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| Description | The yolk sac of a killifish embryo contains a population of deep cells which migrate through the subepithelial space, and this sequence follows one such cell crawling within the normal matrices and cell substrates of an intact embryo. At the two-cell stage, this Fundulus heteroclitus embryo was injected with a GFP-actin plasmid. Expression is mosaic, and the non-expressing cells of the embryo act as a backdrop for the dramatic migrations of these wandering cells. If you watch carefully, you can see a few cells expressing very low amounts of fluorescent actin, giving context to the fact that this deep cell is not migrating on glass. This sequence begins as a deep cell extends a large protrusion out of the plane of focus, and the cell body can be seen to pour into the leading edge. This cell migrates with a structured lamellipodium, which shows retrograde waves of actin as the cell progresses. A thin cortical band of labeled actin can be seen around the entire periphery. The leading edge cytoplasm is clearly distinct from the rest of the cell material. (The cell migrates out of the field of view, and when the movie shifts to follow it, the cell changes its behavior and undergoes circus movements. Free of organized cortical actin, the hyaline cytoplasm that forms the hemispheric bleb protrudes beyond the existing cortex. Once the bleb forms, the fluorescent line of actin is seen to disappear, and a new cortical array is established as the bleb spreads. Near the very end of the sequence, the cell reestablishes a leading edge at the top of the screen, and the cell resumes migration. Fundulus deep cells often switch between structured protrusive migration and circus movements.) I have taught cell migration to large numbers of undergraduates, and this sequence has helped them 'get' actin dynamics. After discussing actin polymerization, actin-binding proteins, and calcium flux, the class could understand why and how a cell has a net rearward flow of actin during lamellipodial extension. Students compare these sequences with those of cells crawling on cover slips, and with EM images, to understand how the actin-rich leading edge really is the engine for cell migration. Deep cells are approximately 20 microns in length, and migrate rapidly with rates up to 18 microns/min. |
| Title | Run Silent, Run Deep |
| Author(s) | Rachel Fink |
| Materials & Methods | Fundulus heteroclitus embryos at the two-celled stage were injected with a GFP-actin plasmid (Clontech), and allowed to develop through epiboly. Fluorescent cells were filmed using a Perkin Elmer spinning disc scan head mounted on a Nikon inverted TE300. Images were collected with 100x objective, N.A. 1.4, using an Orca ER cooled CCD camera. Exposure times were approximately 500 msec. Shutters for illumination were driven by Metamorph software, and images were collected approximately 5 seconds apart. |
| Citation | Fink R. Run silent, run deep. ASCB Image & Video Library. October 2007:VID-34. Available at: http://cellimages.ascb.org |
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| Contributors | Pat Wadsworth (University of Massachusetts at Amherst, Amherst, MA). Work conducted by RF at Mount Holyoke College, South Hadley, MA. |
| Resource Type | Video |
| Digital Format | quicktime; Digitization process: Recompressed with H.264 codec, automatic keyframes selected, and data rate reduced (restricted to 768 kbs optimized for streaming) in Quicktime Pro. New file size: 1.82 Mb; Duration: 00:00:26.02; Dimensions: 650 x 515; FPS: 15; Data rate: 587.46 kbs; Codec: H.264; Software required: Apple Quicktime 7 player: http://www.apple.com/quicktime/download/win.html |
| Original Date | Original resource created on June 23, 2004. |
| Original Format | Original resource: metamorph stack; received from authors as quicktime file; File size: 20.69 Mb; Duration: 00:00:26.00; Dimensions: 650 x 515 pixels; FPS: 10; Data rate: 6.67 Mbs. |
| License Details | Terms for non-commercial use: http://cellimages.ascb.org/cdm4/terms.php; for commercial use contact cellimages@ascb.org. |
| Publisher | The American Society for Cell Biology |
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