Large plasma membrane disruptions are rapidly resealed by Ca2+- dependent vesicle-vesicle fusion events
Journal of Cell Biology 139: 63-74 (1997)

Mark Terasaki, Katsuya Miyake, and Paul L. McNeil

Figure 1, bottom panel
To make a 40 x 10 micron wound in the cell surface, a polylysine coated micropipette was laid against the side of a sea urchin egg and rapidly moved away from the surface. Fluorescein stachyose (FS) was present in the sea water (SW) and was observed by confocal microscopy. The lack of spreading of FS into the egg indicates that the wound is sealed rapidly. Frames were obtained at 1 sec intervals. Movie, 508 Kb

Figure 2
The same wounding procedure used in figure 1, except that the egg was pre-injected with the calcium indicator Ca Green dextran in order to observe the cytosolic Ca concentration at the wound site. A large increase is seen at the time of wounding, but this rapidly diminishes. This is further evidence that the wound heals rapidly. Frames were obtained at 1 sec intervals. Movie, 508 Kb.

Figure 4
The following movie (429K) shows fluorescent dextran in calcium free sea water being injected into a starfish oocyte. The dextran diffuses freely from the injection site. During the injection, the dextran is preceded by an oil cap in the needle that gets expelled first. Interval between each frame is 1 sec.

This movie (363K) shows fluorescent dextran in sea water being injected. The dextran is confined to the injection site, probably due to a rapid fusion of intracellular membranes that encapsulates it. During the injection, the dextran is preceded by an oil cap in the needle that is expelled first. Interval between each frame is 1 sec.

Figure 9
Fluorescein stachyose (FS) was injected into starfish oocytes and then the cytoplasm was pulled up into an injection pipette. The cytoplasm was then extruded into either CFSW or SW. The FS, which is present in the cytosol, diffused away when the cytoplasm was injected into CFSW, but it was trapped when the cytoplasm was injected into SW. This is consistent with Ca induced fusion of intracellular membranes which encloses the cytosol within a new boundary. Injection into CFSW and injection into sw. Images were obtained at 1 sec intervals.

Figure 10
Sea urchin egg cytoplasm was stratified by centrifugation. The centrifuged egg was injected with fluorescein stachyose, and then the clear cytoplasm or the yolk-containing cytoplasm was drawn up into an injection pipette. When the clear cytoplasm was extruded into SW, most of the FS diffused away. When the yolk-containing cytoplasm was extruded into SW, most of the FS was trapped. This suggests that it is the yolk platelets that are stimulated to undergo fusion by calcium. Images obtained at 1 sec intervals, except that the last image of both sequences was 42 seconds after the beginning of the extrusion. Extrusion of clear cytoplasm and extrusion of yolk platelet-containing cytoplasm .

Movie making
The confocal microscope was set to scan continuously, and each frame was recorded on an optical memory disk recorder (OMDR). The OMDR recording was done manually (by hitting the record button every time the scan hit the bottom of the screen) or later, automatically by use of a trigger circuit designed by J. Galbraith.

The OMDR images were captured on a Mac computer with a miroMotion DC20 capture card using "stop motion" in Adobe Premiere at 640 x 480 pixels. The movie clip was made into a quicktime movie (no compression) and then opened as a stack in NIH Image. The images were cropped and scaled by 50% using NIH Image stack macros. The stack was saved as a quicktime movie with no compression and set to play at 6 frames per second. Lastly, the movies were opened in Movie Player (quicktime vers 2.5) and saved as a "self-contained movie, playable on non-Apple computers".

Historical notes pictures of R. Chambers and L.V. Heilbrunn

To contact the authors:

Mark Terasaki Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032

Kats Miyake. Second Department of Anatomy, Fukushima Medical College, Fukushima 960-12, Japan

Paul McNeil Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912

Most of the experiments were done at the Marine Biological Laboratory at Woods Hole, MA. M. Terasaki was associated with the Laboratory of Neurobiology, NINDS, NIH, Bethesda, MD during part of this project.