Davenport NR et al. (2016), Membrane dynamics during cellular wound repair.

XB-ART-52186

Mol Biol Cell 2016 Jul 15;2714:2272-85. doi: 10.1091/mbc.E16-04-0223.

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Membrane dynamics during cellular wound repair.

Davenport NR , Sonnemann KJ , Eliceiri KW , Bement WM .

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Cells rapidly reseal after damage, but how they do so is unknown. It has been hypothesized that resealing occurs due to formation of a patch derived from rapid fusion of intracellular compartments at the wound site. However, patching has never been directly visualized. Here we study membrane dynamics in wounded Xenopus laevis oocytes at high spatiotemporal resolution. Consistent with the patch hypothesis, we find that damage triggers rampant fusion of intracellular compartments, generating a barrier that limits influx of extracellular dextrans. Patch formation is accompanied by compound exocytosis, local accumulation and aggregation of vesicles, and rupture of compartments facing the external environment. Subcellular patterning is evident as annexin A1, dysferlin, diacylglycerol, active Rho, and active Cdc42 are recruited to compartments confined to different regions around the wound. We also find that a ring of elevated intracellular calcium overlaps the region where membrane dynamics are most evident and persists for several minutes. The results provide the first direct visualization of membrane patching during membrane repair, reveal novel features of the repair process, and show that a remarkable degree of spatial patterning accompanies damage-induced membrane dynamics.

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Species referenced: Xenopus laevis
Genes referenced: anxa1 cdc42 dysf fubp1 rho

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	FIGURE 1:. Exocytosis occurs upon wounding. (A) X. laevis oocyte wounded in the presence of extracellular fluorescent dextran (Texas Red dextran). Intrusion of dextran into the cell is initially limited to a region proximal to the wound (arrowheads), but then compartments distal to the wound undergo exocytosis and imbibe dextran (first event indicated by arrow). See Supplemental Movie S1. (B) An oblique view of an oocyte wounded in medium containing FM 1-43. Wound center is out of frame, toward the bottom of the image. Numbered arrowheads indicate sites of exocytosis, highlighted in enlarged insets. See Supplemental Movie S2. (C) Oblique view of an oocyte wounded in the presence of FM 1-43. Arrowheads highlight membranous compartments labeled by FM 1-43 during repair. See Supplemental Movie S3. Times in minutes:seconds, with t = 0:00 corresponding to moment of wounding. Scale bars, 10 μm (A), 5 μm (B, C), 1 μm (B, insets).
	FIGURE 2:. Extracellular dextran has limited access to the cytoplasm of wounded cells. En face view of oocyte prestained with R18 and wounded in the presence of extracellular fluorescent dextran (Oregon Green 488 dextran). Areas highlighted by boxes at t = 0:04 are enlarged to highlight wound dynamics. Enlargement of region 1 (bottom, left) shows fusion of two compartments (arrowheads) before rupture and imbibing of dextran at 0:14–0:16. Arrow shows fragment of compartment after rupture. Enlargement of region 2 (bottom, right) highlights a compartment at the wound edge (arrowheads) that is exposed to dextran upon rupture at 0:10. A fragment of the vesicle is visible after rupture (arrow). See Supplemental Movie S4. Times in minutes:seconds, with t = 0:00 corresponding to moment of wounding. Scale bars, 5 μm (top), 2.5 μm (bottom).
	FIGURE 3:. Vesicle–vesicle fusion at sites of plasma membrane disruption. An oocyte expressing eGFP-PKCβ-C2 (a probe for phosphatidylserine and calcium) was preincubated with R18 and wounded in the presence of fluorescent extracellular dextran (Alexa Fluor 647 dextran). Arrowhead at t = 0:00 denotes R18-labeled vesicle about to undergo fusion. eGFP-C2 probe begins to label R18-stained compartments (arrow at t = 0:02) soon after wounding. eGFP-C2–labeled compartment is contiguous with the plasma membrane (double arrows) and forms a barrier (asterisks) limiting dextran influx. See Supplemental Movie S5. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bar, 5 μm.
	FIGURE 4:. The C2 domain of PKCβ labels intracellular vesicles and wound patch. (A) Oblique, low-magnification view of oocyte expressing eGFP-PKCβ-C2 wounded in the presence of extracellular fluorescent dextran (Texas Red dextran). eGFP-PKCβ-C2 is rapidly recruited to the wound and labels vesicles clustered around its edge. A brightly stained structure (asterisk) of unknown identity appears in the wound pit. See Supplemental Movie S6. (A′) Enlargement of A starting at t = 0:30, showing tightly packed, C2-labeled vesicles at the wound edge (arrows) closely opposed to a nascent patch (green line bordering wound). A large vesicle fuses with the patch and imbibes dextran (0:32) and then fuses with another vesicle (0:36). (B) Cell expressing eGFP-PKCβ-C2 wounded in the presence of extracellular fluorescent dextran (Texas Red dextran). Single arrowheads denote membranous compartments incorporated into the patch; double arrowheads denote a region of the patch that progressively accumulates C2. See Supplemental Movie S7. (C) Oocyte expressing eGFP-PKCβ-C2 and stained with R18 before wounding. The vesicular structures labeled by the C2 probe upon wounding are also labeled by R18 (arrowhead). (C′) Enlargement of C showing that the C2 probe labels most, but not all, compartments labeled by R18 (R18-labeled vesicles free of C2 signal are denoted by the arrowheads). Times in minutes:seconds, with t = 0:00 corresponding to moment of wounding. Scale bars, 5 μm (A, A′, B, C), 2.5 μm (C′).
	FIGURE 5:. Vesicle–vesicle fusion continues through wound repair. (A) Oblique view of oocyte wounded in the presence of extracellular fluorescent dextran (Texas Red dextran) reveals compound exocytosis occurs at the wound edge (i.e., the site of patch formation). Compound exocytotic events are indicated by arrowheads. See Supplemental Movie S8. (B) Oblique, high-magnification view of the edge of a wound in an oocyte expressing eGFP-PKCβ-C2 wounded in the presence of extracellular fluorescent dextran (Alexa Fluor 647 dextran, pseudocolored red). Small, tightly packed vesicles near the wound edge (arrow) acquire C2, fuse with each other (0:48–0:54), and then fuse with another large compartment (double arrows), thereby acquiring dextran (arrowheads). See Supplemental Movie S9. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bars, 5 μm (A), 2 μm (B).
	FIGURE 6:. High-speed imaging of vesicle–vesicle fusion and rupture. (A) Still from a single-focal plane movie showing an oblique view of wounded oocyte expressing eGFP-PKCβ-C2. (A′) Enlargement and movie series from same wound depicted in A, showing growth, rupture, and collapse of vesicles (arrowhead) at the wound edge. Supplemental Movie S10 shows several cycles of growth, rupture, and collapse of vesicles. (B) Still from a high-resolution four-dimensional movie showing wounded oocyte expressing eGFP-PKCβ-C2 forming a large compartment (arrowheads) at the wound edge that was no longer visible several frames later (not shown). See Supplemental Movie S11. (B′) Enlargement of B along the xy- (left) and xz-planes (right) reveals fusion of smaller compartments before rupture of the apical portion of membrane and subsequent collapse of the entire compartment. See Supplemental Movies S12 and S13. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bars, 5 μm.
	FIGURE 7:. Human annexin A1 and dysferlin are recruited to membranous structures at wounds. (A) Low-magnification, en face view of wounded oocyte expressing eGFP-annexin A1. Annexin is recruited to cortical foci (0:18), vesicles at the wound edge (0:38), and a tight ring around the PM (1:18). See Supplemental Movie S14. (B) En face view of oocyte expressing eGFP-annexin A1 wounded in the presence of fluorescent extracellular dextran (Alexa Fluor 647 dextran). Annexin is recruited to foci (arrowheads) and vesicles before dextran incorporation (arrows). At increasing times postwounding, annexin also labels compartments in the wound interior (1:38). See Supplemental Movie S15. (C) Oblique view of a cell expressing eGFP-annexin A1 wounded in the presence of extracellular fluorescent dextran (not shown), showing eGFP-annexin A1 accumulating at a nascent patch (arrowheads). See Supplemental Movie S16. (C′) Still from C showing that eGFP-annexin A1–labeled compartments (green) fuse to form a barrier that excludes extracellular fluorescent dextran (red; Alexa Fluor 647 dextran) from the cytoplasm. (D) Coomassie-stained SDS–PAGE gel showing recombinant FLAG-eGFP-hDysferlin_isoform 1 purified from Sf9 cells. (E) Oocyte wounded after microinjection with recombinant FLAG-eGFP-hDysferlin. (E′) Enlargement of region indicated by an arrowhead in E, showing recruitment of dysferlin to a tight ring at the PM bordering the wound and to vesicles at wound edge. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bars, 5 μm.
	FIGURE 8:. Wound-induced lipid domains persist throughout membrane repair. (A) En face view of wounded eGFP-PKCβ-C1–expressing oocyte. C1 labels both small (arrowhead at t = 2:00) and large (arrow at t = 4:40) membrane structures upon wounding. See Supplemental Movie S17. (B) Cells expressing mRFP-PKCβ-C1 and eGFP-PKCβ-C2 reveal lipid patterning around wounds. C1 accumulates at the leading edge of wounds, whereas C2 is more broadly distributed. See Supplemental Movie S18. (C) A z-view of membrane dynamics after wounding, as seen with mRFP-PKCβ-C1 and eGFP-PKCβ-C2. Remodeling of wound area and patch continues long after the initial membrane disruption and loss of C2, with C1 evident on the presumptive patch, as well as in vesicles surrounding the patch (arrow). See Supplemental Movie S19. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bar, 5 μm.
	FIGURE 9:. C2 accumulation follows elevation of intracellular calcium. Oblique view of wounded oocyte expressing GCaMP5G and BFP-PKCβ-C2. Increased calcium (detected by GCaMP5G) initially extends far from the site of membrane damage before being confined to a ring around the wound edge at later time points. The pattern of C2 recruitment follows that of calcium elevation, although the GCaMP5G signal is broader and more diffuse, whereas C2 is concentrated on the PM and vesicles (arrowheads). See Supplemental Movie S20. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bar, 10 μm.
	FIGURE 10:. Patterning of elevated calcium, Rho, and Cdc42. (A) Low-magnification, en face view of wounded oocyte expressing BFP-wGBD, mCherry-2XrGBD, and GCaMP5G; the ring of calcium elevation overlaps the Rho and Cdc42 activity zones. See Supplemental Movie S21. (A′) Line scan from A, showing positions of active Rho and Cdc42 with respect to calcium. The increased calcium spans both zones, with peak intensity of the GCaMP5G signal closer to the peak of Cdc42 activity than that of Rho. (B) En face views of wounded oocyte expressing mRFP-wGBD and eGFP-rGBD from two different focal planes. z = 0 μm (top row, Supplemental Movie S22) corresponds to a surface view, whereas z = −3 μm (bottom row, Supplemental Movie S23) represents a subcortical view. Active Rho (arrowheads) and Cdc42 (arrows) are present on compartments other than the PM at wounds. Time in minutes:seconds, with t = 0:00 corresponding to the moment of wounding. Scale bars, 10 μm.
	FIGURE 11:. Schematic diagram of membrane dynamics at single-cell wounds. Representative time course of wound-induced membrane dynamics. (A) Resting plasma membrane with proximal intracellular membranous compartments. Double-headed arrows represent incipient membrane fusion events. Upon wounding (B), calcium enters the cell, causing fusion of intracellular vesicles with each other and the PM to form a patching membrane (C). Intracellular compartments continue to fuse with both the patch and the PM distal to the site of damage (D–F). The extracellular face of the patch ruptures outward (E; arrowheads) in a process called “explodosis”; this allows for resolution of a double-membrane structure and formation of a continuous barrier between the cytoplasm and the extracellular environment (F). Membrane fusion and remodeling events occur long after the initial resealing events.

References [+] :

Abreu-Blanco, Cell wound repair in Drosophila occurs through three distinct phases of membrane and cytoskeletal remodeling. 2011, Pubmed