October 1, 2008;
Live imaging of cell protrusive activity, and extracellular matrix assembly and remodeling during morphogenesis in the frog, Xenopus laevis.
Cell motility and matrix assembly have traditionally been studied in isolation because of a lack of suitable model systems in which both can be observed simultaneously. With embryonic tissues from the gastrulating frog Xenopus laevis we observe stages of fibronectin
fibrillogenesis coincident with protrusive activity in the overlying cells. Using live confocal time-lapse images collected from Cy3-tagged fibronectin
and plasma membrane
tethered green fluorescent protein, we describe the movement and the elaboration of a complex fibrillar network undergoing topological rearrangements of fibrils on the surface of an embryonic tissue
. Discrete processes of annealing, polymerization, stretching, breaking, and recoiling are recorded. Elaboration and maintenance of the complex topology of the extracellular matrix appears to require filamentous actin. These findings support a mechanical-model in which cell tractive forces elaborate the complex topological fibrillar network and are part of a homeostatic mechanism for the regulation of the extracellular matrix.
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Figure 1. Live imaging of fibrillogenesis. A: Epithelia-free explants are microdissected from either the animal cap (e, ectoderm) or the marginal zone (n, notochord; s, somite) of gastrulating embryos (d, dorsal anterior midline; bc, blastocoel) and cultured between two sheets of precast agarose (B). C: To collect high resolution confocal time-lapses, the tissue is gently compressed under a glass coverslip fragment (g), sandwiched between two sheets of agarose (the lower sheet as thin as 10 microns), glued in place with silicone grease (s). D,E: A sequence of frames from a confocal time-lapse of fibronectin-rich fibrils (red; labeled by incubation with a nonfunction blocking monoclonal antibody to Xenopus laevis fibronectin, 4H2, conjugated to the fluorophore Cy3) on the ventral surface of the explant (D) and a sequence of frames from the same time-lapses showing a scatter-labeled group of cells expressing RNA encoding a plasma membrane localizing GFP (E; green; GAP-43 GFP). Small fibronectin-rich spines can be seen forming perpendicular to thick fibrils (arrowheads).Download figure to PowerPoint
Figure 2. Remodeling the network topology of fibrils. A–D: Frames from representative time-lapse sequences showing dynamic changes to the organization and topology of the fibrillar network showing fibril growth (A), fibril shortening and thickening (B), trans-fibril annealing (C), and a sequence demonstrating stretching, breaking, and subsequent recoil of the fibril fragments (D). Fibrils are labeled by incubation of explants with a Cy3-coupled mAb to frog fibronectin. Scale bars = 10 μm.Download figure to PowerPoint
Figure 3. Movements of fibrils associated with lamellipodia and perinuclear regions. A: Frames from a representative time-lapse of the ventral surface of the ectoderm showing lamellipodia (green; GAP43-GFP) moving a cluster of fibrils (red; Cy3-coupled mAb). B: A lamellipodia (arrowhead at 6 min) extends from one cell (asterisk) to the ventral surface of the adjacent cell (hash mark) and retracts fully by 8 min elapsed time. C: A group of fibrils is moved by the lamellipodia between 4 and 6 min and subsequently anneals by 8 min. D,E: Frames from a time-lapse sequence showing fibrils gathered to the perinuclear mid-body of a cell (asterisk in E). F: No lamellipodia are seen near these fibrils although they move with the cell over the course of 14 min. The cluster of fibrils moved by the lamellipodia (A,C) and those associated with perinuclear regions of the cell (D,F) are enclosed by yellow circles at the start and end of each sequence.Download figure to PowerPoint
Figure 4. Disrupting cortical actin or cell adhesion rapidly alters fibril topology. A: Frames from a representative time-lapse sequence show cell plasma membrane and fibronectin-rich fibrils on the surface of animal cap ectoderm after addition of 4 μM Cytochalasin D. Within 10 min the complex network is reduced to a few thick cables. These cables remain on the ventral surface of the explant even after the cells have rounded up at 20 min. B: Frames from a time-lapse sequence where 1 mM ethylenediaminetetraacetic acid (EDTA) is added. EDTA also reduces network complexity but remaining cables penetrate deep into the tissue where the fibrils are held between cells (arrows in panel on far right in B).Download figure to PowerPoint