Macropinocytosis-mediated membrane recycling drives neural crest migration by delivering F-actin to the lamellipodium

Individual cell migration requires front-to-back polarity manifested by lamellipodial extension. At present, it remains debated whether and how membrane motility mediates this cell morphological change. To gain insights into these processes, we perform live imaging and molecular perturbation of migrating chick neural crest cells in vivo. Our results reveal an endocytic loop formed by circular membrane flow and anterograde movement of lipid vesicles, resulting in cell polarization and locomotion. Rather than clathrin-mediated endocytosis, macropinosomes encapsulate F-actin in the cell body, forming vesicles that translocate via microtubules to deliver actin to the anterior. In addition to previously proposed local conversion of actin monomers to polymers, we demonstrate a surprising role for shuttling of F-actin across cells for lamellipodial expansion. Thus, the membrane and cytoskeleton act in concert in distinct subcellular compartments to drive forward cell migration.

Cell migration is central to embryogenesis, organogenesis, and cancer metastasis (1, 2). Individual migrating cells rapidly change their shape via cycles of protrusion and retraction (3, 4), raising the question of how a cell distributes its limited amount of membrane to maintain polarized morphology without affecting membrane integrity. Early studies, based on observing the movement of cross-linked antigen on the cell surface, proposed a “membrane flow” model to explain the role of membrane recycling during cell locomotion (5–8). This two-dimensional (2D) model posits that cells undergo clathrin-mediated endocytosis in the rear of the cell, which generates anterograde flow of lipid vesicles and retrograde flow of membrane (Fig. 1A). Such a retrograde membrane flow could enable membrane proteins to generate traction forces against the extracellular matrix (ECM) to push the cell forward; thus, this model in theory explains how mechanical forces could drive cell migration (9, 10). However, other research presents conflicting data (11), likely due to the use of different experimental systems and cell tagging reagents. Moreover, most studies of individual cell motility use cultured cells or simple organisms, such that little is known about how membrane and vesicle motion coordinate to influence three-dimensional (3D) morphological changes of migrating cells in higher vertebrates.Open in a separate windowFig. 1.Circular membrane flow across migrating cells. (A) The 2D “membrane flow” model from top view. Endocytosis at the posterior end produces vesicles that move toward the anterior end (arrows inside the cytoplasm); these vesicles integrate into the lamellipodium and then translate into retrograde membrane flow (arrows outside the cell). (B) The 3D “treadmilling” model from lateral view. F-actin (red) is distributed underneath the plasma membrane. In the basal side of the lamellipodium, actin displays a net polymerization toward the cell’s anterior end. “−” and “+” represents depolymerization and polymerization end of actin, respectively. (C) Schematic illustration of explant culture and imaging. An ∼500-μm-thick transverse slice is dissected through the trunk of virally labeled chicken embryos for confocal time-lapse imaging. Successive movies of individual migrating cells are collected at different positions from the apical to the basal sides of the cell. For quantitative analysis, a coordinate system is used in which 0 denotes the center of the cell body. (D) Live imaging reveals a stereotypical fashion of cell migration (Top). A cell protrudes the lamellipodium (red arrow) and then progressively retracts its body (yellow arrow) toward the anterior end. The cell surface is computationally segmented with each optical slice pseudocolored. The surface area of individual segmented slices is measured, and the results are presented in SI Appendix, Fig. S1 C and D. (Middle) Top view. (Bottom) Lateral view. (Scale bar: 5 µm.) (E–H) Quantification of membrane flow based on the photo-conversion experiment (see SI Appendix, Fig. S1 E–G for details). On the basal side, anterior intensity of the red fluorescence is higher following photo-conversion (t = 15 s) (rank sum test in frame 6, P < 0.001, n = 7 cells) (E), suggesting anterior membrane flow (Schematic, F). This scenario is reversed on the apical side (rank sum test in frame 6, P < 0.001, n = 8 cells) (G and H).In addition to the lipid portion of the plasma membrane that maintains fluidity of the cell boundary, the underlying cytoskeleton provides rigidity to the cell surface (12). A “treadmilling” model was used to explain cytoskeletal regulation of lamellipodial function (13, 14). According to this model, actin polymerization at the cell’s leading edge and actin depolymerization at the back of the network (Fig. 1B) cause relative displacement to the cytosol, which subsequently “pulls” the rounded cell body. Yet, it is unclear whether and how actin turnover on the cell’s basal side is coupled with other actin pools and membrane flow throughout the cells.To address these long-standing cell biology questions in vivo, we directly visualize membrane and cytoskeletal behaviors in migrating neural crest cells at the trunk level of chicken embryos. The neural crest is one of the most migratory of embryonic cell types (15), initiating movement via an epithelial to mesenchymal transition from the neural tube (16). These multipotent cells then migrate throughout the periphery as individuals (17, 18), differentiating into diverse cells types including peripheral neurons, glia, and melanocytes of the skin (19). As neural crest-derived cells are prone to give rise to adult cancers, including melanoma, neuroblastoma, and gliomas, their innate migratory mode appears to be recapitulated during cancer metastasis (16). By combining live imaging with quantitative analysis, we extract dynamic molecular and cellular information about cell migration and utilize perturbation approaches to challenge it.