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Figure 4: Mechanism of TMEM138 and TMEM216 localization and morphological defects in zebrafish knockdowns.
To determine the mechanisms by which TMEM138 and TMEM216 vesicles are localized, the authors labeled TMEM138 and TMEM216 in cells with knocked-down epression of specific vesicle tethering proteins. To test whether a loss of TMEM138 or TMEM216 function results in indistinguishable phenotypes in lower vertebrates lacking TMEM138 and TMEM216 adjacency and co-regulation, the authors examined the morphology of zebrafish with knocked-down expression of these genes.
Panel A: The authors knocked down the expression of genes encoding vesicle tethering proteins using small interfering (si) RNA specific for various vesicle tethering proteins' mRNA. Shown here is the significant impairment of paired movements of TMEM138 and TMEM216 resulting from TRAPPC9 (a specific vesicle tethering protein) knockdown compared to controls with siRNA not specific for TRAPPC9 mRNA. This impaired coordination of vesicular movement was rescued by tagging TRAPPC9 with myc. Images display co-localization (indicative of paired movement) of TMEM138 and TMEM216 in cells with control (non-targetting) siRNA and disrupted protein co-localization in cells with siRNA specific for TRAPPC9 mRNA. The graph quantifies the percent of vesicles demonstrating paired movement of TMEM138 and TMEM216 vesicles under each condition. The cell type used was not stated explicitly, although the cells are presumably human embryonic kidney (IMCD3) cells given that this was the cell type used to generate Figure 3.
Panel B: The authors blocked transcription of either TMEM138, TMEM216, or both using antisense morpholinos in developing zebrafish embryos and observed the resulting morphological defects. Displayed in the images, knockdown of TMEM138, TMEM216, or both resulted in a kinked or curved tail (arrows) and heart edema (arrow heads). However, only TMEM216 knockdown resulted in hydrocephalus (*). The graph and table quantify the percent of embryos with each of these morphological defects.
Data not shown: With data shown in supplemental figures, the authors claim to have demonstrated that movement of TMEM138 and TMEM216 orthologs are also paired in zebrafish.
Conclusions: The authors conclude from these data that coordination of intracellular TMEM138 and TMEM216 localization evolutionarily precedes TMEM138 and TMEM216's co-regulation. Moreover, they conclude that disrupting these proteins results in distinguishable morphological phenotypes in zebrafish, and thus that the similarity in human morphological defects arising from TMEM138 and TMEM216 mutations is a product of their adjacency and co-regulation.
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Lee, J.H., Silhavy, J.L., Lee, J.E., Al-Gazali, L., Thomas, S., Davis, E.E., Bielas, S.L., Hill, K.J., Iannicelli, M., Brancati, F., Gabriel, S.B., Russ, C., Logan, C.V., Sharif, S.M., Bennett, C.P., Abe, M., Hildebrandt, F., Diplas, B.H., Attie-Bitach, T., Katsanis, N., Rajab, A., Koul, R., Sztriha, L., Waters, E.R., Ferro-Novick, S., Woods, C.G., Johnson, C.A., Valente, E.M., Zaki, M.S., Gleeson, J.G. 2012. Evolutionarily Assembled cis-Regulatory Module at a Human Ciliopathy Locus. Science. 335: 966-969.
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