Cytoskeletal live cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics in living cells.
Up to now the options for live cell staining of Actin and Tubulin molecules were limited. Phalloidin derivatives which selectively bind to F-actin are not cell permeable. They can thus only be used in fixed and permeabilized cells. Paclitaxel (Taxol) derivatives binding to microtubules and other cytoskeletal modeling molecules do not show increased fluorescence upon target binding (meaning they are not fluorogenic), anyhow they have a strong impact on tubulin dynamics. The only methods for live cell imaging available so far have been systems based on expression vectors which have to be transfected to target cells. In the case of Actin, a binding protein is coupled to either GFP or RFP and thus is expressed in target cells which leads to a labeling of F-actin. Such an experimental approach can bring major technical hurdles such as transfection efficiency and duration.
The need for "easy-to-use" and non toxic stains, which can pass the cell membrane and directly bind and stain microtubules or F-actin, has been a demand from cell biologists for quite some years now. Up to now phalloidine based dyes were recommended to stain F-actin, but these dyes - although being perfect tools to stain F-actin in fixed cells - cannot be used for living cells as already been mentioned above.
Non-toxic Actin & Tubulin fluorescent stains for live-cell imaging
Cell biologists at tebu-bio have selected Spirochrome's unique fluorescent stains for monitoring Actin and Tubulin easily in living cells. These fluorescent stains (SiR-Actin and SiR-Tubulin) are cell permeable compounds which stain microtubules and F-actin respectively in living cells.
They are compatible with Super-Resolution Microscopy like Stimulated Emission Depletion [STED] and Structured Illumination Microscopy [SIM] and render live cell cytoskeleton dynamics studies convenient:
- No transfection
- No washing steps
- No toxic effects
- Excellent brightness
- Far-red excitation & emission
- Deep tissue penetration and minimal background
- Multiple fluorescent stainings with other dyes possible
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Want to know more about these new SiR actin and Tubulin labelling probes?
This proprietary bright and photostable silicon rhodamine-like (SiR) technology is compatible with most microscopes. It can be used with standard Cy5 settings. The combination of all these properties set SiR-based probes apart from other fluorescent probes. (1) The SiR dyes are coupled to binding components which specifically bind to F-actin (Jasplakinolide natural compound) and Microtubules (Docetaxel) in cells.
SiR derivatives exist in two states - the nonfluorescent spirolactone (OFF state) and a fluorescent zwitterion (ON state). Conjugated to Jasplakinolide or Docetaxel and with an optimized hydrophobic linker between the fluorophore and the targeting ligand, the final product SiR-Actin displays an increase in fluorescence of more than 100-fold upon binding to F-actin, while SiR-tubulin showed an increase of more than tenfold (2).
Sources:
- A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins G. Lukinavičius et al., Nature Chemistry, 5, 132–139 (2013).
- Fluorogenic probes for live-cell imaging of the cytoskeleton, G. Lukinavičius et al., Nature Methods, 11, 731–733 (2014)
SiR-actin (Live Cell Fluorogenic F-actin Labelling Probe) and SiR-tubulin (Live Cell Fluorogenic Microtubule Labelling Probe) are available at 50 nmol separately. A complete Cytoskeleton kit with SiR-actin and SiR-tubulin (50 nmol each) is also available.
Leave your questions or comments here to learn more about these new cytoskeletal stains for fluorescent live cell imaging!