Shimizu K et al. (2001) Mol Cell Biol "Swift is a novel BRCT domain coactivator of Smad2 in transforming growth factor beta signaling."

Gurdon JB, Nielsen SJ, Zorn AM, Bourillot PY, Shimizu K

[Mol Cell Biol, 2001]

Transforming growth factor beta (TGFbeta) signaling is transduced via Smad2-Smad4-DNA-binding protein complexes which bind to responsive elements in the promoters of target genes. However, the mechanism of how the complexes activate the target genes is unclear. Here we identify Xenopus Swift, a novel nuclear BRCT (BRCA1 C-terminal) domain protein that physically interacts with Smad2 via its BRCT domains. We examine the activity of Swift in relation to gene activation in Xenopus embryos. Swift mRNA has an expression pattern similar to that of Smad2. Swift has intrinsic transactivation activity and activates target gene transcription in a TGFbeta-Smad2-dependent manner. Inhibition of Swift activity results in the suppression of TGFbeta-induced gene transcription and defective mesendoderm development. Blocking Swift function affects neither bone morphogenic protein nor fibroblast growth factor signaling during early development. We conclude that Swift is a novel coactivator of Smad2 and that Swift has a critical role in embryonic TGFbeta-induced gene transcription. Our results suggest that Swift may be a general component of TGFbeta signaling.

Bauer, Rosemary et al. MicroPubl Biol

Golden, Andy, Bauer, Rosemary, Nebenfuehr, Benjamin

[MicroPubl Biol, 2019]

KQT-3 is the C. elegansortholog of human KCNQ1, a voltage-gated potassium channel that is implicated in multiple types of Long Q-T cardiac arrhythmias including Jervell and Lange-Nielsen syndrome and Romano-Ward syndrome. In order to assess the possibility of modeling these syndromes in C. elegans, we obtained the tm542deletion allele, which had previously been associated with changes to the defecation cycle (Nehrke et al., 2008, Kwan et al. 2008). Any visible, easily-scored phenotype would be an indication that we could move forward with creating patient-specific missense alleles by CRISPR. Thetm542deletion, a 1001bp deletion generated by chemical mutagenesis that disrupts exons 2-4, appeared to cause a dramatic reduction in pharyngeal pump frequency (Fig 1B). This was an indication that mutations to kqt-3 might be a reasonable way to model Long Q-T arrhythmias in worms. When three patient missense mutations and two smaller deletions yielded only very subtle changes to pharyngeal pumping, we became increasingly suspicious of the tm542deletion strain. This prompted the creation of a full-length, 9004bp deletion allele by CRISPR that starts 69bp upstream of the start codon, ends 66bp after the stop codon, and inserts stop codons in every frame. Surprisingly, complete deletion of this 621 amino acid protein led to virtually no change in pharyngeal pumping (Fig 1B-D). In order to confirm that the pharyngeal pumping phenotype in the kqt-3(tm542) strain was due to extraneous mutations and not a dominant negative effect, we created the full-length deletion of kqt-3by CRISPR in the tm542 strain. This kqt-3(tm542av187) deletion strain phenocopied the original tm542deletion (Fig 1B-D), indicating that other mutations besides the deletion in kqt-3are responsible for the lack of pharyngeal pumping in the tm542strain. This study should serve as a cautionary tale for any lab studying a deletion allele that was generated by chemical mutagenesis; the strain likely contains numerous other mutations. We now routinely generate a full gene deletion by CRISPR as part of any genetic study.

Glock C et al. (2015) Methods Mol Biol "Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior."

Glock C, Gottschalk A, Nagpal J

[Methods Mol Biol, 2015]

Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15-22, 2002; Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352-1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95-109, 2012; Yizhar et al., Neuron 71:9-34, 2011). As C. elegans is transparent, genetically amenable, has a small nervous system mapped with synapse resolution, and exhibits a rich behavioral repertoire, it is especially open to optogenetic methods (White et al., Philos Trans R Soc Lond B Biol Sci 314:1-340, 1986; De Bono et al., Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans, In: Hegemann P, Sigrist SJ (eds) Optogenetics, De Gruyter, Berlin, 2013; Husson et al., Biol Cell 105:235-250, 2013; Xu and Kim, Nat Rev Genet 12:793-801, 2011). Optogenetics, by now an "exploding" field, comprises a repertoire of different tools ranging from transgenically expressed photo-sensor proteins (Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005) or cascades (Zemelman et al., Neuron 33:15-22, 2002) to chemical biology approaches, using photochromic ligands of endogenous channels (Szobota et al., Neuron 54:535-545, 2007). Here, we will focus only on optogenetics utilizing microbial rhodopsins, as these are most easily and most widely applied in C. elegans. For other optogenetic tools, for example the photoactivated adenylyl cyclases (PACs, that drive neuronal activity by increasing synaptic vesicle priming, thus exaggerating rather than overriding the intrinsic activity of a neuron, as occurs with rhodopsins), we refer to other literature (Weissenberger et al., J Neurochem 116:616-625, 2011; Steuer Costa et al., Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity, In: Cambridge S (ed) Photswitching proteins, Springer, New York, 2014). In this chapter, we will give an overview of rhodopsin-based optogenetic tools, their properties and function, as well as their combination with genetically encoded indicators of neuronal activity. As there is not "the" single optogenetic experiment we could describe here, we will focus more on general concepts and "dos and don'ts" when designing an optogenetic experiment. We will also give some guidelines on which hardware to use, and then describe a typical example of an optogenetic experiment to analyze the function of the neuromuscular junction, and another application, which is Ca(2+) imaging in body wall muscle, with upstream neuronal excitation using optogenetic stimulation. To obtain a more general overview of optogenetics and optogenetic tools, we refer the reader to an extensive collection of review articles, and in particular to volume 1148 of this book series, "Photoswitching Proteins."