Modular control of endothelial sheet migration

Abstract

Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood. Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell-cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space. In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell-cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate. Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell-cell coordination.[Keywords: Collective migration; functional genomics; fibroblast growth factor; RNAi; signaling modules] Supplemental material is available at http://www.genesdev.org.

Migration of cells in a social context

“…Contrary to previous work (22,(24)(25)(26)29), these cells form neither 2D sheets nor 3D structures, nor are they highly polarized, and their single-cell migratory behavior is established (13,14,16). The microfluidic cell culture platform hosts independent and isolated culture conditions in each of the isolated 96 polymethylsiloxane (PDMS) chambers (34-nL volume) that mimic physiological conditions more plausibly than traditional cell-culture environments in which concentrations of, for instance, secreted signaling molecules are diluted into large volumes of surrounding fluid.…”

“…Previous work on single-cell migration has focused on isolated (11)(12)(13)(14)(15)(16)(17)(18)(19)(20) or strongly polarized and aligning (21,22) cell types, mostly using population-averaged bulk assays (23) or simple observations in a social context (2,3). However, strongly cross-correlated cell motion and collective substrate deformation has been found to arise in mechanically interlinked cells transmitting forces through both cell-cell linkages and the substrate (24)(25)(26)(27)(28)(29). These studies revealed useful information on cell migration, but because in general the relevant interactions in a social context and their relative importance are not established, migratory behavior of cells in a social context remains as one of the major unresolved problems in biology (30).…”

Direct detection of guidance receptor activity during border cell migration

“…Uniform ligand, or no ligand, could therefore give directional signaling output if a group is not free but organized in an inherently polarized manner. This effect could be responsible for, or contribute to, directional cell movement in other types of collective migration-for example, sheet or slug migration in tissue culture models or in vivo (6,17).…”

RNAi-based functional selection identifies novel cell migration determinants dependent on PI3K and AKT pathways

“…This selection method identified 91 shRNAs that accelerated or impaired migration (Tables 1 and 2). Sixty-two of the 91 identified target genes have been previously associated with cell migration by RNAi screen [8][9][10]13,16 (Supplementary Tables 2 and 3). The remaining 29 target genes had not been previously identified as cell migration regulators by RNAi screen.…”

“…shRNA libraries have been used to perform genetic screens in tissue culture cells for a variety of phenotypes 7 . Recent studies have analysed the closures of scratches in cellular monolayers after growth factor stimulation, using siRNA library targeting kinase and phosphatase genes 8,9 . siRNA-based screening was performed to identify regulators of multiple cell adhesion complex formation 10 , and RNAi screening to identify inhibitors of cell migration using SKOV-3 cells (a highly motile ovarian carcinoma cell line) 11 .…”