Spectral scanning and fluorescence lifetime imaging microscopy (FLIM) enable separation and characterization of C. elegans autofluorescence in the Cuticle and Gut.

C. elegans gut and cuticle produce a disruptive amount of autofluorescence during imaging. Although C.

A Fiji process for quantifying fluorescent puncta in linear cellular structures.

Understanding the cell biology of protein trafficking and homeostasis requires reproducible methods for identifying and quantifying proteins within cells or cellular structures. Imaging protocols for measuring punctate protein accumulation in linear structures, for example the neurites of have relied on proprietary software for a full range of analysis capabilities. Here we describe a set of macros written for the NIH-supported imaging software ImageJ or Fiji (Fiji is Just ImageJ) that reliably identify protein puncta so that they can be analyzed with respect to intensity, density, and width at half-maximum intensity (Full-Width, Half-Maximum, FWHM).

A Fiji protocol for analyzing puncta is a robust tool for measuring GLR-1::GFP accumulation in the ventral nerve cord of .

In DAF-7/TGF-beta signaling regulates development, metabolism, and behavior. In addition loss of leads to an increase of the glutamate receptor GLR-1. In mutants, GLR-1 tagged with GFP (GLR-1::GFP) accumulates in wide puncta along the ventral nerve cord of the animal.

Comparison and agreement between two image analysis tools for quantifying GFP::SNB-1 puncta in mutants of C. elegans.

Quantitative imaging of synaptic vesicle localization and abundance using fluorescently labeled synaptic vesicle associated proteins like GFP::SNB-1 is a well-established method for measuring changes in synapse structure at neuromuscular junctions (NMJ) in . To date, however, the ability to easily and reproducibly measure key parameters at the NMJ - maximum intensity, size of GFP::SNB-1 puncta, density of puncta - has relied on the use of expensive, customizable software that requires coding skills to modify, precluding widespread access and thus preventing standardization within the field. We carried out a comparative evaluation of a new, open-source Fiji puncta plugin versus traditional Igor-based analysis of GFP::SNB-1 imaging data taken of cholinergic motor neurons in the dorsal nerve cord of loss of function mutants in , which encodes a G protein-coupled receptor known to impact GFP::SNB-1 accumulation.

Building a laboratory at a Primarily Undergraduate Institution (PUI).

Scientists who are interested in building research programs at primarily-undergraduate institutions (PUIs) have unique considerations compared to colleagues at research-intensive (R1) institutions. Maintaining a research program at a PUI holds unique challenges that should be considered before prospective faculty go on the job market, as they negotiate a job offer, and after they begin a new position. In this article we describe some of the considerations that aspiring and newly hired faculty should keep in mind as they plan out how they will set up a laboratory as a new Principle Investigator (PI) at a PUI.

Student Annotations of Published Data as a Collaboration between an Online Laboratory Course and the C. elegans Database, WormBase.

Course-based undergraduate research experiences (CUREs) provide the same benefits as individual, mentored faculty research while expanding the availability of research opportunities. One important aspect of CUREs is students' engagement in collaboration. The shift to online learning during the COVID-19 pandemic created an immediate need for meaningful, collaborative experiences in CUREs.

The WD40-Repeat Protein WDR-20 and the Deubiquitinating Enzyme USP-46 Promote Cell Surface Levels of Glutamate Receptors.

Reversible modification of AMPA receptors (AMPARs) with ubiquitin regulates receptor levels at synapses and controls synaptic strength. The conserved deubiquitinating enzyme (DUB) ubiquitin-specific protease-46 (USP-46) removes ubiquitin from AMPARs and protects them from degradation in both and mammals. Although DUBs are critical for diverse physiological processes, the mechanisms that regulate DUBs, especially in the nervous system, are not well understood.

The WD40-repeat protein WDR-48 promotes the stability of the deubiquitinating enzyme USP-46 by inhibiting its ubiquitination and degradation.

Ubiquitination is a reversible post-translational modification that has emerged as a critical regulator of synapse development and function. However, the mechanisms that regulate the deubiquitinating enzymes (DUBs) responsible for the removal of ubiquitin from target proteins are poorly understood. We have previously shown that the DUB ubiquitin-specific protease 46 (USP-46) removes ubiquitin from the glutamate receptor GLR-1 and regulates its trafficking and degradation in We found that the WD40-repeat proteins WDR-20 and WDR-48 bind and stimulate the catalytic activity of USP-46.

Function of the Deubiquitinating Enzyme USP46 in the Nervous System and Its Regulation by WD40-Repeat Proteins.

Posttranslational modification of proteins by ubiquitin regulates synapse development and synaptic transmission. Much progress has been made investigating the role of ubiquitin ligases at the synapse, however very little is known about the deubiquitinating enzymes (DUBs) which remove ubiquitin from target proteins. Although there are far fewer DUBs than ubiquitin ligases encoded by the human genome, it is becoming clear that DUBs have very specific physiological functions, suggesting that DUB activity is tightly regulated .

The CaM Kinase CMK-1 Mediates a Negative Feedback Mechanism Coupling the C. elegans Glutamate Receptor GLR-1 with Its Own Transcription.

Regulation of synaptic AMPA receptor levels is a major mechanism underlying homeostatic synaptic scaling. While in vitro studies have implicated several molecules in synaptic scaling, the in vivo mechanisms linking chronic changes in synaptic activity to alterations in AMPA receptor expression are not well understood. Here we use a genetic approach in C.

Effect of helix length on the stability of the Lac repressor antiparallel coiled coil.

The helix length dependence of the stability of antiparallel four-chain coiled coils is investigated using eight synthetic peptides (Lac21-Lac28) whose sequences are derived from the tetramerization domain of the Lac repressor protein. Previous studies using analytical ultracentrifugation sedimentation equilibrium experiments to characterize Lac21 and Lac28 justifies the use of a two state model to describe the unfolding behavior of these two peptides. Using circular dichroism spectropolarimetry as a measure of tetramer assembly, both chemical and thermal denaturation experiments were carried out to determine thermodynamic parameters.

The WD40-repeat proteins WDR-20 and WDR-48 bind and activate the deubiquitinating enzyme USP-46 to promote the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of Caenorhabditis elegans.

Ubiquitin-mediated endocytosis and degradation of glutamate receptors controls their synaptic abundance and is implicated in modulating synaptic strength. The deubiquitinating enzymes (DUBs) that function in the nervous system are beginning to be defined, but the mechanisms that control DUB activity in vivo are understood poorly. We found previously that the DUB USP-46 deubiquitinates the Caenorhabditis elegans glutamate receptor GLR-1 and prevents its degradation in the lysosome.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis.

Objective: Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence.

The deubiquitinating enzyme USP-46 negatively regulates the degradation of glutamate receptors to control their abundance in the ventral nerve cord of Caenorhabditis elegans.

Ubiquitin-mediated endocytosis and post-endocytic trafficking of glutamate receptors control their synaptic abundance and are implicated in modulating synaptic strength. Ubiquitination is a reversible modification, but the identities and specific functions of deubiquitinating enzymes in the nervous system are lacking. Here, we show that the deubiquitinating enzyme ubiquitin-specific protease-46 (USP-46) regulates the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of Caenorhabditis elegans.

Large-scale SOD1 mutation screening provides evidence for genetic heterogeneity in amyotrophic lateral sclerosis.

Objective: To estimate the frequency of SOD1 mutations in a large referral cohort of familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS) patients from The Netherlands and to compare this frequency with that of other developed countries.

Methods: A total of 451 sporadic and 55 FALS patients were screened for SOD1 mutations. The authors performed PCR amplification of all five coding exons of SOD1 followed by direct DNA sequencing using forward and reverse primers.

Genome-wide association study identifies 19p13.3 (UNC13A) and 9p21.2 as susceptibility loci for sporadic amyotrophic lateral sclerosis.

We conducted a genome-wide association study among 2,323 individuals with sporadic amyotrophic lateral sclerosis (ALS) and 9,013 control subjects and evaluated all SNPs with P < 1.0 x 10(-4) in a second, independent cohort of 2,532 affected individuals and 5,940 controls. Analysis of the genome-wide data revealed genome-wide significance for one SNP, rs12608932, with P = 1.

Interactions between Casein kinase Iepsilon (CKIepsilon) and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

Background: Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates.

Crystal structure of a beta-catenin/BCL9/Tcf4 complex.

The canonical Wnt pathway plays critical roles in embryonic development, stem cell growth, and tumorigenesis. Stimulation of the Wnt pathway leads to the association of beta-catenin with Tcf and BCL9 in the nucleus, resulting in the transactivation of Wnt target genes. We have determined the crystal structure of a beta-catenin/BCL9/Tcf-4 triple complex at 2.

Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics.

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.

Compensating effects on the cytotoxicity of ribonuclease A variants.

Ribonuclease (RNase) A can be endowed with cytotoxic activity by enabling it to evade the cytosolic ribonuclease inhibitor protein (RI). Enhancing its conformational stability can increase further its cytotoxicity. Herein, the A4C/K41R/G88R/V118C variant of RNase A was created to integrate four individual changes that greatly decrease RI affinity (K41R/G88R) and increase conformational stability (A4C/V118C).