Netrin-1 Signals Through Protein Kinases in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are a family of signaling proteins involved in developmental processes such as neuronal guidance and angiogenesis. The best characterized netrin, netrin-1, signals through a number of different receptors. When acting as a chemoattractant, netrin-1 primarily signals through the DCC receptor and associated protein tyrosine kinase and MAP kinase signaling pathways. When acting as a chemorepellent, netrin-1 signals through the UNC5 receptor, which involves recruitment of the protein tyrosine phosphatase, SHP2. While netrins are ubiquitously expressed throughout the animal kingdom, our laboratory was the first to describe a netrin-1 like protein in Tetrahymena. This netrin-1 like protein is secreted from Tetrahymena and acts as a chemorepellent. In our current study, we describe signaling through netrin-1 in this organism. Netrin-1 signaling is inhibited by the tyrosine kinase inhibitor, hypericin, and by the broad-spectrum kinase in hibitor, apigenin, both acting in the micromolar range.. We are conducting further studies to determine whether netrin-1 signaling results in changes to the phosphorylation state of intracellular proteins

Mapping Netrin Signaling in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrin family of proteins, found throughout the animal kingdom, are well known for their roles in developmental signaling. Netrin-1, the best-studied member of this family, signals through four receptor types in vertebrates: the UNC-5 family, DCC, neogenin, and DSCAM. We have previously characterized a netrin-1-like protein in the ciliated protozoan, Tetrahymena thermophila. This protein is secreted from Tetrahymena, and functions as a chemorepellent. Since a netrin-like protein is produced by this organism, we hypothesized that some components of the vertebrate netrin signaling pathway might also be present in Tetrahymena. Through immunolocalization on the plasma membrane of the cell, we have found that Tetrahymena appear to have a UNC-5 like protein, as well as proteins that are immunologically similar to neogenin. A homolog of src-1, a tyrosine kinase involved in vertebrate netrin-1, is also present in Tetrahymena. Future experiments will allow us to make more comparisons between netrin signaling in Tetrahymena with netrin signaling in the animal kingdom, and will allow us to determine the suitability of Tetrahymena as a model system for this particular pathway

Netrin-3 Signals Through Serine Phosphorylation in Tetrahymena thermophila

The netrin family of proteins are structurally related to laminin and, while first discovered in the nematode Caenorhabditis elegans, are now known to be present in species throughout the animal kingdom, including humans. These proteins also have a wide variety of roles that include inhibition of apoptosis, chemorepulsion, and axonal guidance. Due to the results of previous studies involving netrin-1 in vertebrate systems, the current prevailing assumption is that netrins, when acting as chemorepellents, signal using tyrosine kinases. However, data that we gathered through phosphoserine-targeting ELISA assays and immunofluorescence microscopy demonstrates that the netrin-3 peptides signal Tetrahymena thermophila through serine phosphorylation instead, causing the ciliate protists to avoid netrin-3 peptides in response. Treatment with netrin-3 peptides also seems to cause mitotic inhibition in Tetrahymena, which can be reversed by addition of a serine kinase inhibitor. This new information suggests that netrin-3 may have physiological roles that have previously been unexplored

Netrin-3 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrins are a family of signaling proteins expressed throughout the animal kingdom. Netrins play important roles in developmental processes such as axonal guidance and angiogenesis. Netrin-1, for example, can act as either a chemoattractant or a chemorepellent for axonal growth cones depending upon the concentration of the protein as well as the cell type. Netrin-1 acts as a growth factor in some mammalian cell types and is also expressed by some tumor cells. Netrin-3 appears to share some signaling apparatus with netrin-1, but is less widely expressed, and its physiological roles are much less understood. Netrin-3 is also used as a biomarker for some cancers as well as traumatic kidney injury. Tetrahymena thermophila are free-living, eukaryotic, ciliated protozoas used as a model system for studying chemorepellents and chemoattractants because their swimming behavior is readily observable under a microscope. We have previously found that netrin-1 peptide acts as a chemorepellent in Tetrahymena thermophila at concentrations ranging from micromolar to nanomolar. However, netrin-1 peptide does not affect growth in Tetrahymena at these concentrations. In our current study, we have found that related peptides, netrin-3 peptide (H-19 and C-19; Santa Cruz Biotechnology), act as chemorepellents in Tetrahymena thermophila at concentrations at or below 1 μg/ml. The same concentration of netrin-3 peptide reduces growth of Tetrahymena cultures by approximately 75%. We are currently conducting further studies to determine the mechanism through which these peptides are signaling

DDX3X and specific initiation factors modulate FMR1 repeat‐associated non‐AUG‐initiated translation

A CGG trinucleotide repeat expansion in the 5′ UTR of FMR1 causes the neurodegenerative disorder Fragile X‐associated tremor/ataxia syndrome (FXTAS). This repeat supports a non‐canonical mode of protein synthesis known as repeat‐associated, non‐AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate‐based screen of eukaryotic initiation factors and RNA helicases in cell‐based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5′UTR. These include the DEAD‐box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat‐induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat‐associated neurodegeneration.SynopsisFragile X‐associated tremor/ataxia syndrome is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical for FMR1 RAN translation.Knockdown of the RNA helicase DDX3X selectively suppresses FMR1 RAN translation in Drosophila melanogaster, cultured HeLa cells, and in vitro translation assays.DDX3X knockdown reduces CGG repeat‐associated toxicity in Drosophila and mammalian neurons.Eukaryotic initiation factors that modulate RNA‐RNA secondary structure (DDX3X, EIF4B, EIF4H) or start codon fidelity (EIF1, EIF5) impact FMR1 RAN translation.FXTAS is caused by CGG repeat‐associated non‐AUG (RAN) translation that initiates within the 5′UTR of FMR1. A candidate‐based screen identified several initiation factors—DDX3X/Belle, eIF4B, eIF4H, eIF1, and eIF5—critical for FMR1 RAN translation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/1/embr201847498.reviewer_comments.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/2/embr201847498-sup-0001-Appendix.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/3/embr201847498_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151325/4/embr201847498.pd

Netrin-3 Avoidance and Mitotic Inhibition in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e Involves Intracellular Calcium and Serine/Threonine Kinase Activity

Netrins are a family of signaling proteins ubiquitously expressed throughout the animal kingdom. While netrin-1 has been well characterized, other netrins, such as netrin-3, remain less well understood. In our current study, we characterize the behavior of two netrin-3 peptides, one derived from the N-terminal and one derived from the C-terminal of netrin-3. Both peptides cause avoidance behavior and mitotic inhibition in Tetrahymena thermophila at concentrations as low as 0.5 micrograms (μg) per milliliter. These effects can be reversed by addition of the calcium chelator, EGTA; the intracellular calcium chelator, BAPTA-AM, or the serine/threonine kinase inhibitor, apigenin. The broad spectrum tyrosine kinase inhibitor, genistein, has no effect on netrin-3 signaling, indicating that netrin-3 signaling in this organism uses a different pathway than the previously described netrin-1 pathway. Further studies will allow us to better describe the netrin-3 signaling pathway in this organism

Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall

Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology

Search for gravitational-lensing signatures in the full third observing run of the LIGO-Virgo network

Gravitational lensing by massive objects along the line of sight to the source causes distortions of gravitational wave-signals; such distortions may reveal information about fundamental physics, cosmology and astrophysics. In this work, we have extended the search for lensing signatures to all binary black hole events from the third observing run of the LIGO--Virgo network. We search for repeated signals from strong lensing by 1) performing targeted searches for subthreshold signals, 2) calculating the degree of overlap amongst the intrinsic parameters and sky location of pairs of signals, 3) comparing the similarities of the spectrograms amongst pairs of signals, and 4) performing dual-signal Bayesian analysis that takes into account selection effects and astrophysical knowledge. We also search for distortions to the gravitational waveform caused by 1) frequency-independent phase shifts in strongly lensed images, and 2) frequency-dependent modulation of the amplitude and phase due to point masses. None of these searches yields significant evidence for lensing. Finally, we use the non-detection of gravitational-wave lensing to constrain the lensing rate based on the latest merger-rate estimates and the fraction of dark matter composed of compact objects

Search for eccentric black hole coalescences during the third observing run of LIGO and Virgo

Despite the growing number of confident binary black hole coalescences observed through gravitational waves so far, the astrophysical origin of these binaries remains uncertain. Orbital eccentricity is one of the clearest tracers of binary formation channels. Identifying binary eccentricity, however, remains challenging due to the limited availability of gravitational waveforms that include effects of eccentricity. Here, we present observational results for a waveform-independent search sensitive to eccentric black hole coalescences, covering the third observing run (O3) of the LIGO and Virgo detectors. We identified no new high-significance candidates beyond those that were already identified with searches focusing on quasi-circular binaries. We determine the sensitivity of our search to high-mass (total mass M>70 M⊙) binaries covering eccentricities up to 0.3 at 15 Hz orbital frequency, and use this to compare model predictions to search results. Assuming all detections are indeed quasi-circular, for our fiducial population model, we place an upper limit for the merger rate density of high-mass binaries with eccentricities 0<e≤0.3 at 0.33 Gpc−3 yr−1 at 90\% confidence level