Browsing by Author "Garrity, Deborah M., committee member"

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Open Access
An investigation of the molecular complexities that regulate molting in decapod crustaceans
(Colorado State University. Libraries, 2015) Pitts, Natalie Lynn, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Tjalkens, Ronald B., committee member; Tsunoda, Susan, committee member
Molting in decapod crustaceans is regulated by the interaction of two hormones, molt inhibiting hormone (MIH) and ecdysteroids. Ecdysteroids are steroid hormones secreted from the molting gland or Y-organ (YO) and fluctuations in hemolymph ecdysteroid titers regulate progression through the molt cycle. Secretion of ecdysteroids is controlled by the peptide hormone MIH, which is synthesized and released from the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). The field of crustacean endocrinology has mainly focused on understanding the molecular underpinnings of MIH’s action on ecdysteroid production in the YO. The goal of this dissertation was to examine how MIH synthesis and secretion from the XO/SG complex contributes to molt cycle progression. Blackback land crabs, Gecarcinus lateralis, were induced to molt via autotomy of five or more walking legs (multiple limb autotomy or MLA). ESG were collected from intermolt, premolt, and post-molt animals and changes in expression of Gl-MIH and mTOR signaling pathway components were investigated. There was a significant effect of molt stage on Gl-MIH and mTOR signaling pathway gene expression in the ESG of G. lateralis. Continuous elevation of MIH transcript abundance during pre and post molt indicates that MIH titers in the hemolymph are not regulated by changes in transcript abundance. Molting also significantly increased expression of Gl-Akt, Gl-mTOR, Gl-Rheb, and Gl-S6K in one or more molt stages. Akt inhibits the tuberous sclerosis complex allowing for the activation of Rheb. Rheb is a GTPase that binds and activates the mechanistic target of rapamycin (mTOR). mTOR activates S6 kinase (S6K), increasing protein synthesis. ESG of naturally molting green crabs, Carcinus maenas, were also collected from intermolt, early premolt, and post molt animals. Molting had little effect on gene expression in C. maenas, confirming previous findings that molt progression is regulated post transcriptionally. This dissertation identifies a novel nitric oxide (NO) binding protein in the SG of C. maenas. The hypothesis is that NO negatively regulates MIH secretion from the SG thereby controlling molt progression. This unidentified endogenous binding protein allows NO to be present in the SG for a prolonged period and can therefore continually regulate neuropeptide release. Localization of the enzyme that produces NO (nitric oxide synthase; NOS) and MIH in the SG of C. maenas, G. lateralis, and Metacarcinus magister is consistent with the hypothesis that NO is a regulator of neuropeptide release in the crustacean SG. The second goal of this dissertation was to explore why some crustacean populations or individuals within a population are refractory to molt induction. The Bodega Bay population of C. maenas is refractory to molt induction techniques and a similar phenomenon is observed in G. lateralis. Some G. lateralis induced to molt via MLA did not enter premolt 90 days post induction and were classified as "blocked." These animals underwent a second molt induction technique, eyestalk ablation (ESA), and YO, brain (Br), and thoracic ganglia (TG) were collected at 1, 3, and 7 days post ESA. Gene expression of MIH and mTOR signaling pathway genes was examined in all three tissues (see Figure 3 for MIH signaling pathway components and there interactions). Results from this experiment suggested that a similar mechanism of molt resistance exists between C. maenas and G. lateralis. ESA did not increase hemolymph ecdysteroid titers of blocked animals, whereas ESA significantly increased ecdysteroid titers in control and intermolt animals. Gl-MIH expression in the ESG and expression of many Gl-MIH signaling components in the YO were upregulated in blocked animals, suggesting that the blocked animals were in a "hyper-repressed" state, and therefore resistant to molt induction by ESA and MLA. In both species, MIH is expressed in the Br and TG. The hypothesis is that MIH secretion from these other central nervous system (CNS) tissues contributes to a resistance to molt induction techniques. Expression of MIH signaling pathway genes is unchanged in the Br and TG in response to ESA. These data suggest that MIH does not activate a signaling pathways in CNS tissues but like the ESG, MIH is synthesized and secreted from these tissues. This experiment also supports the growing body of literature that mTOR inhibition activates downstream transcription factors which are important in maintaining energy homeostasis in times of environmental stress.
Open Access
Breaking the MF curse: the regulatory role of the Methyl farnesoate – MEKRE93 pathway in crustacean molting
(Colorado State University. Libraries, 2025) Bentley, Vanessa Leah, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Montgomery, Taiowa, committee member; Reist, Noreen, committee member
Ecdysis, or the active shedding of the exoskeleton, is critical for arthropod growth, development, and/or regeneration of lost or damaged appendages. The antagonistic interaction between the steroid hormone 20-hydroxyecdysone (20-E) and the sesquiterpenoid juvenile hormone (JH) control insect molting and development, respectively. On the other hand, crustacean molting is primarily regulated through the endocrine crosstalk between 20-E and the neuropeptide molt-inhibiting hormone (MIH). MIH secretion by the X-organ, sinus gland complex (XO) inhibits Y-organ (YO) synthesis of 20-E. Molting is initiated by the decrease in MIH titers thereby allowing increasing 20-E concentrations in the hemolymph. The molting process is divided into different stages where the YO exhibits different phenotypic states: intermolt (IM)– basal, early premolt (EP)– activated, mid premolt (MP)– committed, late premolt (LP)– committed/repressed, ecdysis (E)– repressed, and postmolt (PM)– repressed/basal. The mechanistic target of rapamycin (mTOR) and transforming growth factor beta (TGF-β) signaling leads to YO activation and commitment, respectively. However, the YO transition to- and from- the repressed state is unknown. Methyl farnesoate (MF), commonly referred to as the crustacean JH, is produced by the mandibular organ (MO) and is suppressed by the mandibular organ-inhibiting hormone (MOIH). MF regulates several physiological processes in crustaceans including metamorphosis, development, reproduction, morphogenesis, and molting; however, the underlying mechanism remains unknown and thereby is considered to be "cursed". The differential effects MF has on molting and ecdysteroidogenesis is hypothesized to be regulated through the Methoprene tolerant– Krüppel homolog 1– E93 (MEKRE93) transcriptional cascade. Using bioinformatic approaches, the components of the MF signaling pathway were identified in the European green shore crab (Carcinus maenas) and the blackback land crab (Gecarcinus lateralis) YO transcriptomes, including the MF/JH receptor Methoprene tolerant (Met), the zinc finger transcription factor Krüppel homolog 1 (Kr-h1), Ecdysone response gene 93 (E93), Steroid receptor coactivator (Src), and transcription comediators CREB–binding protein (CBP) and C-terminal–binding protein (CtBP). Additionally, genes encoding for the MF synthetic pathway enzymes were also identified in the YO transcriptomes including 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesoic acid O-methyltransferase (FAMeT), and FAMeT2. Additionally, a FAMeT2 transcript was also identified in the YO and contains an unconventional domain organization compared to annotated FAMeT. Nonetheless, phylogenetic analysis of each gene was overall highly conserved across pancrustaceans (and occaisionally panarthropodans). Furthermore, in vitro assays showed that C. maenas and G. lateralis YOs were responsive to JH-mimics (e.g. pyriproxyfen, fenoxycarb, methoprene, and hydroprene), but not to MF. Taken altogether, these data suggest that the YO can respond to MF and may have its own MF-innate system serving as an autocrine factor to regulate the YO by acting through a MEKRE93 transcriptional network that can be mediated by coregulators.
Open Access
Clustering of non-conducting Kv2.1 channels induces endoplasmic reticulum/plasma membrane junctions and forms cell-surface trafficking hubs
(Colorado State University. Libraries, 2014) Fox, Philip Douglas, author; Tamkun, Michael M., advisor; Amberg, Gregory C., committee member; Vigh, Jozsef, committee member; Tsunoda, Susan, committee member; Garrity, Deborah M., committee member
The voltage-gated K+ channel, Kv2.1, is expressed widely in the mammalian CNS, where it carries the majority of the delayed-rectifier current. The Kv2.1 current facilitates high-frequency action potential firing by promoting the repolarization of the membrane potential and subsequent recovery of voltage-gated Na+ channels from inactivation. Furthermore, Kv2.1 displays a unique cell-surface localization to dense, micron-sized clusters which are sensitive to neuronal insults such as glutamate excitotoxicity. The following dissertation presents original research extending our knowledge of the Kv2.1 K+ channel. The majority of Kv2.1 channels are held in a non-conducting state which is incapable of fluxing K+ in response to membrane potential depolarization. These non-conducting channels tend to localize to the micron-sized clusters which distinguish Kv2.1. Non-conducting, clustered Kv2.1 channels remodel the cortical endoplasmic reticulum (cER) into tight connections with the plasma membrane (PM), likely through a direct interaction. Trafficking of membrane proteins, both exo- and endocytosis are localized to the perimeter of the Kv2.1-induced ER/PM contacts by virtue of remodeling the cER underneath the Kv2.1 clusters. Thus the clustering of Kv2.1 functions to bring protein trafficking and intermembrane signaling together at the neuronal soma.
Open Access
Genomics and transcriptomics of the molting gland (Y-organ) in the blackback land crab, Gecarcinus lateralis
(Colorado State University. Libraries, 2016) Martin, Lindsay, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Yao, Tingting, committee member
Molting is required for growth and development in crustaceans. In the blackback land crab Gecarcinus lateralis, molting is stimulated by ecdysteroids, hormones produced in the Y-organ (YO). Throughout the molting cycle, the YO demonstrates phenotypic plasticity. The phenotypic plasticity is correlated with the stages of the molt cycle, during which YO ecdysteroid production varies. During intermolt, the longest stage of the molt cycle, the circulating ecdysteroid titers are low and molting is suppressed. In preparation for molting, the YO increases ecdysteroid production during premolt. Circulating ecdysteroids continue to rise, dropping right before the ecdysis and remaining low in the subsequent postmolt period. During the molt cycle, the YO's sensitivity to inhibitory cues also varies, which contributes to ecdysteroid fluctuations. To better understand how changes in gene expression modulate the YO's phenotypic plasticity, a YO transcriptome from five molt stages was generated. Using over 5.6 million reads from Illumina, 229,278 contigs were assembled to comprise the reference transcriptome. By comparing expression levels of the transcripts between the molt stages, 13,189 unique differentially expressed contigs were identified in G. lateralis. Based on differential expression, insect hormone biosynthesis and oxidative phosphorylation pathways were enriched, validating the YO transcriptome identity. Using GO enrichment, MAP kinase was identified as a possible candidate gene for regulating YO ecdysteroid synthesis. To complement and validate the transcriptome, claw muscle genomic DNA was sequenced and assembled using 2.6 million reads. 375,152 scaffolds ≥ 500 bp were built, with an N50 of 1,841 bp. Using k-mer frequencies, the genome size was estimated to be 3.07 Gb, similar to mammalian vertebrates. The median gene size of G. lateralis was approximated to be 6,300 bp; the disparity between the median estimate and the N50 prohibited further computational analysis. Genome scaffolds were sufficient in length for manual comparison. Alignment of the transcriptome and genome sequences of the Rheb gene showed 100% nucleotide alignment in the open reading frame, and extended the sequence by 7.7 fold, including the identification of four introns. The sequence comparison validated both genome and transcriptome assemblies and extended the gene sequence. Next-generation sequencing provided us with a global perspective of molecular variations within the YO throughout the molt cycle. We hypothesize variations in gene expression regulate YO phenotypic plasticity by varying ecdysteroid production. YO transitions throughout molting are essential for regulation. YO activation and commitment, both corresponding to increased ecdysteroids, are required to induce ecdysis. YO repression, during which circulating ecdysteroid titers are low, is needed to prevent precocious molting. Identifying changes in gene expression and key regulatory elements correlating with variations in YO phenotype will increase our understanding of molt cycle regulation, which is critical for crustacean development, growth, and repair.
Open Access
Global analysis of mRNA decay rates and RNA-binding specificity reveals novel roles for CUGBP1 and PARN deadenylase in muscle cells
(Colorado State University. Libraries, 2011) Lee, Jerome Edward, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Garrity, Deborah M., committee member; Curthoys, Norman P., committee member
Type I Myotonic Dystrophy (DM1) is characterized by myotonia, cardiac conduction defects, muscle wasting, and insulin resistance. In patient muscle cells expression and function of the RNA-binding proteins CUGBP1 and MBNL1 are disrupted, resulting in altered mRNA metabolism at the levels of splicing and translation. Intriguingly, despite strong evidence for CUGBP1 being a regulator of mRNA turnover in humans and other organisms, the possibility that defects in mRNA decay contribute to DM1 pathogenesis has not been investigated to date. As such, we sought to further characterize the roles of CUGBP1 and its partner, the deadenylase PARN, in mRNA decay in mouse C2C12 muscle cells. The TNF message, which encodes a cytokine known to cause muscle wasting and insulin resistance when over-expressed, was stabilized by depletion of CUGBP1. The normally rapid decay of the TNF mRNA was also disrupted in cells treated with phorbol ester and this coincided with phosphorylation of CUGBP1. These findings provided impetus to undertake a global analysis of mRNA decay rates in muscle cells. Our investigation revealed that GU- and AU-rich sequence elements are enriched in labile transcripts, which encode cell cycle regulators, transcription factors, and RNA-processing proteins. Transcripts specifically bound to CUGBP1 in myoblasts are linked with processes such as mRNA metabolism, protein targeting to the endoplasmic reticulum, cytoskeletal organization, and transcriptional regulation, all of which have implications for muscle cell biology. Consistent with this, CUGBP1 depletion profoundly altered the formation of myotubes during differentiation. Finally we investigated whether PARN, which interacts with CUGBP1 and mediates rapid deadenylation of TNF in HeLa cell extracts, also plays a role in mediating mRNA decay in muscle. We identified 64 mRNA targets whose decay was dependent on PARN. Moreover, deadenylation of the Brf2 mRNA was impaired in PARN knock-down cells supporting that this mRNA is directly and specifically targeted for decay by PARN. Taken together our findings demonstrate that CUGBP1 and PARN are critical regulators of decay for specific sets of transcripts in muscle cells. It seems likely that some or all of the CUGBP1 targets we have identified may be affected in myotonic dystrophy. Defective mRNA turnover could be linked with defects in myogenesis, TNF over-expression, muscle wasting and/or ER stress, all of which have been documented in DM1.
Open Access
Molecular regulation of growth and molting in decapod crustaceans
(Colorado State University. Libraries, 2014) Mudron, Megan Reese, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Curthoys, Norman P., committee member
The green shore crab, Carcinus maenas, is a highly invasive species that inhabits coastal temperate zones worldwide. The reaction of C. maenas to acute temperature change was determined in six tissues (heart, gill, thoracic ganglion, eyestalk ganglion, Y-organ, and claw muscle) using genetic markers for temperature-induced metabolic stress, including HSP70, AMPKγ, mTOR, and Rheb. Animals were exposed to temperatures between 5° and 30°C for 1 or 2 h. mRNA levels in six tissues were quantified by quantitative RT-PCR (qPCR). The results indicate that C. maenas tolerated a wide temperature range, requiring 2-h exposures at 5 °C and 30 °C to affect tissue-specific changes in gene expression. Cm-HSP70 expression was robustly increased at 30 °C in all tissues. Ecdysteroids produced from the molting gland (Y-organ or YO) induce molting in decapod crustaceans. Reduction in molt-inhibiting hormone (MIH) activates the YO and animals enter premolt. At mid-premolt, YOs transition to the committed state, during which ecdysteroid production increases further. In blackback land crab (Gecarcinus lateralis), a tropical decapod species, SB1431542, an inhibitor of Activin receptors, decreases hemolymph ecdysteroid titers in premolt animals, suggesting that an Activin-like transforming-growth factor (TGF-β) is produced by the activated YO and drives the transition of the YO to the committed state. Myostatin (Gl-Mstn) is an Activin-like factor that is highly expressed in skeletal muscle. Rapamycin lowers hemolymph ecdysteroid titers by inhibiting mTOR, which controls global translation of mRNA into protein. Endpoint RT-PCR established that Gl-Mstn was expressed in the YO, not just muscle tissue. YOs were harvested from intact (intermolt) animals and from animals at 1, 3, 5, 7, and 14 days post-ESA. Quantitative PCR was used to quantify the effects of molt induction by eyestalk ablation (ESA) on gene expression. Expression of mTOR components peaked at 3 days post-ESA, which is consistent with the increased activity required for activation of the YO. Gl-Mstn expression also peaked at 3 days post-ESA, which is before the transition to the committed state at 7 days post-ESA. These results indicate that mTOR components are involved in activation of the YO, and Mstn is involved in transitioning the YO to the committed state.
Open Access
Role of mechanistic Target of Rapamycin (mTOR) signaling in the crustacean molting gland
(Colorado State University. Libraries, 2012) Abuhagr, Ali Moftah M., author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Reddy, Anireddy N., committee member; Curthoys, Norman P., committee member
Regulation of the molt cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). Molt-inhibiting hormone (MIH), secreted by the XO/SG complex, suppresses production of molting hormone (ecdysteroids) by a pair of YOs. In the blackback land crab, Gecarcinus lateralis, molting can be induced by eyestalk ablation (ESA) or autotomy of 5 or more walking legs (multiple leg autotomy or MLA). During the molt cycle, the YO transitions through four physiological states: "basal" state at postmolt and intermolt; "activated" state at early premolt; "committed" state at mid premolt and "repressed" state at late premolt. The basal to activated state transition is triggered by a transient reduction in MIH; the YOs hypertrophy, but remain sensitive to MIH. The main hypothesis is that up-regulation of mechanistic Target of Rapamycin (mTOR) signaling, which controls global translation of mRNA into protein, is necessary for YO hypertrophy and ecdysteroidogenesis. cDNAs encoding mTOR, Rheb, Akt (protein kinase B) and p70 S6 kinase (S6k) were cloned from blackback land crab, G. lateralis, and green shore crab, Carcinus maenas. All four genes were expressed in all tissues examined. mTOR appears to be involved in YO activation in early premolt, as rapamycin inhibited YO ecdysteroidogenesis in vivo and in vitro. In addition, the expression of Gl-elongation factor 2 (EF2), Gl-mTOR, and Gl-Akt increased significantly in YOs from premolt, suggesting that an increase in protein synthetic capacity is necessary for YO activation. A putative transforming growth factor-beta (TGFâ) appeared to be involved in the transition of the YO from the activated to committed state, as SB431542, an Activin receptor antagonist, lowered hemolymph ecdysteroid titers in mid premolt animals and abrogated the premolt increases in Gl-EF2, Gl-mTOR, and Gl-Akt mRNA levels. By contrast, molting had no effect on Cm-EF2, Cm-mTOR, Cm-Rheb, Cm-Akt, and Cm-S6k expression in C. maenas YOs. Unlike G. lateralis, adult C. maenas was refractory to ESA. ESA caused a small increase in hemolymph ecdysteroid titers, but animals did not immediately enter premolt. Some ES-ablated animals molted after many months, but most failed to molt at all. We hypothesized that other regions of the nervous system, specifically the brain and/or thoracic ganglion, were secondary source(s) of MIH. Nested endpoint RT-PCR showed that MIH transcript was present in brain and thoracic ganglion of intermolt crabs. Sequencing of the PCR product confirmed its identity as MIH. Real time PCR was used to quantify the effects of ESA on MIH expression in brain and thoracic ganglion on C. maenas red and green color morphs. ESA had little effect on MIH transcript levels, indicating that MIH was not regulated transcriptionally by the loss of the eyestalks. The data suggest that MIH secreted by neurons in the brain and thoracic ganglion is sufficient to prevent molt induction when the primary source of MIH is removed by ESA. There was also no effect of ESA on the expression of Gl-EF2 and mTOR signaling components in C. maenas YOs.