Table 1. Probes for live imaging of Rho GTPases and the cytoskeleton in Xenopus.
A non-exhaustive list of validated probes available for live imaging in Xenopus. References for pCS2+-based plasmids are listed. “*” denotes that the plasmid is available through Addgene.
| Probe based on: | pCS2+ vectors, citations, availability through Addgene (*) |
|
|---|---|---|
| Active Rho GTPase Probes | ||
| Active Rho | rGBD (GBD of Rhotekin) |
GFP†-rGBD (Benink and Bement 2005)* 3xGFP-rGBD (Bement et al. 2015) mCherry-2xrGBD (Davenport et al. 2016) |
| Active Cdc42 | wGBD (GBD of N-WASP) |
GFP-wGBD (Sokac et al. 2003)* mRFP-wGBD (Benink and Bement 2005)* |
| Active Rac‡ | pGBD (GBD of PAK3) |
GFP-pGBD (Benink and Bement 2005; Miller and Bement 2009)* |
| Actin Probes | ||
| Filamentous (F-) actin | UtrCH (Calponin Homology domain of Utrophin) |
mRFP-UtrCH (Burkel et al. 2007)* GFP-UtrCH (Burkel et al. 2007)* mCherry-UtrCH (Miller and Bement 2009) |
| Globular (G-) and F-actin§ | Lifeact (First 17 amino acids of abp140 from S. cerevisiae) |
Lifeact-GFP (Riedl et al. 2008; Bement et al. 2015; Higashi et al. 2016) Lifeact-mRFP (Riedl et al. 2008; Bement et al. 2015; Higashi et al. 2016) |
| Microtubule Probes | ||
| Polymerized microtubules | EMTB (Ensconsin microtubule binding domain) |
EMTB-3xGFP (Miller and Bement 2009)* EMTB-mCherry (Miller and Bement 2009) EMTB-2xmCherry (vonDassow et al. 2009) |
| Total tubulin** | α-tubulin | GFP-tubulin (Woolner et al. 2008) mCherry-tubulin (Woolner et al. 2008) |
| Microtubule plus ends | EB3 (End binding 3) |
EB3-GFP (Shindo et al. 2008) |
| Membrane Probes | ||
| Membrane | Farnesylation (Farnesylation sequence from human Ras) |
GFP-farnesyl (Reyes et al. 2014) mCherry-farnesyl (Reyes et al. 2014) |
| Membrane | Myristolation (2xmyristolation sequence from Lyn kinase) |
Mem-TagBFP (Higashi et al. 2016) |
| DNA Probe | ||
| Chromatin | H2B (Histone 2B) |
mCherry-H2B (Reyes et al. 2014) |
†
GFP refers to Enhanced GFP (EGFP).
‡
pGBD can bind to Rac and Cdc42, so controls to determine specificity of response should be performed (Breznau et al. 2015).
§
Lifeact binds to F-actin with ~30-fold higher affinity than G-actin (Riedl et al. 2008).
**
Using fluorescently-tagged tubulin may result in poor signal over background; however, EMTB binds microtubules in a cell cycle-dependent manner, meaning that signal becomes significantly weaker during mitosis.