Collagens in avian neural crest development: distribution in vivo and migration-promoting ability in vitro

This study examines the spatiotemporal distribution of collagen (Col) types I-V and IX during neural crest development in vivo and their ability to support neural crest cell movement in vitro. Col I, III and IV were widespread throughout the embryo, including the neural crest migratory pathways, whereas Col II, V and IX preferentially localized to regions from which migrating neural crest cells were absent. Col I-IV and IX occurred both in association with basement membranes and within interstitial matrices, whereas Col V only was detected in juxtaposition to basement membranes. Although initially distributed throughout the rostrocaudal extent of the somitic sclerotome, Col I and III rearranged to the caudal portion with progressive neural crest cell migration through the rostral portion of the sclerotome. This rearrangement does not occur in neural crest-ablated embryos, suggesting that it is a direct consequence of neural crest cell migration. The perinotochordal matrix, avoided by neural crest cells, contained a metameric Col II/IX immunoreactivity along the rostrocaudal axis which alternated with that of Col I and III. In contrast, Col IV and V were not observed in this matrix, but lined the basement membranes of the notochord and ventrolateral neural tube. To determine their functional significance for neural crest cell migration in vivo, purified collagens were tested for their ability to promote neural crest cell motility in vitro. Neural crest cell migration on isolated collagens was most pronounced on Col I and IV, whereas Col II, V and the triple-helical fragment of Col VII were unable to support cell motility. Substrata created by copolymerization of Col I and fibronectin, or Col I and laminin-nidogen, supported cell motility better than Col I alone, whereas both Col V and a cartilage-type chondroitin sulfate proteoglycan reduced cell movement on Col I. Fibronectin bound to pre-immobilized monomeric Col I, II or V had a reduced ability to support neural crest cell movement when compared to fibronectin alone. A similar reduction was seen for Col IV bound to the low density heparan sulfate proteoglycan from the EHS mouse tumor. The results demonstrate that Col I-IX are differentially distributed in the early avian embryo. During neural crest development several of these collagens undergo dynamic reorganizations that correlate with the migration of neural crest cells. Furthermore, various collagens possess distinct abilities to support neural crest cell migration in vitro, and their migration-promoting activity can be modulated by their conformation and/or association with other matrix components

Molecular mechanisms of neural crest cell attachment and migration on types I and IV collagen

We have examined the mechanisms involved in the interaction of avian neural crest cells with collagen types I and IV (Col I and IV) during their adhesion and migration in vitro. For this purpose native Col IV was purified from chicken tissues, characterized biochemically and ultrastructurally. Purified chicken Col I and Col IV, and various proteolytic fragments of the collagens, were used in quantitative cell attachment and migration assays in conjunction with domain-specific collagen antibodies and antibodies to avian integrin subunits. Neural crest cells do not distinguish between different macromolecular arrangements of Col I during their initial attachment, but do so during their migration, showing a clear preference for polymeric Col I. Interaction with Col I is mediated by the α1β1 integrin, through binding to a segment of the α1(I) chain composed of fragment CNBr3. Neural crest cell attachment and migration on Col IV involves recognition of conformation-dependent sites within the triple-helical region and the noncollagenous, carboxyl-terminal NC1 domain. This recognition requires integrity of inter- and intrachain disulfide linkages and correct folding of the molecule. Moreover, there also is evidence that interaction sites within the NC1 domain may be cryptic, being exposed during migration of the cells in the intact collagen as a result of the prolonged cell-substratum contact. In contrast to Col I, neural crest cell interaction with Col IV is mediated by β1-class integrins other than α1β1

Cell Origin of Tumors and the Persistence of Cancer Propagating Cells in Tumor Lesions

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Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development

In this study, we describe the distribution of various classes of proteoglycans and their potential matrix ligand, hyaluronan, during neural crest development in the trunk region of the chicken embryo. Different types of chondroitin and keratan sulfate proteoglycans were recognized using a panel of monoclonal antibodies produced against specific epitopes on their glycosaminoglycan chains. A heparan sulfate proteoglycan was identified by an antibody against its core protein. The distribution of hyaluronan was mapped using a biotinylated fragment that corresponds to the hyaluronan-binding region of cartilage proteoglycans. Four major patterns of proteoglycan immunoreactivity were observed. (1) Chondroitin-6-sulfate-rich proteoglycans and certain keratin sulfate proteoglycans were absent from regions containing migrating neural crest cells, but were present in interstitial matrices and basement membranes along prospective migratory pathways such as the ventral portion of the sclerotome. Although initially distributed uniformly along the rostrocaudal extent of the sclerotome, these proteoglycans became rearranged to the caudal portion of the sclerotome with progressive migration of neural crest cells through the rostral sclerotome and their aggregation into peripheral ganglia. (2) A subset of chondroitin/keratan sulfate proteoglycans bearing primarily unsulfated chondroitin chains was observed exclusively in regions where neural crest cells were absent or delayed from entering, such as the perinotochordal and subepidermal spaces. (3) A subset of chondroitin/keratan sulfate proteoglycans was restricted to the perinotochordal region and, following gangliogenesis, was arranged in a metameric pattern corresponding to the sites where presumptive vertebral arches form. (4) Certain keratan sulfate proteoglycans and a heparan sulfate proteoglycan were observed in basement membranes and in an interstitial matrix uniformly distributed along the rostrocaudal extent of the sclerotome. After gangliogenesis, the neural crest-derived dorsal root and sympathetic ganglia contained both these proteoglycan types, but were essentially free of other chondroitin/keratan-proteoglycan subsets. Hyaluronan generally colocalized with the first set of proteoglycans, but also was concentrated around migrating neural crest cells and was reduced in neural crest-derived ganglia. These observations demonstrate that proteoglycans have diverse and dynamic distributions during times of neural crest development and chondrogenesis of the presumptive vertebrae. In general, chondroitin/keratan sulfate proteoglycans are abundant in regions where neural crest cells are absent, and their segmental distribution inversely correlates with that of neural crest-derived ganglia

centrifugal assay for fluorescence based cell adhesion adapted to the analysis of ex vivo cells and capable of determining relative binding strengths

Cell adhesion assays are widely used to identify novel cellula r ligands, novel cell surface receptors for these ligands and to eluc i date the mechanisms responsible for the underlying cellular and molecular interactions. We report here the development of a nove l centrifugal assay for fluorescence-based cell adhesion (CAFCA ) that offers a number of advantages over the currently available a s says. CAFCA is based on two centrifugation steps: one to allow fo r the synchronization of the initial cell-substratum contact and one to enable both a defined removal force to be exerted onto the cells fo r displacement of unbound cells and determination of the relativ e binding strengths of adhering cells. The fluorescently tagged cell s are monitored in specifically devised, disposable microplate assem blies by a two-sided fluorescence detection through the computer-in terfaced SPECTRAF LUO Rmicroplate fluorometer. The assay i s rapid, accurate, reproducible and adaptable to small numbers o f delicate primary cells that can ideally be labeled with the fluoro chrome calcein AM (or analogous vital fluorescent dyes). Mos t uniquely, CAFCA provides (i )means of assessing the precise num ber of cells bound to a given substratum out of the total amount o f cells contained within the population to be analyzed and (ii )a mean s of establishing the attachment strengths (i.e., dynes/cell) in a high number of samples/conditions simultaneously. CAFCA is therefor e expected to make a substantial methodological and conceptual con tribution to the range of available assays aimed at examining cellu lar interactions in vitro and promises the potential of being able to transpose automated versions of these tests for routine use in labo ratories

Avian neural crest cell migration is diversely regulated by the two major hyaluronan-binding proteoglycans PG-M/versican and aggrecan

It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r)of 360–530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to ‘attract’ moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve ‘haptotactic-like’ motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells

paving the path for invasion the polyedric role of lasp1 in cancer

Although usually referred to as a structural actin-binding protein, LIM and SH3 domain-containing protein may actually be dynamically involved in the control of a wide spectrum of cellular processes, by virtue of its interaction with several molecular partners. Alongside being ubiquitously expressed in physiological conditions, LIM and SH3 domain-containing protein is overexpressed in a growing number of human cancers, in which it may actively contribute to their aggressiveness by promoting cell proliferation and migration. In view of the recent findings, implicating the protein in cancer progression, we discuss here the most relevant discoveries highlighting the role of this versatile protein in various human tumors. The correlation between LIM and SH3 domain-containing protein expression levels in cancer and the poor outcome and metastatic behavior of tumors denotes the clinical significance of this protein and hints its potential value as a new cancer prognostic or even diagnostic biomarker. This may be decisive not only to optimize existing pharmacological regimes but also to delineate novel, more efficacious therapeutic and/or preventive approaches

Methylation Dynamics of RASSF1A and Its Impact on Cancer

5-methyl cytosine (5mC) is a key epigenetic mark entwined with gene expression and the specification of cellular phenotypes. Its distribution around gene promoters sets a barrier for transcriptional enhancers or inhibitor proteins binding to their target sequences. As a result, an additional level of regulation is added to the signals that organize the access to the chromatin and its structural components. The tumor suppressor gene RASSF1A is a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway. RASSF1A action stimulates mitotic arrest, DNA repair and apoptosis, and controls the cell cycle and cell migration. De novo methylation of the RASSF1A promoter has received much attention due to its increased frequency in most cancer types. RASSF1A methylation is preceded by histones modifications and could represent an early molecular event in cell transformation. Accordingly, RASSF1A methylation is proposed as an epigenetic candidate marker in many cancer types, even though an inverse correlation of methylation and expression remains to be fully ascertained. Some findings indicate that the epigenetic abrogation of RASSF1A can promote the alternative expression of the putative oncogenic isoform RASSF1C. Understanding the complexity and significance of RASSF1A methylation is instrumental for a more accurate determination of its biological and clinical role. The review covers the molecular events implicated in RASSF1A methylation and gene silencing and provides a deeper view into the significance of the RASSF1A methylation patterns in a number of gastrointestinal cancer types

Hyaluronan–CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell–substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9–specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA–CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells

Microvascular pericytes involvement in experimental autoimmune encephalomyelitis

In the CNS, pericytes are microvessel wall-encircling cells that, together with endothelial cells, perivascular glial endfeet and basement membrane, form the blood-brain barrier (BBB). Dysfunction of the BBB and migration of autoreactive T lymphocytes into the CNS are histopathological hallmarks of both Multiple Sclerosis (MS), a chronic demyelinating disease, and experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. The proteoglycan NG2, which has been described to accumulate within MS plaques and at spinal cord (SC) injury sites, is a primary component of pericytes, engaged in pericyte/endothelial cell interaction, proliferation and migration. To explore the role of NG2-expressing pericytes during neuroinflammation and BBB dysfunction, pericyte coverage (pericyte number/vessel length) and density (pericyte number/tissue volume) ratios were studied in brain microvessels by immunohistochemistry and laser confocal microscopy using specific pericyte markers, NG2, RGS5, and CD13. The observations were made in mice affected by MOG-induced chronic EAE with two different genetic C57BL/6 backgrounds: wild type (WT) and homozygous NG2 null (NG2-/-). In literature, NG2-/- mice did not exhibit gross phenotypic or vascular alterations, whereas our results demonstrated an unaltered pericyte density associated with slightly decreased pericyte coverage index and pericyte/endothelial cell ratio. These observations were confirmed in NG2-/- EAE-affected mice, that showed an attenuated disease severity and demyelination, and a milder BBB leakage and leukocyte infiltration, as compared with EAE WT. Taken together these results lend support to the idea of a direct involvement of NG2 proteoglycan in pericyte-endothelial cell interactions essential for the preservation of a proper BBB function