I use GFP-tubulin or GFP-jupiter, in fly. It works very well in mitosis.

did you ever try LifeAct-GFP or LifeAct-RFP from ibidi?

Thanks for the quick responses, I'll look into those options. :)

Molecular Probes/invitrogen/Life technologies sells rfp or gfp tubulin and rfp or gfp actin in a insect virus designed to transfect mammalian cells. They work great in neonatal cardiomyocytes.

I use YFP tubulin, GFP tubulin in transfection

For actin, Lifeact works well in my experience, although has been reported to perturb cell behavior in long-term experiments.. F-tractin is also supposed to be excellent (Schell MJ, Erneux C, Irvine RF (2001) Inositol 1,4,5-trisphosphate 3-kinase A associates with F-actin and dendritic spines via its N terminus. J Biol Chem 276: 37537–37546. The latter has been picked up by Clare Waterman's lab, one of the best groups in cytoskeletal imaging.

You can try using any of the following (by simply transfecting the FP-tagged constructs) : utrophin, lifeact,ezrin-c-term domain.

I've used mCherry-LifeAct for 4-6h expressions after microinjection, I've also tried it by transfection and works fine, not sure if it has effects on the dynamics, nothing big that we noticed at least. We got it originally from Roland Wedlich-Söldner, now has been made commercially. If not the Utrophin actin binding domain has been also used a lot in live imaging, you can get it from Bill Bemets lab http://bement.molbio.wisc.edu/

I'm working with plant cells, which are quite different to animal/ human systems. I have experience mainly on immunohistochemistry and less in live cell imaging. However to my best knowledge for microtubules in plants, GFP-MAP4 constructs work very well, with minimal or no disturbance of intracellular dynamics.

In plant systems, many folks are also using GFP-I/LWEQ modules (such as the domain from talin) to follow F-actin distributions.

Hi, Jurgen,

you can also use actin and tubulin antibodies. Several companies sales very specific antibodies, albeit in the fixing cells only. You can use immunolocalization and this have some advanteges versus FP fusion, especially for objects other than cultured cells.

There are also a couple of actin-specifci dyes (Alexa- conjugated), but you shoudl consider that these dyes may have effect on cell development itself,

Good luck!

Thanks again for the suggestions, I'll soon have some time to get to the bench again and when I do I'll share my experiences. My team works with plant cells, but it's always good to have an open mind and learn from all models, yeasts, fungi, animals...and also plants.

Hi, Jurgen, for plant cell antibody is a best decision, you do not need to transform plants with any markers. Well, you can not do living experiments, but in fixing cells it is work excellent. You can also combine cytoskeleton with cellulose fibril orientation by cellulose co-stain. Good luck!

Labelling actin filaments with fluorescent reporter proteins is more problematic than for microtubules. Nevertheless, many problems also occur when using fluorescent reporters of microtubules. The GFP-MAP4 and GFP-MBD constructs driven with the 35S promoter can appear to be benign but in various mutant backgrounds, the microtubules form prominent bundles and since the same effect is not observed when using other reporters (or immunofluorescence) the reporter is responsible for the defect. Some of the GFP-tagged tubulin constructs, such as the p35S::GFP-TUB reporter from the Hashimoto lab work without apparent phenotype but unfortunately, they only work in some tissues or organs, and do not appear to label microtubules in root cells. We recently got around this by using the ubiquitin 1 promoter to drive expression of a RFP-TUB6 (see Ambrose et al., 2011 A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis. Nature Comm. 2:430). This reporter works well throughout the root and is a good option for co-expression with a green-fluorescent reporter.

So, the versatile marker used for  labelling actin filaments that didn't disturb the cytoskeleton dynamics in vivo without affecting any cellular processes such as secretion as well as endocytic movement is Life-act. It is working properly in any system.