Structure Function Analysis of sma-10: A Novel Regulator of TGB-β Signaling
Writer: Kelvin Liao
Date: Spring 2016
Citation: Liao, K. & Padgett, R. (2016). Structure Function Analysis of sma-10: A Novel Regulator of TGB-β Signaling. Rutgers Research Review, 1(1).
My name is Kelvin Liao and I am currently a sophomore in the School of Arts and Sciences Honors Program. I work as an undergraduate researcher in Dr. Richard Padgett's lab at the Waksman Institute of Microbiology. I am majoring in Molecular Biology and Biochemistry and minoring in Computer Science. After I complete my undergraduate education, I intend to pursue medical training to become a doctor. Currently, I am undecided on which medical specialty I will pursue. For now, I am focusing on giving myself the skills and experiences that are integral to success in the health professions as a whole. To this end I see the work I do in Dr. Padgett's lab as an opportunity to challenge myself, develop my analytical thinking, and mature as both a researcher and student.
The research conducted in Dr. Padgett's lab can be classified as either developmental or molecular genetics. Though both fields are heavily intertwined, each has its own distinct focuses. Developmental genetics is concerned with how an organism's genes regulate its growth and development throughout its life cycle. Molecular genetics studies the structure and function of genes at a molecular level. Research in developmental genetics relies heavily on molecular genetics because the process of discerning how a gene regulates growth and development is entirely dependent on elucidating the molecular functions of the gene, how it interacts with other genes, and how its expression can be affected. As such, the majority of research in developmental and molecular genetics revolves around more than just the study of the structure-function of individual genes; it explores the structure-function of entire pathways that regulate the activity of multiple genes.
The pathways that are relevant to my research belong to a class of pathways known as the transforming growth factor-beta-like (TGF-β) pathways. TGF-β pathways have been and continue to be heavily studied in both Caenorhabditis elegans (C. elegans) and Drosophila model organisms. Researchers are interested in the TGF-β family because its pathways are highly conserved across all animal phyla, from sponges to humans. The TGF-β pathway, like most signaling pathways, is first activated by the binding of a signaling ligand to a cell's surface receptors. From there, the signal is transduced into the cell's nucleus where it activates specific gene transcription. In the TGF-β pathway, the proteins responsible for signal transduction are known as SMADs [a portmanteau of the Drosophila protein MAD (mothers against decapentaplegic) and the C. elegans protein SMA (small body size)]. TGF-β is responsible for regulating cell growth, patterning, and death in essentially all vertebrate tissues (Ashcroft et al., 1999).
I am interested in the structure-function of the TGF-β regulator sma-10. The sma-10 gene encodes a protein that is a member of the Leucine-rich repeats and immunoglobulin-like domains family of proteins (LRIG). It functions to regulate the TGF-β signaling pathway that controls body size in C. elegans (Gur et al., 2004). The mammalian homologue to sma-10, LRIG1, has also been shown to encode a protein that binds vertebrate receptors, indicating an evolutionary conservation (Savage-Dunn, 2003). Though not definitely known, the cytoplasmic tail of the SMA-10 protein is likely involved in the regulation of TGF-β receptor trafficking into the cell. This study is being conducted to examine the role, if any, of sma-10's cytoplasmic tail in regulating the trafficking of TGF-β receptors and to determine whether the short tail is required for SMA-10 function.
To achieve this, I constructed two truncations of the sma-10 gene. The first lacked the DNA sequences that code for the cytoplasmic tail region. This construct was labelled sma-10 Δcyto. The second lacked the DNA sequences that code for the transmembrane region of SMA-10 in addition to the cytoplasmic tail region mentioned previously. This construct was labelled sma-10 ΔTMΔcyto. Both constructs were built to include a Green Fluorescent Protein (GFP) tag. When expressed, the GFP gene produces a protein that fluoresces bright green when exposed to ultraviolet light. Each of these DNA constructs was then injected in vivo into C. elegans embryos, integrating the mutant gene into their genomes. These truncated genes were then expressed in the hypodermes of the sma-10 mutant animals. Animals expressing the truncated sma-10 were then selected for by the visualization of GFP in their hypodermes. Body size measurements of the mutant animals will be recorded through the use of epifluorescent microscopy. In the final part of the experiment, localization and accumulation patterns of TGF-β receptors in mutant sma-10 background will be imaged using confocal microscopy and examined.
Currently, I am quantifying the body size differences between the wild type and mutant sma-10 animals. Qualitative observation of the sma-10 ΔTMΔcyto animals has already shown the effects of the truncated sma-10 gene on C. elegans. The sma-10 ΔTMΔcyto animals are indeed shorter in body length than their wild type counterparts. Normal trafficking of sma-10 results in worms exhibiting the normal body size phenotype. The small body size phenotype that is apparent in the sma-10 ΔTMΔcyto animals indicates a mistrafficking of sma-10 somewhere in the worm's TGF-β pathway. My preliminary qualitative observations confirm my hypothesis that the cytoplasmic tail region of the SMA-10 protein is necessary for its normal participation in the TGF-β pathway. Further quantitative analyses will be necessary in solidifying this claim and will be carried out as mentioned in the previous section. Upon completion of this project I will be able to identify either the cytoplasmic tail alone or the transmembrane region and cytoplasmic tail in conjunction as necessary in SMA-10's function.
The work I am doing in Dr. Padgett's lab is fundamental toward understanding how the TGF-β pathway functions under normal conditions and how it can be affected by mutations in its relevant genes. A comprehensive understanding of the TGF-β pathway is essential in combatting such developmental disorders as congenital heart disease and cancer where TGF-β pathways are known to be involved. I put my work into perspective by appreciating the fact that although I am far removed from the realms of drug development and clinical research, my findings are vital in driving subsequent research in these fields.
- Ashcroft, G. S., X. Yang, et al. (1999). Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat. Cell. Biol., 1(5), 260-266.
- Gur, G., Rubin, C., Katz, M., Amit, I., Citri, A., Nilsson, J., ... Yarden, Y. (2004). LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation. The EMBO Journal, 23(16), 3270-3281.
- Savage‐Dunn, C., Maduzia, L. L., Zimmerman, C. M., Roberts, A. F., Cohen, S., Tokarz, R., and Padgett, R. W. (2003). Genetic screen for small body size mutants in C. elegans reveals many TGFβ pathway components. Genesis, 35(4), 239-247.