The mechanisms and dynamics of αvβ3 integrin clustering in living cells

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility

Régulation différentielle du métabolisme des phosphoinositides par les intégrines avb3 et avb5 lors de la migration des cellules musculaires lisses

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

A specific endoplasmic reticulum export signal drives transport of stem cell factor (Kitl) to the cell surface

Stem cell factor, also known as Kit ligand (Kitl), belongs to the family of dimeric transmembrane growth factors. Efficient cell surface presentation of Kitl is essential for the migration, proliferation, and survival of melanocytes, germ cells, hemopoietic stem cells, and mastocytes. Here we demonstrate that intracellular transport of Kitl to the cell surface is driven by a motif in the cytoplasmic tail that acts independently of the previously described basolateral sorting signal. Transport of Kitl to the cell surface is controlled at the level of the endoplasmic reticulum (ER) and requires a C-terminal valine residue positioned at a distance of 19-36 amino acids from the border between the transmembrane and cytoplasmic domains. Deletion or substitution of the valine with other hydrophobic amino acids results in ER accumulation and reduced cell surface transport of Kitl at physiological expression levels. When these mutant proteins are overexpressed in the ER, they are transported by bulk flow to the cell surface albeit at lower efficiency. A fusion construct between Kitl and the green fluorescent protein-labeled extracellular domain of a temperature-sensitive mutant of vesicular stomatitis virus G protein revealed the valine-dependent recruitment into coat protein complex II-coated ER exit sites and vesicular ER to Golgi transport in living cells. Thus the C-terminal valine defines a specific ER export signal in Kitl. It is responsible for the capture of Kitl at coat protein complex II-coated ER exit sites, leading to subsequent cell surface transport under physiological conditions

Dimerization of Kit-ligand and efficient cell-surface presentation requires a conserved Ser-Gly-Gly-Tyr motif in its transmembrane domain

Kit-ligand (Kitl), also known as stem cell factor, is a membrane-anchored, noncovalently bound dimer signaling via the c-kit receptor tyrosine kinase, required for migration, survival, and proliferation of hematopoietic stem and germ cells, melanocytes, and mastocytes. Despite its fundamental role in morphogenesis and stem cell biology, the mechanisms that regulate Kitl dimerization are not well understood. By employing cell-permeable cross-linker and quantitative bimolecular fluorescence complementation of wild-type and truncated forms of Kitl, we determined that Kitl dimerization is initiated in the endoplasmic reticulum and mediated to similar levels by the transmembrane and the extracellular growth factor domain. Further biochemical and mutational analysis revealed a conserved Ser-Gly-Gly-Tyr-containing motif that is required for transmembrane domain dimerization and efficient cell-surface expression of Kitl. A novel intracellular capture assay with the Kitl transmembrane domain as bait revealed specific interactions with Kitl, but not with unrelated transmembrane proteins. During evolution, the transmembrane dimerization motif appeared in Kitl at the transition from teleosts to tetrapods, which correlates with the emergence of Kitl as a supporter of stem cell populations. Thus, transmembrane-mediated association of membrane-anchored growth factors consists of a novel mechanism to improve paracrine signaling and morphogenesis