LifeAct Dye
LifeAct is a 17 amino acid recombinant peptide that stains filamentous actin (F-actin) structures of eukaryotic living or fixed cells.[1] The peptide is a registered trademark of ibidi GmbH.[2] There are several types and combinations of LifeAct that can be utilized depending on the cell type, protocol, and purpose of the analysis.
Lifeact amino acid sequence
[edit]Lifeact 17 amino acid sequence is MGVADLIKKFESISKEE.[1]
Types
[edit]- LifeAct Plasmid
- LifeAct mRNA
- LifeAct Adenovirus
- LifeAct Lentivirus
- LifeAct Protein
Chemistry
[edit]LifeAct-TagGFP2 being the most widely used fluorescent variant compared to other LifeAct constructs is composed of the first 17 amino acid from the Saccharomyces cerevisiae Abp140, an actin-binding protein. The Abp140 is highly conserved among Saccharomyces cerevisiae and other closely related organisms.[3] The 17 amino acid fragment of Abp140 was genetically fused to GFP and fluoresces green when it binds the F-actin structures of living and fixed cells, allowing for visualization of cell mechanics under microscopes. Previous experiments involving the analysis of cell mechanics had depended on fluorescently labeled phalloidin and actin GFP fusion proteins obtained from utrophin in Xenopus laevis and ABP120 in Dictyostelium discoideum.[4][5] However, due to their large protein size, markers such as phalloidin and GFP fusion proteins are limited to cells that can be transfected and tend to compete with their orthologous protein. These localization markers affect cellular mechanical properties and F-actin structures, thus making these markers unreliable.[6] An alternative to these markers is Life Act-TagGFP2, which is a much smaller protein and does not affect cell mechanics. Cells synthesize LifeAct-TagGFP2 in a short period of time at a cost-effective making it suitable as an in vivo marker.[1]
Applications in biomedical research
[edit]LifeAct peptides have been used as a universal marker for F-actin visualization in biomedical research. An experiment conducted by Sawant et al. utilized LifeAct GFP to visualize the migration of control border cells in the ovaries of Drosophila flies, in order to determine how cells move in terms of small and large collectives during development and cancer.[7] Lifeact labels F-actin in border cells and adjacent follicle cells allowed for the detailed examination of border cell membranes and protrusions. Studies regarding the degradation of actin cytoskeleton due to aging relied on LifeAct for the analysis of cytoskeletal organization as a function of age. Transgenic lines that expressed the LifeAct in various tissues of C. elegans were primarily used for imaging.[8]
References
[edit]- ^ a b c Riedl J, Crevenna AH, Kessenbrock K, Yu JH, Neukirchen D, Bista M, Bradke F, Jenne D, Holak TA, Werb Z, Sixt M, Wedlich-Soldner R (July 2008). "Lifeact: a versatile marker to visualize F-actin". Nature Methods. 5 (7): 605–7. doi:10.1038/nmeth.1220. PMC 2814344. PMID 18536722.
- ^ "LifeAct-TagGFP2 Protein | Actin Visualization in Living Cells | ibidi". ibidi.com. Retrieved 2018-09-05.
- ^ Noma A, Yi S, Katoh T, Takai Y, Suzuki T, Suzuki T (June 2011). "Actin-binding protein ABP140 is a methyltransferase for 3-methylcytidine at position 32 of tRNAs in Saccharomyces cerevisiae". RNA. 17 (6): 1111–9. doi:10.1261/rna.2653411. PMC 3096043. PMID 21518805.
- ^ "Actin Staining Techniques - Actin staining protocols, Actin stain, Actin probe, Acti-stain 488 phalloidin, Acti-stain 555 phalloidin, Acti-stain 535 phalloidin, Acti-stain 670 phalloidin, Actin stain, Actin -stain488". www.cytoskeleton.com. Retrieved 2018-09-05.
- ^ Hsu ST, Cabrita LD, Fucini P, Dobson CM, Christodoulou J (May 2009). "Structure, dynamics and folding of an immunoglobulin domain of the gelation factor (ABP-120) from Dictyostelium discoideum". Journal of Molecular Biology. 388 (4): 865–79. doi:10.1016/j.jmb.2009.02.063. PMID 19281823.
- ^ Sliogeryte K, Thorpe SD, Wang Z, Thompson CL, Gavara N, Knight MM (January 2016). "Differential effects of LifeAct-GFP and actin-GFP on cell mechanics assessed using micropipette aspiration". Journal of Biomechanics. 49 (2): 310–7. doi:10.1016/j.jbiomech.2015.12.034. PMC 4769141. PMID 26792287.
- ^ Sawant K, Chen Y, Kotian N, Preuss KM, McDonald JA (August 2018). "Rap1 GTPase promotes coordinated collective cell migration in vivo". Molecular Biology of the Cell. 29: mbcE17120752. doi:10.1091/mbc.E17-12-0752. PMC 6249841. PMID 30156466.
- ^ Higuchi-Sanabria R, Paul JW, Durieux J, Benitez C, Frankino PA, Tronnes SU, Garcia G, Daniele JR, Monshietehadi S, Dillin A (August 2018). "Spatial regulation of the actin cytoskeleton by HSF-1 during aging". Molecular Biology of the Cell. 29: mbcE18060362. doi:10.1091/mbc.E18-06-0362. PMC 6254583. PMID 30133343.