Endothelial cells assemble two distinct α6β4-containing vimentin-associated structures: roles for ligand binding and the β4 cytoplasmic tail

Abstract

The alpha6beta4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the alpha6beta4 integrin. Because endothelial cells lose expression of alpha6beta4 in culture, we expressed recombinant alpha6beta4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing alpha6beta4. Using immunofluorescence microscopy, we found that recombinant alpha6beta4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the absence of an exogenous laminin substrate. This localization is regulated by an intracellular mechanism, because the beta4 cytoplasmic domain is sufficient to direct a reporter domain (IL-2R) to the fibrillar structures independently of recombinant alpha6beta4. In addition, this IL-2R-beta4 chimera is sufficient to recruit the intermediate filament-associated protein HD1/plectin to these fibrillar structures and this also occurs in the absence of recombinant alpha6beta4. The fibrillar localization pattern, as well as the recruitment of HD1/plectin, requires the first and second fibronectin type III repeats and the connecting segment of the beta4 tail. In addition, when endothelial cells are provided a laminin 5-rich matrix, recombinant alpha6beta4 redistributes from the fibrillar structure to type I hemidesmosome-like structures. The beta4 cytoplasmic domain can also direct a reporter domain to these type I hemidesmosome-like structures; however, this process is dependent upon the expression of recombinant alpha6beta4 Biochemical analysis indicates that both the fibrillar and the type I hemidesmosome-like structures are associated with the vimentin intermediate filament cytoskeleton. Thus, the results illustrate that endothelial cells have the essential components necessary to assemble at least two distinct alpha6beta4-containing and vimentin-associated structures on their basal surface and that the alpha6beta4 cytoplasmic tail and the availability of specific alph6beta4 ligands regulate receptor localization to these structures.

The vimentin cytoskeleton: when polymer physics meets cell biology

“…Collagen I is the most abundant extracellular matrix protein, and recent studies show that cell adhesion to collagen via β1 integrins is dependent on the actin cross-linking protein filamin A and vimentin [68,119,120]. The effects of vimentin on focal adhesion assembly remains largely unclear, in part because vimentin does not generally concentrate sufficiently at these sites to be evident by immunofluorescence imaging the way it does at desmosomes or hemidesmosomes that engage different transmembrane receptors [121,122]. An exception to this rule is the finding that in the lung cancer cell line A549, complexes containing β4 integrin cluster along vimentin at the cell periphery.…”

HLA Class I Molecules Partner with Integrin β 4 to Stimulate Endothelial Cell Proliferation and Migration

“…1E). Indeed, the integrin b 4 subunit mediates the adherence of cells to the ECM by forming type II hemidesmosomes in endothelial cells (23). Incubation of endothelial cells with W6/32 showed that HLA-I was also localized in a punctuate pattern on the basal surface of the cells.…”

Contribution of Endothelial Laminin-Binding Integrins to Cellular Processes Associated with Angiogenesis

“…We observed the polarized localization of α6β4 during the migratory phase of endothelial tube morphogenesis in planar co-culture, similar to the polarized endothelial expression of α6β4 at the tips of endothelial sprouts during neovascularization in human neonatal foreskins [ 40 ]. We had previously demonstrated that α6β4 associates with the vimentin intermediate filament by mechanisms that require the β4 subunit cytoplasmic domain [ 47 , 48 ]. Interestingly, recent studies demonstrated that α6β4 localizes with vimentin in puncta in lamellipodia at the leading edge, to promote the migration of epithelial cells [ 49 ].…”