Morphogenesis, as illustrated by melanocyte migration and homing to the skin, requires cadherin adhesion, integrin-dependent migration and Kit-ligand growth factor signaling. However, it is not known how Kit ligand regulates integrin or cadherin-dependent intraepidermal melanocyte behavior. To answer this question, we developed specific 2-dimensional (2D) and 3D culture systems analyzing how soluble or immobilized Kit-ligand-regulated melanocyte migration on vitronectin and laminin, or within a monolayer of kidney epithelial cells. In a 2D system, soluble Kit ligand stimulated integrin-dependent melanoblast migration and chemotaxis and accelerated integrin turnover. In contrast, immobilized, but not soluble, Kit ligand, enhanced integrin-dependent melanocyte spreading on suboptimal laminin concentrations. In 3D, membrane-bound Kit ligand induced intraepithelial melanocyte proliferation, survival, and tight adhesion to epithelial cells, while cleavable Kit ligand was less effective. In contrast, melanocyte motility was independent of membrane-bound Kit ligand, but increased in the presence of the cleavable Kit-ligand isoform. Transmembrane-dimerization or basolateral-targeting mutants of Kit ligand altered intraepithelial melanocyte localization, survival, and adhesion to epithelial cells. These data and the identification of c-kit/Kit-ligand clusters at cell contacts suggest that membrane-bound Kit ligand captures cell surface-expressed c-kit, providing mechanical anchoring and survival signaling within intraepithelial niches, and thereby defining a new mechanism for melanocyte homeostasis and requirement for environmental niches.

Kit ligand (KitL) and its tyrosine kinase receptor c-kit are critical for germ cells, melanocytes, mastocytes, and hematopoietic stem cells. Alternative splicing of KitL generates membrane-bound KitL (mb-KitL) or soluble KitL, providing survival or cell migration, respectively. Here we analyzed whether c-kit can function both as an adhesion and signaling receptor to mb-KitL presented by the environmental niche. At contacts between fibroblasts and MC/9 mast cells, mb-KitL, and c-kit formed ligand/receptor clusters that formed stable complexes, which resisted dissociation by c-kit blocking mAbs and provided cell anchorage under physiological shear stresses. Clusters recruited tyrosine-phosphorylated proteins and induced spatially restricted F-actin polymerization. Mutational analysis of c-kit demonstrated kinase-independent mb-KitL/c-kit clustering, anchorage, F-actin polymerization, and Tyr567-dependent cluster phosphorylation. Kinase inhibition of c-kit by imatinib reduced cluster coalescence, but allowed cluster phosphorylation and F-actin polymerization, which required PI3K recruitment and a newly identified juxtamembrane residue. Synergies between integrin and c-kit-mediated spreading and adhesion of MC/9 cells were studied in vitro on immobilized-KitL/fibronectin surfaces. While c-kit blocking antibodies prevented spreading, imatinib blocked spreading induced by soluble- but not immobilized KitL. Thus, "mechanical" activation of c-kit provides signaling, niche-anchorage, and synergies with integrin-mediated adhesion, which is independent of kinase function and resistant to c-kit kinase inhibitors.-

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.

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