RESUMEN
BACKGROUND: Caenorhabditis elegans is a model organism used to study gene, protein, and cell influence on function and behavior. These studies frequently require C. elegans to be immobilized for imaging or laser ablation experiments. There are a number of known techniques for immobilizing worms, but to our knowledge, there are no comprehensive studies of the various agents in common use today. NEW METHOD: This study determines the relationship between concentration, immobilization time, exposure time, and recovery likelihood for several immobilization agents. The agents used in this study are 1-Phenoxy-2-propanol, levamisole, sodium azide, polystyrene beads, and environmental cold shock. These tests are conducted using a humidified chamber to keep chemical concentrations consistent. Each of these agents is also tested to determine if they exhibit stress-related after effects using the gcs-1, daf-16, hsp-4, hif-1, hsp-16.2, and tmem-135 stress reporters. RESULTS: We present a range of quick mount immobilization and recovery conditions for each agent tested. This study shows that, under controlled conditions, 1-Phenoxy-2-propanol shows significant stress from the daf-16 reporter. While 1-Phenoxy-2-propanol and sodium azide both create stress related after effects with long term recovery in the case of the hsp-16.2 reporter. COMPARISON WITH EXISTING METHODS: This study shows that commonly used concentrations of immobilizing agents are ineffective when evaporation is prevented. CONCLUSIONS: To improve reproducibility of results it is essential to use consistent concentrations of immobilizing agents. It is also critically important to account for stress-related after effects elicited by immobilization agents when designing any experiment.
RESUMEN
Auxiliary subunits are often needed to tailor K+ channel functional properties and expression levels. Many auxiliary subunits have been identified for mammalian Slo1, a high-conductance K+ channel gated by voltage and Ca2+. Experiments with heterologous expression systems show that some of the identified Slo1 auxiliary subunits can also regulate other Slo K+ channels. However, it is unclear whether a single auxiliary subunit may regulate more than one Slo channel in native tissues. BKIP-1, an auxiliary subunit of C. elegans SLO-1, facilitates SLO-1 membrane trafficking and regulates SLO-1 function in neurons and muscle cells. Here we show that BKIP-1 also serves as an auxiliary subunit of C. elegans SLO-2, a high-conductance K+ channel gated by membrane voltage and cytosolic Cl- and Ca2+. Comparisons of whole-cell and single-channel SLO-2 currents in native neurons and muscle cells between worm strains with and without BKIP-1 suggest that BKIP-1 reduces chloride sensitivity, activation rate, and single-channel open probability of SLO-2. Bimolecular fluorescence complementation assays indicate that BKIP-1 interacts with SLO-2 carboxyl terminal. Thus, BKIP-1 may serve as an auxiliary subunit of SLO-2. BKIP-1 appears to be the first example that a single auxiliary subunit exerts opposite effects on evolutionarily related channels in the same cells.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Subunidades de Proteína/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Citosol/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismoRESUMEN
Neurons communicate through chemical synapses and electrical synapses (gap junctions). Although these two types of synapses often coexist between neurons, little is known about whether they interact, and whether any interactions between them are important to controlling synaptic strength and circuit functions. By studying chemical and electrical synapses between premotor interneurons (AVA) and downstream motor neurons (A-MNs) in the Caenorhabditis elegans escape circuit, we found that disrupting either the chemical or electrical synapses causes defective escape response. Gap junctions between AVA and A-MNs only allow antidromic current, but, curiously, disrupting them inhibits chemical transmission. In contrast, disrupting chemical synapses has no effect on the electrical coupling. These results demonstrate that gap junctions may serve as an amplifier of chemical transmission between neurons with both electrical and chemical synapses. The use of antidromic-rectifying gap junctions to amplify chemical transmission is potentially a conserved mechanism in circuit functions.