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  1. Article ; Online: An

    Myles, Krista M / Ragle, James Matthew / Ward, Jordan D

    microPublication biology

    2023  Volume 2023

    Abstract: ... C. ... ...

    Abstract C. elegans
    Language English
    Publishing date 2023-10-02
    Publishing country United States
    Document type Journal Article
    ISSN 2578-9430
    ISSN (online) 2578-9430
    DOI 10.17912/micropub.biology.000996
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Split-GFP lamin as a tool for studying

    Gregory, Ellen F / Ragle, James Matthew / Ward, Jordan D / Starr, Daniel A

    microPublication biology

    2023  Volume 2023

    Abstract: We engineered a fluorescent fusion protein ... ...

    Abstract We engineered a fluorescent fusion protein of
    Language English
    Publishing date 2023-12-13
    Publishing country United States
    Document type Journal Article
    ISSN 2578-9430
    ISSN (online) 2578-9430
    DOI 10.17912/micropub.biology.001022
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article ; Online: A spontaneous

    Myles, Krista M / Vo, An A / Ragle, James Matthew / Ward, Jordan D

    microPublication biology

    2023  Volume 2023

    Abstract: The auxin-inducible degron (AID) system is a widely-used system for conditional protein depletion. During the course of an experiment, we depleted the nuclear hormone receptor transcription factor NHR-23 to study molting, and we recovered a spontaneous ... ...

    Abstract The auxin-inducible degron (AID) system is a widely-used system for conditional protein depletion. During the course of an experiment, we depleted the nuclear hormone receptor transcription factor NHR-23 to study molting, and we recovered a spontaneous suppressor allele that bypassed the L1 larval arrest caused by NHR-23 depletion. These mutants also failed to deplete a BFP::AID reporter in the strain background, suggesting a broader defect in the AID system. These animals carried an in-frame 18 base pair insertion that produced a 6 amino acid repeat in TIR1. The larval arrest in these animals could be restored by expressing a wild-type
    Language English
    Publishing date 2023-10-15
    Publishing country United States
    Document type Journal Article
    ISSN 2578-9430
    ISSN (online) 2578-9430
    DOI 10.17912/micropub.biology.000994
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: Efficient generation of a single-copy

    Vo, An A / Levenson, Max T / Ragle, James Matthew / Ward, Jordan D

    microPublication biology

    2021  Volume 2021

    Abstract: The auxin-inducible degron (AID) system is a widely used system to conditionally deplete proteins. Using CRISPR/Cas9-based genome editing ... ...

    Abstract The auxin-inducible degron (AID) system is a widely used system to conditionally deplete proteins. Using CRISPR/Cas9-based genome editing in
    Language English
    Publishing date 2021-08-03
    Publishing country United States
    Document type Journal Article
    ISSN 2578-9430
    ISSN (online) 2578-9430
    DOI 10.17912/micropub.biology.000425
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  5. Article ; Online: An

    Myles, Krista M / Clancy, John C / Johnson, Londen C / Ashley, Guinevere / Manzano, Jesus / Ragle, James Matthew / Ward, Jordan D

    microPublication biology

    2023  Volume 2023

    Abstract: ... C. ... ...

    Abstract C. elegans
    Language English
    Publishing date 2023-10-18
    Publishing country United States
    Document type Journal Article
    ISSN 2578-9430
    ISSN (online) 2578-9430
    DOI 10.17912/micropub.biology.000993
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  6. Article ; Online: Experimental considerations for study of C. elegans lysosomal proteins.

    Clancy, John C / Vo, An A / Myles, Krista M / Levenson, Max T / Ragle, James Matthew / Ward, Jordan D

    G3 (Bethesda, Md.)

    2023  Volume 13, Issue 4

    Abstract: Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and ... ...

    Abstract Lysosomes are an important organelle required for the degradation of a range of cellular components. Lysosome function is critical for development and homeostasis as dysfunction can lead to inherited genetic disorders, cancer, and neurodegenerative and metabolic diseases. The acidic and protease-rich environment of lysosomes poses experimental challenges. Many fluorescent proteins are quenched or degraded, while specific red fluorescent proteins can be cleaved from translational fusion partners and accumulate. While studying MLT-11, a Caenorhabditis elegans molting factor that localizes to lysosomes and the cuticle, we sought to optimize several experimental parameters. We found that, in contrast to mNeonGreen fusions, mScarlet fusions to MLT-11 missed cuticular and rectal epithelial localization. Rapid sample lysis and denaturation were critical for preventing MLT-11 fragmentation while preparing lysates for western blots. Using a model lysosomal substrate (NUC-1), we found that rigid polyproline linkers and truncated mCherry constructs do not prevent cleavage of mCherry from NUC-1. We provide evidence that extended localization in lysosomal environments prevents the detection of FLAG epitopes in western blots. Finally, we optimize an acid-tolerant green fluorescent protein (Gamillus) for use in C. elegans. These experiments provide important experimental considerations and new reagents for the study of C. elegans lysosomal proteins.
    MeSH term(s) Animals ; Caenorhabditis elegans Proteins/genetics ; Caenorhabditis elegans Proteins/metabolism ; Caenorhabditis elegans/metabolism ; Lysosomes/metabolism ; Green Fluorescent Proteins/metabolism
    Chemical Substances Caenorhabditis elegans Proteins ; lysosomal proteins ; Green Fluorescent Proteins (147336-22-9)
    Language English
    Publishing date 2023-02-03
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 2629978-1
    ISSN 2160-1836 ; 2160-1836
    ISSN (online) 2160-1836
    ISSN 2160-1836
    DOI 10.1093/g3journal/jkad032
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  7. Article ; Online: MFP1/MSD-1 and MFP2/NSPH-2 co-localize with MSP during

    Morrison, Kayleigh N / Uyehara, Christopher M / Ragle, James Matthew / Ward, Jordan D / Shakes, Diane C

    microPublication biology

    2021  Volume 2021

    Abstract: Until recently, the only verified component of Fibrous Bodies (FBs) ... ...

    Abstract Until recently, the only verified component of Fibrous Bodies (FBs) within
    Language English
    Publishing date 2021-07-22
    Publishing country United States
    Document type Journal Article
    ISSN 2578-9430
    ISSN (online) 2578-9430
    DOI 10.17912/micropub.biology.000427
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  8. Article ; Online: NHR-23 and SPE-44 regulate distinct sets of genes during Caenorhabditis elegans spermatogenesis.

    Ragle, James Matthew / Morrison, Kayleigh N / Vo, An A / Johnson, Zoe E / Hernandez Lopez, Javier / Rechtsteiner, Andreas / Shakes, Diane C / Ward, Jordan D

    G3 (Bethesda, Md.)

    2022  Volume 12, Issue 11

    Abstract: Spermatogenesis is the process through which mature male gametes are formed and is necessary for the transmission of genetic information. While much work has established how sperm fate is promoted and maintained, less is known about how the sperm ... ...

    Abstract Spermatogenesis is the process through which mature male gametes are formed and is necessary for the transmission of genetic information. While much work has established how sperm fate is promoted and maintained, less is known about how the sperm morphogenesis program is executed. We previously identified a novel role for the nuclear hormone receptor transcription factor, NHR-23, in promoting Caenorhabditis elegans spermatogenesis. The depletion of NHR-23 along with SPE-44, another transcription factor that promotes spermatogenesis, caused additive phenotypes. Through RNA-seq, we determined that NHR-23 and SPE-44 regulate distinct sets of genes. The depletion of both NHR-23 and SPE-44 produced yet another set of differentially regulated genes. NHR-23-regulated genes are enriched in phosphatases, consistent with the switch from genome quiescence to post-translational regulation in spermatids. In the parasitic nematode Ascaris suum, MFP1 and MFP2 control the polymerization of Major Sperm Protein, the molecule that drives sperm motility and serves as a signal to promote ovulation. NHR-23 and SPE-44 regulate several MFP2 paralogs, and NHR-23 depletion from the male germline caused defective localization of MSD/MFP1 and NSPH-2/MFP2. Although NHR-23 and SPE-44 do not transcriptionally regulate the casein kinase gene spe-6, a key regulator of sperm development, SPE-6 protein is lost following NHR-23+SPE-44 depletion. Together, these experiments provide the first mechanistic insight into how NHR-23 promotes spermatogenesis and an entry point to understanding the synthetic genetic interaction between nhr-23 and spe-44.
    MeSH term(s) Animals ; Female ; Male ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/genetics ; Caenorhabditis elegans Proteins/metabolism ; Mutation ; Sperm Motility ; Semen/metabolism ; Spermatogenesis/genetics ; Transcription Factors/genetics
    Chemical Substances Caenorhabditis elegans Proteins ; Transcription Factors
    Language English
    Publishing date 2022-08-29
    Publishing country England
    Document type Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
    ZDB-ID 2629978-1
    ISSN 2160-1836 ; 2160-1836
    ISSN (online) 2160-1836
    ISSN 2160-1836
    DOI 10.1093/g3journal/jkac256
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  9. Article ; Online: The mIAA7 degron improves auxin-mediated degradation in Caenorhabditiselegans.

    Sepers, Jorian J / Verstappen, Noud H M / Vo, An A / Ragle, James Matthew / Ruijtenberg, Suzan / Ward, Jordan D / Boxem, Mike

    G3 (Bethesda, Md.)

    2022  Volume 12, Issue 10

    Abstract: Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an ... ...

    Abstract Auxin-inducible degradation is a powerful tool for the targeted degradation of proteins with spatiotemporal control. One limitation of the auxin-inducible degradation system is that not all proteins are degraded efficiently. Here, we demonstrate that an alternative degron sequence, termed mIAA7, improves the efficiency of degradation in Caenorhabditiselegans, as previously reported in human cells. We tested the depletion of a series of proteins with various subcellular localizations in different tissue types and found that the use of the mIAA7 degron resulted in faster depletion kinetics for 5 out of 6 proteins tested. The exception was the nuclear protein HIS-72, which was depleted with similar efficiency as with the conventional AID* degron sequence. The mIAA7 degron also increased the leaky degradation for 2 of the tested proteins. To overcome this problem, we combined the mIAA7 degron with the C. elegans AID2 system, which resulted in complete protein depletion without detectable leaky degradation. Finally, we show that the degradation of ERM-1, a highly stable protein that is challenging to deplete, could be improved further by using multiple mIAA7 degrons. Taken together, the mIAA7 degron further increases the power and applicability of the auxin-inducible degradation system. To facilitate the generation of mIAA7-tagged proteins using CRISPR/Cas9 genome engineering, we generated a toolkit of plasmids for the generation of dsDNA repair templates by PCR.
    MeSH term(s) Animals ; Caenorhabditis elegans/metabolism ; Humans ; Indoleacetic Acids/metabolism ; Indoleacetic Acids/pharmacology ; Nuclear Proteins/genetics ; Nuclear Proteins/metabolism ; Proteolysis
    Chemical Substances Indoleacetic Acids ; Nuclear Proteins
    Language English
    Publishing date 2022-08-27
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 2629978-1
    ISSN 2160-1836 ; 2160-1836
    ISSN (online) 2160-1836
    ISSN 2160-1836
    DOI 10.1093/g3journal/jkac222
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  10. Article ; Online: NHR-23 activity is necessary for C. elegans developmental progression and apical extracellular matrix structure and function.

    Johnson, Londen C / Vo, An A / Clancy, John C / Myles, Krista M / Pooranachithra, Murugesan / Aguilera, Joseph / Levenson, Max T / Wohlenberg, Chloe / Rechtsteiner, Andreas / Ragle, James Matthew / Chisholm, Andrew D / Ward, Jordan D

    Development (Cambridge, England)

    2023  Volume 150, Issue 10

    Abstract: Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important ... ...

    Abstract Nematode molting is a remarkable process where animals must repeatedly build a new apical extracellular matrix (aECM) beneath a previously built aECM that is subsequently shed. The nuclear hormone receptor NHR-23 (also known as NR1F1) is an important regulator of C. elegans molting. NHR-23 expression oscillates in the epidermal epithelium, and soma-specific NHR-23 depletion causes severe developmental delay and death. Tissue-specific RNAi suggests that nhr-23 acts primarily in seam and hypodermal cells. NHR-23 coordinates the expression of factors involved in molting, lipid transport/metabolism and remodeling of the aECM. NHR-23 depletion causes dampened expression of a nas-37 promoter reporter and a loss of reporter oscillation. The cuticle collagen ROL-6 and zona pellucida protein NOAH-1 display aberrant annular localization and severe disorganization over the seam cells after NHR-23 depletion, while the expression of the adult-specific cuticle collagen BLI-1 is diminished and frequently found in patches. Consistent with these localization defects, the cuticle barrier is severely compromised when NHR-23 is depleted. Together, this work provides insight into how NHR-23 acts in the seam and hypodermal cells to coordinate aECM regeneration during development.
    MeSH term(s) Animals ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/genetics ; Caenorhabditis elegans Proteins/metabolism ; Epithelium/metabolism ; Extracellular Matrix/metabolism ; Receptors, Cytoplasmic and Nuclear/metabolism
    Chemical Substances Caenorhabditis elegans Proteins ; Receptors, Cytoplasmic and Nuclear ; NHR-23 protein, C elegans
    Language English
    Publishing date 2023-05-22
    Publishing country England
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
    ZDB-ID 90607-4
    ISSN 1477-9129 ; 0950-1991
    ISSN (online) 1477-9129
    ISSN 0950-1991
    DOI 10.1242/dev.201085
    Database MEDical Literature Analysis and Retrieval System OnLINE

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