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    ItemOpen Access
    Data associated with "Improved methods for single-molecule fluorescence in situ hybridization and immunofluorescence in Caenorhabditis elegans embryos"
    (Colorado State University. Libraries, 2021) Parker, Dylan
    Visualization of gene products in Caenorhabditis elegans has provided insights into the molecular and biological functions of many novel genes in their native contexts. Single-molecule Fluorescence In Situ Hybridization (smFISH) and Immunofluorescence (IF) visualize the abundance and localization of mRNAs and proteins, respectively, allowing researchers to elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol has challenges in the C. elegans embryo. smFISH protocols suffer from high initial costs and can photobleach rapidly. Immunofluorescence requires technically challenging permeabilization and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH in C. elegans embryos either in sequence or simultaneously. We also present protocols to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols include 1) improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, 2) a streamlined, in-tube approach for antibody staining that negates freeze-cracking, 3) a previously validated protocol to perform the cost-reducing smiFISH (single molecule inexpensive FISH) adaptation, 4) slide preparation using empirically determined optimal antifade products, and 5) straightforward quantification and data analysis methods. Figure 1 shows a schematic workflow for each protocol. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols and optimizations simplify existing protocols for single-molecule RNA and protein detection. Moreover, simultaneous, high-resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development.
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    ItemOpen Access
    Dataset associated with "mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage"
    (Colorado State University. Libraries, 2020) Parker, Dylan, M.; Winkenbach, Lindsay, P.; Boyson, Samuel, P.; Saxton, Matthew, N.; Daidone, Camryn; Al-Mazaydeh, Zainab, A.; Nishimura, Marc, T.; Mueller, Florian; Osborne Nishimura, Erin
    Caenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription. This presents an opportunity to study mechanisms of post-transcriptional regulatory control. In seeking the mechanisms behind this patterning, we discovered that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage), and P-bodies (associated with RNA processing). Transcripts differed in their dependence on 3'UTRs and RNA Binding Proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling these, we untangled a long-standing question: Are mRNAs directed to P granules for translational repression or do they accumulate there as a downstream step? We found translational repression preceded P granule localization and could occur independent of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. Overall, we show transcripts important for germline development are directed to P granules by translational repression, and this, in turn, directs their accumulation in the progenitor germ lineage where their repression can ultimately be relieved.
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    ItemOpen Access
    Dataset associated with "Multicolor single-molecule tracking of mRNA interactions with RNP granules"
    (Colorado State University. Libraries, 2018) Moon, Stephanie; Morisaki, Tatsuya; Khong, Anthony; Lyon, Kenneth; Parker, Roy; Stasevich, Timothy
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    ItemOpen Access
    Dataset associated with “An in-frame deletion mutation in the degron tail of auxin co-receptor IAA2 confers resistance to the herbicide 2,4-D in Sisymbrium orientale”
    (Colorado State University. Libraries, 2021) de Figueiredo, Marcelo R. A.; Küpper, Anita; Malone, Jenna M.; Petrovic, Tijana; de Figueiredo, Ana Beatriz T. B.; Campagnola, Grace; Peersen, Olve B.; Prasad, Kasavajhala V.S.K.; Patterson, Eric L.; Reddy, Anireddy S. N.; Kubeš, Martin F.; Napier, Richard; Dayan, Franck E.; Preston, Christopher; Gaines, Todd A.
    The natural auxin indole-3-acetic acid (IAA) is a key regulator of many aspects of plant growth and development. Synthetic auxin herbicides such as 2,4-D mimic the effects of IAA by inducing strong auxinic signaling responses in plants. To determine the mechanism of 2,4-D resistance in a Sisymbrium orientale (Indian hedge mustard) weed population, we performed a transcriptome analysis of 2,4-D-resistant (R) and -susceptible (S) genotypes that revealed an in-frame 27-nucleotide deletion removing 9 amino acids in the degron tail (DT) of the auxin co-receptor Aux/IAA2 (SoIAA2). The deletion allele co-segregated with 2,4-D resistance in recombinant inbred lines. Further, this deletion was also detected in several 2,4-D resistant field populations of this species. Arabidopsis transgenic lines expressing the SoIAA2 mutant allele were resistant to 2,4-D and dicamba. The IAA2-DT deletion reduced binding to TIR1 in vitro with both natural and synthetic auxins, causing reduced association and increased dissociation rates. This novel mechanism of synthetic auxin herbicide resistance assigns a new in planta function to the DT region of this Aux/IAA co-receptor for its role in synthetic auxin binding kinetics and reveals a potential biotechnological approach to produce synthetic auxin resistant crops using gene editing.