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MBC Video for Molecular Biology of the Cell: Volume 0, Issue 2003 -- Page E02-03-0163

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Figure 2. Small localized wounds in the squid fin nerve and giant axon. Different size wounds were made in a fin nerve of the squid with either a 25X 0.8NA multi-immersion lens (A-C) or a 63X 1.2NA (D-F) water immersion lens. Arrowheads point to the wound in the transmitted light images A and D. The corresponding fluorescent scar is shown in the x-y (C and F) and y-z (B and E) planes. The corresponding volumes for these fluorescent scars are 76, 10.9, and 376.1 µm3 (B,C) and 10.9 and 0.253 µm3 (E,F). G) Spot wound in the squid giant axon using a 63X 1.2NA water immersion lens. To confirm that damage was confined to the wound plane axonal transport was monitored by taking time series of Rhodamine 123 labeled mitochondria at the wound plane, 4 µm above and 4 µm below the wound plane (see movies above.mov and below.mov). The volume of the wound was 13 µm3. Arrowheads indicate the wound site in the out of plane images. Scale bar for all images = 10 µm.

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• below.mov

Figure 7. Wounding of the nuclear envelope of starfish oocytes. A) A multiphoton line scan was done across the nuclear envelope. This fluorescence image was obtained by multiphoton excitation at 800 nm. The nuclear interior has less autofluorescence than cytoplasm and is visible as the dark oval. The fluorescent scar is present at the site of the wound. The surface of the oocyte contains autofluorescent organelles. Scale bar = 20 µm. B) Oocytes were injected in the cytoplasm with either rhodamine 70 kD dextran or fluorescein 500 kD dextran then the nuclear envelope was wounded by multiphoton line scan as above. The entry of fluorescence dextran in the nucleus was monitored. The fluorescence level at the start was set to 0 and was normalized to twice the starting cytoplasmic fluorescence because the nuclear space for soluble markers is about twice that in cytoplasm due to volume exclusion by yolk platelets (Terasaki, 1994). C) Collapse of the nucleus after a line scan wound at the nuclear envelope. Images were taken every 30 sec (see collapse.mov). The first image, shown on the left, was taken 13 min after the wound. The image on the right was taken at 50 min, after the nucleus had collapsed. Scale bar = 20 µm. D) Oocytes with a collapsed nucleus are still able to undergo meiotic divisions. The same oocyte shown in the previous panel was exposed to the maturation hormone 2.0 hrs after the nuclear envelope wound. Images were taken every 30 sec beginning 2 min after application of the hormone (see division.mov). The left panel was taken at 91 min, soon after the first polar body had formed (black arrowhead; inset shows polar body at twice magnification). The small circle within the egg cytoplasm is the oil drop from the injection of 70 kD rhodamine dextran which helps in visualizing the nuclear envelope for wounding. The right panel was taken at 4.0 hrs, and shows the egg pronucleus (white arrowhead). Scale bars = 20 µm.

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