[
Genetics,
2020]
Evolutionary relationships between prodomains in the TGF- family have gone unanalyzed due to a perceived lack of conservation. We developed a novel approach, identified these relationships, and suggest hypotheses for new regulatory mechanisms in TGF- signaling. First, a quantitative analysis placed each family member from flies, mice, and nematodes into the Activin, BMP, or TGF- subfamily. Second, we defined the prodomain and ligand via the consensus cleavage site. Third, we generated alignments and trees from the prodomain, ligand, and full-length sequences independently for each subfamily. Prodomain alignments revealed that six structural features of 17 are well conserved: three in the straitjacket and three in the arm. Alignments also revealed unexpected cysteine conservation in the "LTBP-Association region" upstream of the straitjacket and in 8 of the bowtie in 14 proteins from all three subfamilies. In prodomain trees, eight clusters across all three subfamilies were present that were not seen in the ligand or full-length trees, suggesting prodomain-mediated cross-subfamily heterodimerization. Consistency between cysteine conservation and prodomain clustering provides support for heterodimerization predictions. Overall, our analysis suggests that cross-subfamily interactions are more common than currently appreciated and our predictions generate numerous testable hypotheses about TGF- function and evolution.
[
J Mol Evol,
2008]
TGFbeta and Wnt pathways play important roles in the development of animals from sponges to humans. In both pathways posttranslational modification as a means of regulating their function, such as lysine modification by ubiquitination and sumoylation, has been observed. However, a gap exists between the immunological observation of posttranslational modification and the identification of the target lysine. To fill this gap, we conducted a phylogenetic analysis of lysine conservation and context in TGFbeta and Wnt pathway receptors and signal transducers and suggest numerous high-probability candidates for posttranslational modification. Further comparison of results from both pathways suggests two general features for biochemical regulation of intercellular signaling: receptors are less frequent targets for modification than signal transduction agonists, and a lysine adjacent to an upstream hydrophobic residue may be a preferred context for modification. Overall the results suggest numerous applications for an evolutionary approach to the biochemical regulation of developmental pathways, including (1) streamlining of the identification of the target lysine, (2) determination of when members of a multigene family acquire distinct activities, (3) application to any conserved protein family, and (4) application to any modification of a specific amino acid.
[
J Mol Evol,
2010]
The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand-receptor pairs likely facilitated the expansion of this pathway's capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFbeta signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand-receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFbeta pathway. From a practical perspective, the former mechanism limits the investigator's ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer.
[
Proc Natl Acad Sci U S A,
1996]
Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood, Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown, We have identified three Caenorhabditis elegans genes,
sma-2,
sma-3, and
sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene
daf-4, indicating that they are required for
daf-4-mediated developmental processes, We show that
sma-2 functions in the same cells as
daf-4, consistent with a role in transducing signals from the receptor, These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358], The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction, In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates, These analyses suggest that dwarfins are conserved cytoplasmic
[
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."