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First published online 13 June 2006
doi: 10.1242/jcs.03006

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Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site

Misaki Marine Biological Station, University of Tokyo, 1024 Ko-Ajiro, Misaki, Miura, Kanagawa 238-0225, Japan

View larger version (72K): [in a new window]   Fig. 1. Disruption of the cell membrane induces disassembly and reassembly of MTs. (A) A PtK2 cell expressing -tubulin-EGFP was wounded by a glass needle in 1.8 mM Ca2+ Ringer's solution. The asterisk indicates the site of cell membrane disruption. The membrane disruption initially induced MT disassembly around the wound site. Arrows indicate examples of disassembled MTs. (B,C) Plus-end tips of disassembled MTs approached the wound site in 1.8 mM Ca2+ Ringer's solution. Colored dots indicate selected MT ends.

View larger version (166K): [in a new window]   Fig. 2. Visualization of EB1-GFP reveals the approach of MT plus-end tips toward the wound site. A PtK2 cell expressing EB1-GFP was wounded in 1.8 mM Ca2+ Ringer's solution. The asterisk indicates the site of the cell membrane disruption. A slight increase in the number of bright EB1-GFP `comets' was observed throughout the cell. Furthermore, numerous EB1 comets appeared around the wound site within 30 seconds of membrane disruption (arrowheads). Some comets approached the wound site (arrows). Movies are available in supplementary material (Movies 1 and 2).

View larger version (77K): [in a new window]   Fig. 3. Disassembly and reassembly of MTs upon cell membrane disruption is dependent on extracellular Ca2+ concentration. (A) A PtK2 cell expressing -tubulin-EGFP was wounded in 0.4 mM-Ca2+ Ringer's solution. The asterisk indicates the site of cell membrane disruption. The cell membrane disruption induced MT disassembly around the wound site, but the propagation of the MT disassembled area was slower than in 1.8 mM Ca2+ Ringer's solution. Arrows indicate disassembled MTs. (B,C) MT ends did not approach the wound site in 0.4 mM Ca2+ Ringer's solution. Colored dots indicate selected MT ends.

View larger version (115K): [in a new window]   Fig. 4. Recruitment of EB1 to the MTs upon cell membrane disruption is dependent on Ca2+. A PtK2 cell expressing EB1-GFP was wounded in 0.4 mM-Ca2+ Ringer's solution. The asterisk indicates the site of cell membrane disruption. A slight increase in the number of bright EB1-GFP comets was observed throughout the cell, however there was no obvious increase in the number of EB1-comets and directional movement of the comets around the wound site in this condition. A movie is available in supplementary material (Movie 3).

View larger version (128K): [in a new window]   Fig. 5. Disassembly and reassembly of MTs upon cell membrane disruption are independent of PKC activity. A PtK2 cell expressing EB1-GFP was wounded in 1.8 mM Ca2+ Ringer's solution containing 1 µM Gö-6976. The asterisk indicates the site of cell membrane disruption. Numerous EB1-GFP comets appeared within 30 seconds of membrane disruption (arrowheads). Some comets approached the wound site (arrows). A movie is available in supplementary material (Movie 4). A slight increase in the number of bright EB1-GFP comets was also observed throughout the cell.

View larger version (74K): [in a new window]   Fig. 6. Cell membrane disruption stimulates the delivery of NBD lipids toward the wound site along MTs in a PKC- and temperature-dependent manner. PtK2 cells were incubated with 5 µM NBD C6-ceramide/BSA for 30 minutes at 4°C, washed, warmed to 37°C for 30 minutes and wounded with a glass needle. Arrows indicate the sites of cell membrane disruption. Left panel, before wounding; right panel, 5 minutes after wounding. (A) control; (B) 1 µM Gö-6976; (C) 1 µg/ml nocodazole; (D) 20°C. Images are inverted for easier visualization.

View larger version (18K): [in a new window]   Fig. 7. Nocodazole treatment blocked membrane resealing at the second wound. (A) Cells were loaded with 1 µM calcein-AM for 1 hour in the presence or absence of 1 µg/ml nocodazole and wounded twice (arrows). Membrane disruption is indicated by the loss of calcein fluorescence. Bars mark the completion times of membrane resealing. Units of fluorescent intensity were normalized to 100% before the initial wounding. (B) Comparison of membrane resealing rates at the initial and second wounds. The resealing rates were defined as the inverse of the resealing time in seconds. For cells that failed to reseal, the rate was defined as zero. Values are the means ± s.e.m.

View larger version (29K): [in a new window]   Fig. 8. Summary of MT rearrangement and membrane traffic toward the wound site. Ca2+ influx induced by cell membrane disruption initially stimulates MT disassembly around the wound site. Within 30 seconds of membrane disruption, EB1 is recruited to the MTs especially around the wound site, and stimulates elongation of MTs. Apparently vesicles are transported from the region of the TGN toward the wound site to refill the vesicle pool that was depleted by the initial wound.

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