Membrane Potentials,Synaptic Responses,Neuronal Circuitry,Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell''s membrane potential and which we address in these experiments are: (1) Ion concentration of K⁺ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.     

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution^1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions ^7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin ¹⁸, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH < 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.     

Phosphoramidate derivates as controlled-release prodrugs of l-Dopa

l-Dopa has continued to be a mainstay in the symptomatic treatment of Parkinson’s disease (PD). However, extensive peripheral metabolism, a short systemic circulation half-life and development of motor complications called dyskinesia prevents its long-term utilization as a PD therapeutic. Herein, we report a series of phosphoramidate derivatives of l-Dopa and controlled release of l-Dopa at pH 7.4 and 3. The kinetic data for the release of l-Dopa support our hypothesis that a proximal carboxylic acid can promote the pH-triggered hydrolysis of the phosphoramidate PN bond. As expected, esterification of the proximal carboxylic acid protects the scaffold from rapid release at low pH. This latter observation is particularly noteworthy as it suggests that the phosphoramidate-based l-Dopa?conjugate scaffold can be adapted for oral administration as an ester prodrug.     

Differences in soil characteristics between field and forest may influence the distribution of an invasive earthworm

In northern North America, invasive earthworms (including the nightcrawler Lumbricus terrestris) have been dispersing from points of introduction and dramatically affecting soil structure, soil food webs, and forest floor dynamics. However, little is known about the factors influencing the local distribution of invasive earthworms south of the Wisconsinan glaciation. Earthworms were sampled at suspected sites of introduction near Mountain Lake Biological Field Station, Virginia, USA. The density of invasive earthworms decreased as distance from suspected sites of introduction increased; native earthworms displayed the opposite relationship. However, the distance that L. terrestris was found into the forest was less than expected given dispersal rates calculated from more northern invasions. We also found correlations among population densities of L. terrestris and physical–chemical properties of the soil, and differences between field and forest soils in terms of temperature, moisture, and soil chemical properties. We conducted two experiments to analyze some factors possibly responsible for the observed distribution: (1) temperature and moisture, and (2) soil type (field vs. forest) and food resources. Our results suggest that L. terrestris may not disperse as far into forested habitats of the Southern Appalachians compared to northern forests due to local physical‐chemical soil characteristics.     

Cellular characterization of the primosome and rep helicase in processing and restoration of replication following arrest by UV-induced DNA damage in Escherichia coli

Following arrest by UV-induced DNA damage, replication is restored through a sequence of steps that involve partial resection of the nascent DNA by RecJ and RecQ, branch migration and processing of the fork DNA surrounding the lesion by RecA and RecF-O-R, and resumption of DNA synthesis once the blocking lesion has been repaired or bypassed. In vitro, the primosomal proteins (PriA, PriB, and PriC) and Rep are capable of initiating replication from synthetic DNA fork structures, and they have been proposed to catalyze these events when replication is disrupted by certain impediments in vivo. Here, we characterized the role that PriA, PriB, PriC, and Rep have in processing and restoring replication forks following arrest by UV-induced DNA damage. We show that the partial degradation and processing of the arrested replication fork occurs normally in both rep and primosome mutants. In each mutant, the nascent degradation ceases and DNA synthesis initially resumes in a timely manner, but the recovery then stalls in the absence of PriA, PriB, or Rep. The results demonstrate a role for the primosome and Rep helicase in overcoming replication forks arrested by UV-induced damage in vivo and suggest that these proteins are required for the stability and efficiency of the replisome when DNA synthesis resumes but not to initiate de novo replication downstream of the lesion.     

The basic region of the diaphanous-autoregulatory domain (DAD) is required for autoregulatory interactions with the diaphanous-related formin inhibitory domain

Mammalian diaphanous-related (mDia) formins act as Rho GTPase effectors during cytoskeletal remodeling. Rho binding to mDia amino-terminal GTPase-binding domains (GBDs) causes the adjacent Dia-inhibitory domain (DID) to release the carboxyl-terminal Dia-autoregulatory (DAD) domain that flanks the formin homology-2 (FH2) domain. The release of DAD allows the FH2 domain to then nucleate and elongate nonbranched actin filaments. DAD, initially discovered as a region of homology shared between a phylogenetically divergent set of formin proteins, is comprised of a core motif, MDXLLXL, and an adjacent region is comprised of numerous basic residues, typically RRKR in the mDia family. Here, we show that these specific amino acids within the basic region of DAD contribute to the binding of DID and therefore the maintenance of the mDia autoregulatory mechanism. In addition, expression of full-length versions of mDia2 containing amino acid substitutions in either the DAD core or basic regions causes profound changes in the F-actin architecture, including the formation of filopodia-like structures that rapidly elongate from the cell edge. These studies further refine our understanding of the molecular contribution of DAD to mDia control and the role of mDia2 in the assembly of membrane protrusions.