specific rearrangements in fish.
Throughout the animal kingdom, syndecan extracellular domains have undergone rapid change and elements of the cytoplasmic domains have been very conserved. The four syndecan genes of vertebrates are syntenic across tetrapods, and synteny of the syndecan-2 and -3 genes is apparent between tetrapods and fish. In vertebrates, each of the four family members are encoded from paralogous genomic regions in which members of the matrilin family are also syntenic between tetrapods and fish. This genomic organization appears to have been set up after the divergence of urochordates (Ciona) and vertebrates. The syndecan-1 gene appears to have been lost relatively early in the fish lineage. These conclusions provide the basis for a new model of syndecan evolution in vertebrates and a new perspective for analyzing the roles of syndecans in cells and whole organisms.
PMCID: PMC1464127  PMID: 16620374
20.  Functional Role of Syndecan-1 Cytoplasmic V Region in Lamellipodial Spreading, Actin Bundling, and Cell Migration 
Molecular Biology of the Cell  2005;16(8):3678-3691.
Cell protrusions contribute to cell motility and migration by mediating the outward extension and initial adhesion of cell edges. In many cells, these extensions are supported by actin bundles assembled by the actin cross-linking protein, fascin. Multiple extracellular cues regulate fascin and here we focus on the mechanism by which the transmembrane proteoglycan, syndecan-1, specifically activates lamellipodial cell spreading and fascin-and-actin bundling when clustered either by thrombospondin-1, laminin, or antibody to the syndecan-1 extracellular domain. There is almost no knowledge of the signaling mechanisms of syndecan-1 cytoplasmic domain and we have tested the hypothesis that the unique V region of syndecan-1 cytoplasmic domain has a crucial role in these processes. By four criteria—the activities of N-cadherin/V region chimeras, syndecan-1 deletion mutants, or syndecan-1 point mutants, and specific inhibition by a membrane-permeable TAT-V peptide—we demonstrate that the V region is necessary and sufficient for these cell behaviors and map the molecular basis for its activity to multiple residues located across the V region. These activities correlate with a V-region-dependent incorporation of cell-surface syndecan-1 into a detergent-insoluble form. We also demonstrate functional roles of syndecan-1 V region in laminin-dependent C2C12 cell adhesion and three-dimensional cell migration. These data identify for the first time specific cell behaviors that depend on signaling through the V region of syndecan-1.
PMCID: PMC1182307  PMID: 15930135
21.  Molecular phylogeny of the kelch-repeat superfamily reveals an expansion of BTB/kelch proteins in animals 
BMC Bioinformatics  2003;4:42.
The kelch motif is an ancient and evolutionarily-widespread sequence motif of 44–56 amino acids in length. It occurs as five to seven repeats that form a β-propeller tertiary structure. Over 28 kelch-repeat proteins have been sequenced and functionally characterised from diverse organisms spanning from viruses, plants and fungi to mammals and it is evident from expressed sequence tag, domain and genome databases that many additional hypothetical proteins contain kelch-repeats. In general, kelch-repeat β-propellers are involved in protein-protein interactions, however the modest sequence identity between kelch motifs, the diversity of domain architectures, and the partial information on this protein family in any single species, all present difficulties to developing a coherent view of the kelch-repeat domain and the kelch-repeat protein superfamily. To understand the complexity of this superfamily of proteins, we have analysed by bioinformatics the complement of kelch-repeat proteins encoded in the human genome and have made comparisons to the kelch-repeat proteins encoded in other sequenced genomes.
We identified 71 kelch-repeat proteins encoded in the human genome, whereas 5 or 8 members were identified in yeasts and around 18 in C. elegans, D. melanogaster and A. gambiae. Multiple domain architectures were identified in each organism, including previously unrecognised forms. The vast majority of kelch-repeat domains are predicted to form six-bladed β-propellers. The most prevalent domain architecture in the metazoan animal genomes studied was the BTB/kelch domain organisation and we uncovered 3 subgroups of human BTB/kelch proteins. Sequence analysis of the kelch-repeat domains of the most robustly-related subgroups identified differences in β-propeller organisation that could provide direction for experimental study of protein-binding characteristics.
The kelch-repeat superfamily constitutes a distinct and evolutionarily-widespread family of β-propeller domain-containing proteins. Expansion of the family during the evolution of multicellular animals is mainly accounted for by a major expansion of the BTB/kelch domain architecture. BTB/kelch proteins constitute 72 % of the kelch-repeat superfamily of H. sapiens and form three subgroups, one of which appears the most-conserved during evolution. Distinctions in propeller blade organisation between subgroups 1 and 2 were identified that could provide new direction for biochemical and functional studies of novel kelch-repeat proteins.
PMCID: PMC222960  PMID: 13678422
22.  A Role for Syndecan-1 in Coupling Fascin Spike Formation by Thrombospondin-1 
The Journal of Cell Biology  2001;152(6):1169-1182.
An important role of cell matrix adhesion receptors is to mediate transmembrane coupling between extracellular matrix attachment, actin reorganization, and cell spreading. Thrombospondin (TSP)-1 is a modulatory component of matrix expressed during development, immune response, or wound repair. Cell adhesion to TSP-1 involves formation of biochemically distinct matrix contacts based on stable fascin spikes. The cell surface adhesion receptors required have not been identified. We report here that antibody clustering of syndecan-1 proteoglycan specifically transduces organization of cortical actin and fascin bundles in several cell types. Transfection of COS-7 cells with syndecan-1 is sufficient to stimulate cell spreading, fascin spike assembly, and extensive protrusive lateral ruffling on TSP-1 or on syndecan-1 antibody. The underlying molecular mechanism depends on glycosaminoglycan (GAG) modification of the syndecan-1 core protein at residues S45 or S47 for cell membrane spreading and on the VC2 region of the cytoplasmic domain for spreading and fascin spike formation. Expression of the VC2 deletion mutant or GAG-negative syndecan-1 showed that syndecan-1 is necessary in spreading and fascin spike formation by C2C12 cells on TSP-1. These results establish a novel role for syndecan-1 protein in coupling a physiological matrix ligand to formation of a specific matrix contact structure.
PMCID: PMC2199199  PMID: 11257118
cell adhesion; extracellular matrix; proteoglycan; actin; protrusions
23.  Cell-Matrix Adhesions Differentially Regulate Fascin Phosphorylation 
Molecular Biology of the Cell  1999;10(12):4177-4190.
Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members α, γ, and λ, and PKCα localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCα activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin α5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.
PMCID: PMC25751  PMID: 10588651
24.  Characterization of Cell–Matrix Adhesion Requirements for the Formation of Fascin Microspikes 
Molecular Biology of the Cell  1997;8(11):2345-2363.
Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell–substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix requirements for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent on fibronectin, vitronectin, collagen IV, or platelet factor 4. However, both fascin microspikes and focal contacts were assembled by cells adherent on laminin-1. Using mixed substrata containing different proportions of TSP-1, and fibronectin, fascin microspike formation by H9c2 and C2C12 cells was found to be reduced on substrata containing 25% fibronectin and abolished on substrata containing 75% fibronectin. Adhesion to intermediate mixtures of TSP-1 and fibronectin resulted in coassembly of fascin microspikes and focal contacts, colocalization of fascin with actin stress fiber bundles and altered distributions of β1 integrins, cortical α-actinin, and tropomyosin. In cells adherent on 50% TSP-1:50% fibronectin, GRGDSP peptide treatment decreased focal contact assembly and altered cytoskeletal organization but did not inhibit microspike assembly. Treatment with chondroitin sulfate A or p-nitrophenol β-d-xylopyranoside decreased microspike formation and modified cytoskeletal organization but did not inhibit focal contact formation. In polarized migratory and postmitotic C2C12 cells, fascin microspikes and ruffles were localized at leading edges and TSP matrix deposition was also concentrated in this region. Depletion of matrix TSP by heparin treatment correlated with decreased microspike formation and cell motility. Thus, the balance of adhesive receptors ligated at the cell surface during initial cell–matrix attachment serves to regulate the type of substratum adhesion contact assembled and subsequent cytoskeletal organization. A role for fascin microspikes in cell motile behavior is indicated.
PMCID: PMC25712  PMID: 9362073
25.  Cell-Adhesive Responses to Tenascin-C Splice Variants Involve Formation of Fascin Microspikes 
Molecular Biology of the Cell  1997;8(10):2055-2075.
Tenascin-C is an adhesion-modulating matrix glycoprotein that has multiple effects on cell behavior. Tenascin-C transcripts are expressed in motile cells and at sites of tissue modeling during development, and alternative splicing generates variants that encode different numbers of fibronectin type III repeats. We have examined the in vivo expression and cell adhesive properties of two full-length recombinant tenascin-C proteins: TN-190, which contains the eight constant fibronectin type III repeats, and TN-ADC, which contains the additional AD2, AD1, and C repeats. In situ hybridization with probes specific for the AD2, AD1, and C repeats shows that these splice variants are expressed at sites of active tissue modeling and fibronectin expression in the developing avian feather bud and sternum. Transcripts incorporating the AD2, AD1, and C repeats are present in embryonic day 10 wing bud but not in embryonic day 10 lung. By using a panel of nine cell lines in attachment assays, we have found that C2C12, G8, and S27 myoblastic cells undergo concentration-dependent adhesion to both variants, organize actin microspikes that contain the actin-bundling protein fascin, and do not assemble focal contacts. On a molar basis, TN-ADC is more active than TN-190 in promoting cell attachment and irregular cell spreading. The addition of either TN-190 or TN-ADC in solution to C2C12, COS-7, or MG-63 cells adherent on fibronectin decreases cell attachment and results in decreased organization of actin microfilament bundles, with formation of cortical membrane ruffles and retention of residual points of substratum contact that contain filamentous actin and fascin. These data establish a biochemical similarity in the processes of cell adhesion to tenascin-C and thrombospondin-1, also an “antiadhesive” matrix component, and also demonstrate that both the adhesive and adhesion-modulating properties of tenascin-C involve similar biochemical events in the cortical cytoskeleton. In addition to these generic properties, TN-ADC is less active in adhesion modulation than TN-190. The coordinated expression of different tenascin-C transcripts during development may, therefore, provide appropriate microenvironments for regulated changes in cell shape, adhesion, and movement.
PMCID: PMC25670  PMID: 9348542

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