Abstract
The rearward positioning of the nucleus is a characteristic feature of most migrating cells. Studies using wounded monolayers of fibroblasts and myoblasts have shown that this positioning is actively established before migration by the coupling of dorsal actin cables to the nuclear envelope through nesprin-2G and SUN2 linker of nucleoskeleton and cytoskeleton (LINC) complexes. During nuclear movement, these LINC complexes cluster along the actin cables to form adhesive structures known as transmembrane actin-associated nuclear (TAN) lines. Here we described experimental procedures for studying nuclear movement and TAN lines using wounded monolayers of fibroblasts and myoblasts, the acquisition of data using immunofluorescence microscopy and live-cell imaging, and methods for data analysis and quantification.
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Abbreviations
- CH:
-
Calponin homology
- DAPI:
-
4′,6-Diamidino-2-phenylindole
- GFP :
-
Green fluorescent protein
- KASH:
-
Klarsicht, Anc1 and Syne homology
- LINC:
-
Linker of nucleoskeleton and cytoskeleton
- LPA:
-
Lysophosphatidic acid
- MEFs:
-
Mouse embryo fibroblasts
- miniN2G:
-
Mini-nesprin -2G
- MT:
-
Microtubule
- MRCK:
-
Myotonic dystrophy-related, Cdc42-binding kinase
- SUN:
-
Sad1 and Unc83
- TAN lines:
-
Transmembrane actin-associated nuclear lines
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Acknowledgments
The methods described in this paper were developed with support by NIH grants R01GM099481 and R01AR068636.
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Chang, W., Antoku, S., Gundersen, G.G. (2016). Wound-Healing Assays to Study Mechanisms of Nuclear Movement in Fibroblasts and Myoblasts. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_17
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DOI: https://doi.org/10.1007/978-1-4939-3530-7_17
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