Bves and NDRG4 regulate directional epicardial cell migration through autocrine extracellular matrix deposition

Directional cell movement is universally required for tissue morphogenesis. Although it is known that cell/matrix interactions are essential for directional movement in heart development, the mechanisms governing these interactions require elucidation. Here we demonstrate that a novel protein/protein interaction between blood vessel epicardial substance (Bves) and N-myc downstream regulated gene 4 (NDRG4) is critical for regulation of epicardial cell directional movement, as disruption of this interaction randomizes migratory patterns. Our studies show that Bves/NDRG4 interaction is required for trafficking of internalized fibronectin through the “autocrine extracellular matrix (ECM) deposition” fibronectin recycling pathway. Of importance, we demonstrate that Bves/NDRG4-mediated fibronectin recycling is indeed essential for epicardial cell directional movement, thus linking these two cell processes. Finally, total internal reflectance fluorescence microscopy shows that Bves/NDRG4 interaction is required for fusion of recycling endosomes with the basal cell surface, providing a molecular mechanism of motility substrate delivery that regulates cell directional movement. This is the first evidence of a molecular function for Bves and NDRG4 proteins within broader subcellular trafficking paradigms. These data identify novel regulators of a critical vesicle-docking step required for autocrine ECM deposition and explain how Bves facilitates cell-microenvironment interactions in the regulation of epicardial cell–directed movement

Identification of a novel intracellular interaction domain essential for Bves function.

While Blood vessel epicardial substance (Bves) confers adhesive properties, the molecular mechanism of regulating this activity is unknown. No predicted functional motifs in this highly conserved integral membrane protein, other than the transmembrane domain, have been identified. Here, we report for the first time that Bves interacts with itself through an intracellular interaction domain that is essential for its intercellular adhesion activity. Glutathione-S-transferase (GST) pull-down and SPOTs analyses mapped this domain to amino acids 268-274 in the intracellular C-terminus. Site-directed mutagenesis revealed that lysines 272 and 273 are essential for homodimerization and cell adhesion. Human corneal cells transfected with wild-type Bves trafficked the protein to the cell surface, assembled junction complexes and formed epithelial sheets. In contrast, cells expressing Bves mutated at these positions did not form continuous epithelial sheets or maintain junctional proteins such as ZO-1 and E-cadherin at the membrane. A dramatic reduction in transepithelial electrical resistance was also observed indicating a functional loss of tight junctions. Importantly, expression of mutated Bves in epithelial cells promoted the transformation of cells from an epithelial to a mesenchymal phenotype. This study is the first to demonstrate the essential nature of any domain within Bves for maintenance of epithelial phenotype and function

Sequences of primers used to generate Bves constructs

<p>Note: Bold and underline characters show the mutation sites and hyphen shows the deletion site</p><p>S, Sense ; AS, Antisense ; T an, annealing temperature</p

Immunostaining with anti-cytokeratin and anti-vimentin.

<p>Immunostaining with anti-cytokeratin (panels A through D), anti-vimentin (panels E through H) and merge (panels I through L) are given. A, E, I, Parental cells show high levels of cytokeratin and little vimentin staining. Transfection with WT Bves (B, F, J) shows similar staining patterns as parental cells. In contrast, cell lines expressing KK-Mut Bves (C, G, K) have a subpopulation of vimentin-positive cells and KK-Del Bves expressing cells (D, H, L) show nearly complete conversion to a vimentin-positive phenotype. (Scale Bar 100 µm)</p

Detection of endogenous and transfected Bves in human corneal epithelial cells (HCE).

<p>Anti-Bves staining (top panels, A–D), anti-Flag staining (middle panels, E–H) and merge (bottom panels, I–L) are shown. A, Endogenous Bves is detected at the parental cell membrane in confluent monolayers. E, Parental cells do not react with anti-Flag. B, Endogenous Bves is expressed in WT Bves transfected cells. F, Transfected WT Flag-tagged Bves traffics to the cell membrane J, where it co-localizes with anti-Bves staining, which detects endogenous protein. C, G, K, Transfection of KK-Mut Bves and D, H, L, KK-Del Bves show a general loss of membrane staining for both endogenous and transfected protein. Inset K, importantly, sporadic green staining at the membrane is seen in merged images with transfected KK-Mut Bves suggesting localization of endogenous but not transfected protein. (Scale Bar 100 µm)</p

Expression of ZO-1 and E-cadherin in HCE cells.

<p>A, E, Parental HCE cells B, F, HCE cell lines transfected with WT Bves, C, G, KK-Mut Bves, and D, H, KK-Del Bves were stained with ZO-1 in panels A through D and E-cadherin in panels E through H. Note the loss of peripheral ZO-1 and E-cadherin staining in KK-Mut Bves and KK-Del Bves cells. (Scale Bar 100 µm)</p

Bves constructs and GST pull-down assay.

<p>A, Diagram of GST-fusion proteins and deletion constructs. GST C- and N-terminal Bves proteins and serially deleted Flag-tagged Bves proteins. B, GST pull-down assay with C- or N-terminal Bves. Bacterial lysates of GST-Bves fusion proteins were reacted with COS-7 cell lysates transfected with Flag-tagged WT Bves and analyzed with Western blots using an anti-Flag antibody. The potential effects of glycosylation were also tested. Control band (WT Bves protein with or without glycosylation) are shown on lane a and b. Only GST C-terminal Bves reacted in this assay regardless of glycosylation state (lanes d and g) C, Serial deletion analysis of C-terminal interaction. GST C-terminal Bves was reacted with Del-5 Bves, Del-4 Bves and Del-3 Bves and processed for Western blotting to detect interactions. Del-5 Bves and Del-4 Bves (lanes a and b) interact with the GST C-terminal Bves while Del-3 Bves does not.</p

SPOTs protein mapping.

<p>A, Diagram of SPOTs blot (synthesized peptides). Twenty 13-mer peptides (SPOTs) were synthesized from the sequence of C-terminal Bves (amino acids 232-301) and fixed on a cellulose membrane for reaction with WT Bves or Del-3 Bves. B, Spots binding assay with WT Bves. A binding with WT Bves is clearly detected with peptides 11-13. Peptide 10 is likely a false positive, since the signal appears circumferential to the spot of protein. An additional unpredicted reaction is detected with peptide 18. C, Spots binding assay with Del-3 Bves. No binding of Del-3 Bves is detected with peptides 11-13 while peptide 18 is reactive suggesting a non-specific or interaction domain-independent association. D, Alanine substitution analysis. Numbers over the amino acid symbols are the amino acid number of WT Bves. Peptide 12, that showed positive binding at panel B was substituted at each amino acid position with alanine and incubated with WT Bves cell lysate. Substitution of lysines at positions aa 272 and aa 273 with alanine abolished binding with WT Bves protein.</p

L-cell aggregation assay.

<p>Aggregation assays with various cell lines were performed using a standard hanging-drop method. A, Parental L-cells. B, L-cells transfected with WT Bves, C, KK-Mut Bves and D, KK-Del Bves. (Scale Bar 50 µm)</p

Identification of a novel Bves function: regulation of vesicular transport

Blood vessel/epicardial substance (Bves) is a transmembrane protein that influences cell adhesion and motility through unknown mechanisms. We have discovered that Bves directly interacts with VAMP3, a SNARE protein that facilitates vesicular transport and specifically recycles transferrin and β-1-integrin. Two independent assays document that cells expressing a mutated form of Bves are severely impaired in the recycling of these molecules, a phenotype consistent with disruption of VAMP3 function. Using Morpholino knockdown in Xenopus laevis, we demonstrate that elimination of Bves function specifically inhibits transferrin receptor recycling, and results in gastrulation defects previously reported with impaired integrin-dependent cell movements. Kymographic analysis of Bves-depleted primary and cultured cells reveals severe impairment of cell spreading and adhesion on fibronectin, indicative of disruption of integrin-mediated adhesion. Taken together, these data demonstrate that Bves interacts with VAMP3 and facilitates receptor recycling both in vitro and during early development. Thus, this study establishes a newly identified role for Bves in vesicular transport and reveals a novel, broadly applied mechanism governing SNARE protein function