Johnson, Jacque-Lynne F. et al. (2009) International Worm Meeting "Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule severing activity during both meiosis and mitosis."

Mains, Paul E., Raharjo, Eko, McNally, Francis J., McNally, Karen, Lu, Chenggang, Johnson, Jacque-Lynne F.

[International Worm Meeting, 2009]

The MEI-1/MEI-2 katanin microtubule-severing complex is required for meiotic spindle formation in C. elegans. While essential for meiosis, MEI-1/MEI-2 must be quickly downregulated to prevent interference with formation of the first mitotic spindle. Post-meiotic inactivation of MEI-1 is accomplished by two independent protein degradation pathways. One pathway requires MEL-26 as a substrate-specific adaptor for the CUL-3 based E3 ubiquitin ligase, while a parallel pathway requires phosphorylation of MEI-1 by MBK-2 kinase. MBK-2 mediated degradation of MEI-1 appears to be coupled with meiotic exit after which MEL-26 completes the degradation process. A phosphorylation system involving Protein Phosphatase 4 also acts independently of these degradation systems to regulate meiotic and mitotic MEI-1 activity (Han et al, 2009). The parallel MEL-26 and MBK-2 systems explain how MEI-1 microtubule-severing activity is degraded during mitosis, but how is MEI-1 degradation delayed until its essential meiotic function is complete. Stitzel et al (2006) showed that MBK-2 activity is held in check by EGG-3 until meiosis is completed. Our work reveals that the MEL-26 branch of the MEI-1 degradation pathway is regulated in part by MEL-26 protein accumulation. Affinity-purified antibodies revealed that MEL-26 is present at low (but nonzero) levels until meiosis is complete, after which MEL-26 levels increase substantially, paralleling the increase in post-meiotic MEI-1 degradation. During meiosis, MEL-26 levels are kept low by another type of ubiquitin ligase containing CUL-2, and by an upstream activator of ubiquitin ligase activity, RFL-1. Thus, a cascade of proteolysis, triggered at fertilization, results in the correct timing of MEI-1 activity. However, premature meiotic activity of either MEL-26 or MBK-2 (independently or together) is not sufficient to block spindle formation, indicating that additional factors are rate limiting for the downregulation of MEI-1. While it was initially thought that MEL-26 did not play a role during meiosis, we find the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity in the meiotic spindle. In addition, our results demonstrate that while RFL-1 has additional essential targets, MEI-1 is the only essential target for MEL-26 and possibly for the E3 ubiquitin ligase CUL-3.

Jacque-Lynne F Johnson et al. (2003) International Worm Meeting "Post-meiotic inhibition of MEI-1 microtubule-severing by MEL-26."

Paul E Mains, Martin A Srayko, Jacque-Lynne F Johnson

[International Worm Meeting, 2003]

In the newly fertilized C. elegans embryo, the meiotic and mitotic spindles form in the same cytoplasm. mei-1/mei-2 (katanin) form a microtubule-severing complex required for the meiotic spindle and must be inhibited post-meiotically by mel-26 for mitotic spindle formation(1,2). MEI-1/MEI-2 localize to the meiotic chromatin, spindles and spindle poles of the meiotic spindle(3). In mel-26(lf) the mitotic spindle is small and mis-oriented due to ectopic MEI-1/MEI-2 mitotic microtubule severing, indicating that MEL-26 inhibits MEI-1/MEI-2 after meiosis(1).mel-26 has a mutant phenotype similar to rfl-1(4). rfl-1 is a component of the E1 in the ned-8 ubiquitin-like (UBL) pathway, which activates the ubiquitin (UBQ) protein degradation pathway. These two mutants are also similar in their genetic interactions with mei-1 (2,4), suggesting that MEL-26 may inhibit MEI-1 by targeting it for degradation. MEI-1 contains a PEST motif, commonly found among proteins subject to UBQ-mediated degradation. In a mei-1(gf) allele, this motif is abolished and the protein refractory to MEL-26 regulation.Yeast two-hybrid interactions between MEL-26, MEI-1 and MEI-2 reinforce the genetic conclusions and indicate the interaction between MEL-26 and MEI-1 is direct. MEI-1 interacts with itself, MEL-26 and MEI-2. However MEI-1(gf) does not interact with MEL-26 but still binds MEI-1(+) and MEI-2. This suggests that MEI-1(gf) forms a microtubule-severing complex that cannot be inhibited by MEL-26.We are interested in determining how MEL-26 inhibits MEI-1 and how MEL-26 expression is regulated. MEL-26 displays both cytoplasmic and membrane localization in the early embryo that appears after meiosis is completed, thus MEL-26 cannot inhibit MEI-1 during meiosis. Preliminary results indicate MEL-26 expression at the 1-4 cell stage is disrupted in a UBL pathway mutant, suggesting MEL-26 itself may be regulated by degradation(5). We are characterizing the temporal expression pattern of MEL-26 and looking for changes in MEL-26 expression in UBQ and UBL pathway mutants.(1)Srayko, et al, 2000. Genes Dev.14:1072-1084 (2)Dow and Mains,1998. Genetics 150:119-128 (3)Clark-Maguire and Mains,1994. JCB 126(1)199-209 (4)Kurz, et al, 2002. Science 295:1294-1298 (5)Galan and Peter,1999. PNAS 96:9124-9129

Jacque-Lynne F Johnson et al. (2002) West Coast Worm Meeting "Characterizing MEL-26, the post-meiotic inhibitor of MEI-1/MEI-2 microtubule severing."

Jacque-Lynne F Johnson, Paul E Mains, Martin A Srayko

[West Coast Worm Meeting, 2002]

A number of genes have been identified that have an essential role in the oocyte meiotic and early mitotic spindle formation. We focus on mei-1 and mei-2, which are essential for the meiotic spindle and mel-26, which is required for proper mitotic spindle formation. mel-26 encodes a novel 44.5 kDa protein with the exception of two conserved domains. The N-terminal MATH (meprin and TRAF homology) domain is known to be required for proper folding of activable enzymes (1). The centrally located BTB (Bric a Brac, Tramtrack and Broad Complex) domain, implicated in protein-protein interactions (2), has been shown to mediate both homomeric and heteromeric protein dimerisation (3).

Nguyen, Phuong Anh T et al. (2011) International Worm Meeting "The role of cilia in cGMP signaling in the AFD thermosensory neuron."

Leroux, Michel R, Johnson, Jacque-Lynne, Nguyen, Phuong Anh T

[International Worm Meeting, 2011]

Primary cilia act as the antennae of eukaryotic cells, being able to transduce light, mechanical and chemical stimuli from the environment. The importance of these organelles in human physiology and development is increasingly recognized, given their involvement in different signaling pathways and implication in human disorders (ciliopathies) that affect essentially all organs. We have just found through phylogenetic analysis that various cGMP signaling proteins are present in essentially all eukaryotes except for those that have secondarily lost cilia during evolution. As cyclic nucleotides are found in prokaryotes, this finding suggests that cGMP signaling could have been the first signal transduction pathway adopted by primary cilia during its emergence as a sensory device, preceding the advent of other ciliary signaling pathways in vertebrates. A prime example of the cilia-cGMP signaling connection can be gleaned in vertebrate photoreceptors; this specialized cilium concentrate cGMP signaling proteins essential for phototransduction, including guanylyl cyclases that produce cGMP, phosphodiesterases that break down cGMP, and cGMP-dependent cation channels. How the cGMP signaling cascade is established in the ciliary compartment is not understood, however. A well-studied behavior in C. elegans that involves cGMP signaling is thermotaxis. In this behavior, temperature is sensed mainly by AFD neurons, each of which possesses a cilium of unknown function. Our lab has showed that ciliary mutants are defective in thermotaxis. Because of the close link between cilia and cGMP signaling, we hypothesized that ciliary proteins are required to establish and maintain cGMP signaling in AFD. Indeed, we discovered that at AFD dendritic endings, cGMP signaling components are localized to different compartments, and proper localizations require specific ciliary proteins acting cell-autonomously. Microscopy studies of ciliary proteins in AFD suggest that its membranous 'fingers' are a bona fide ciliary compartment, because whereas most ciliary proteins are found in the canonical cilium, some are also present at the base of the fingers. This suggests ciliary proteins might form a barrier or 'transport hub' separating the finger membrane from the dendritic membrane. Our research will help establish the AFD neuron as a model system analogous to the ciliary photoreceptor, probing the important connection between ciliary trafficking and cGMP signaling.

Johnson, Christina K. et al. (2021) MicroPubl Biol

Bianchi, Laura, Johnson, Christina K., Miller III, David M.

[MicroPubl Biol, 2021]

For Johnson, CK; Miller, DD; Bianchi, L (2021). Effect of the protease plasmin on C. elegans hyperactive DEG/ENaC channels MEC-4(d) and UNC-8(d). microPublication Biology. 10.17912/micropub.biology.000412.

Johnson TE (2005) J Gerontol A Biol Sci Med Sci "Genes, phenes, and dreams of immortality: the 2003 Kleemeier Award lecture."

Johnson TE

[J Gerontol A Biol Sci Med Sci, 2005]

The 2002 Kleemeier Award from the Gerontological Society of America was awarded to Thomas E. Johnson, PhD, of the University of Colorado at Boulder. Dr. Johnson was the pioneer who first applied genetic analyses to the study of the aging processes in Caenorhabditis elegans and who introduced the nematode as an aging model. Longer life span was chosen as a surrogate marker for slowed aging. Here Dr. Johnson describes his role(s) in the isolation of age-1, the first longevity mutant, which can more than double the life span and which slows the rate of aging more than twofold. He also reviews research suggesting conservation of function and applicability to intervention by pharmacological targeting of the Age-1 pathway. Current work by biotechnology companies targets this and other basic discoveries in an attempt to postpone human aging.

van der Burght, Servaas et al. (2019) International Worm Meeting "Formation of a salt-sensing compartment at the tip of cilia."

Li, Chunmei, van der Burght, Servaas, Jansen, Gert, Leroux, Michel, Johnson, Jacque-Lynne, Rademakers, Suzanne

[International Worm Meeting, 2019]

Cells and organisms detect cues in their environment using sensory organelles, called primary cilia, in which signaling proteins exhibit specific localizations. The mechanisms that determine their localization within cilia, and the significance of these localizations, are largely unknown. We identify a unique ciliary localization for the receptor-type guanylate cyclase GCY-22, which plays a role in chemotaxis to NaCl, and unravel the mechanisms that control its ciliary localization. We generated a GFP knock-in of GCY-22 and discovered its prominent localization to the ciliary tip, and periciliary membrane compartment (PCMC) of one ciliated neuron, ASER. FRAP experiments showed that these two pools of GCY-22 are very stable but exhibit motility within the respective subcellular domains. Quantification experiments suggest the presence of more than 10,000 GCY-22::GFP molecules within the ciliary tip. To determine how GCY-22::GFP is transported to its ciliary destinations, we used time-lapse microscopy. We observed vesicular transport in the dendrite and particles coinciding with intraflagellar transport (IFT) in the cilium. Dual-color imaging suggested that IFT partially maintains GCY-22::GFP tip localization by targeting the protein to the tip. This localization is altered upon disruption of either BBS-8, an IFT cargo adaptor protein or OSM-3, a specialized IFT anterograde motor. Furthermore, we observed an abrupt collapse of the GCY-22::GFP ciliary tip compartment after treatment with sodium azide, indicating that an ATP-dependent process maintains this ciliary tip localization. Additionally, we investigated which proteins are involved in trafficking of GCY-22::GFP to the cilium. We found that GCY-22::GFP does not enter the cilium in a mutant lacking DAF-25, the ortholog of the poorly studied mammalian ankyrin repeat and MYND domain-containing protein Ankmy2. Interestingly, the daf-25 mutant animals do not respond to NaCl, but the localization and behavioral phenotype could be rescued by disrupting the transition zone protein MKS-5, a core component of a ciliary gate. Finally, GCY-22::GFP deletion and chimeric constructs showed that the C-terminal RD3-associated and dimerization domains are important for ciliary tip localization and that loss of tip localization results in reduced chemotaxis to lower NaCl concentrations. Our findings reveal that a cGMP-producing signaling protein functions within a distinct ciliary domain and that it uses several protein transport machineries to reach and maintain its singular subcellular localization.

Murakami S et al. (1996) West Coast Worm Meeting "LIFE-EXTENSION PATHWAY IN C.ELEGANS"

Dames SA, Johnson TE, Murakami S, Tedesco PM, Duhon SA

[West Coast Worm Meeting, 1996]

Life extension is conferred either by mutants extending adult life (Age), including age-1, daf-2, daf-23, daf-28, spe-26, and an allele of clk-1. Another mode of life extension is conferred by mutants delaying developmental stage (Clk), including clk-1, clk-2, clk-3, gro-1(Wong et al., 1995; Lakowski and Hekimi, 1996). Three groups have identified a life-extension pathway formed by some Daf mutations (Dorman et al., 1995; Larsen et al., 1995; Murakami and Johnson, 1996). We found that the pathway is not limited to Daf mutations but includes other types of the Age mutants (Murakami and Johnson, 1996). Interestingly, the pathway confers UV resistance, suggesting that UV resistance is a good indicator of Age. It is also consistent with the hypothesis that stress resistance is a rate limiting factor determining longevity. We are also testing whether the Age-pathway can confer other types of stress such as H2O2 and heat.

Hooper C (1991) The Journal of NIH Research "Old-aged mutant ninja nematodes' cool anti-age gene."

Hooper C

[The Journal of NIH Research, 1991]

Cowabugna, dudes! Those lean, gene-revealing machines have scored a most totally excellent victory in the battle to understand aging. We are, of course, talking about mutant ninja nematodes here. At a conference on aging in January at Cold Spring Harbor's Banbury Center, Thomas Johnson of the Institute for Behavioral Genetics at the University of Colorado in Boulder brought some dudes and dudettes from Capitol Hill up to date on the latest awesome achievements of the bodacious beasts know as Caenorhabditis elegans.

Cowden C et al. (1986) Worm Breeder's Gazette "a CCK-like neuropeptide in Ascaris."

Stretton AOW, Cowden C

[Worm Breeder's Gazette, 1986]

Our aim is to obtain peptides of known sequence from Ascaris in order to determine their mode of action using electrophysiological methods. Since McIntire and Horvitz (C. elegans CSH Abstracts 1985) showed that cholecystokinin-like immunoreactivity (CCK-LI) is present in certain neurons in C. elegans, we are initially investigating the role of a CCK-like peptide (CCK-LP) in the nervous system of Ascaris using anti-CCK8 antisera to detect and localize CCK-LI. Using the methods developed by Johnson (see Johnson and Stretton, Soc. Neurosci. Abstr. 9: 302; Sithigorngul, Johnson and Stretton, C. elegans CSH Abstracts 1985) we find that in Ascaris, CCK-LI is concentrated in 2 cells (AVF cells) in the ventral nerve cord, in 3 cells in the ventral ganglion, in 4 processes in the ventral cord, and in 2 processes in the lateral line. Thus the CCK-LP is concentrated in a small minority of the 180 neurons present in the anterior region of adult Ascaris.We have developed a procedure for extracting the CCK- LP from C. elegans and fractionating the extract on a C18 cartridge. High voltage paper electrophoresis shows that added radioiodinated CCK8 is chemically intact after this extraction and after fractionation. The CCK-LP was separated from more hydrophilic components with a 20-40% gradient of acetonitrile on reversed phase HPLC. RIA's detected CCK-LI associated with a peak of optical density. Addition of authentic CCK8 (non-sulfated) to the sample showed that the RIA-positive peak was close to, but distinctly separate from, CCK8. Assuming that the specific immunoreactivity of nematode CCK-LP and mammalian CCK8 is the same, we can obtain 100 pmoles from 60g of C. elegans. From this crude estimate, it seems that the levels of recoverable peptide are sufficient for amino acid sequence determination which is now our top priority.